B GIO DC V O TO TRNG I HC CN TH

NG THANH PHONG

PHN LP V TUYN CHN VI KHUN C NH M PSEUDOMONAS SPP.

BN CHO CY LA CAO SN VNG NG BNG SNG CU LONG

LUN N TIN S

Chuyn ngnh VI SINH VT HC M s 62 42 40 01

Ngi hng dn khoa hc PGS. TS. CAO NGC IP

Cn Th - 2012

B GIO DC V O TO TRNG I HC CN TH

NG THANH PHONG

PHN LP V TUYN CHN VI KHUN C NH M PSEUDOMONAS SPP.

BN CHO CY LA CAO SN VNG NG BNG SNG CU LONG

LUN N TIN S

Chuyn ngnh VI SINH VT HC M s 62 42 40 01

Cn Th - 2012

i

CNG HA X HI CH NGHA VIT NAM

c lp T do Hnh phc ------------------------------------

LI CAM OAN

Ti xin cam oan cng trnh nghin cu Phn lp v tuyn chn vi khun c

nh m Pseudomonas spp. bn cho cy la cao sn vng ng bng sng Cu

Long l ca ring ti. Cc s liu, kt qu nu trong lun n l trung thc v cha

tng c ngi khc cng b trong bt k cng trnh no khc.

Tc gi lun n

Ng Thanh Phong

ii

LI CM N

Xin chn thnh cm n Thy - PGS.TS. Cao Ngc ip, ngi tn tm

hng dn khoa hc, t vn v gip ti tip cn cc kha cnh khoa hc cho s thnh

cng ca ti nghin cu sinh.

Xin chn thnh cm n Ban Gim Hiu Trng i Hc Cn Th, Ban lnh

o Vin Nghin Cu v Pht Trin Cng Ngh Sinh Hc, Ban ch nhim Khoa Khoa

Hc T Nhin, Phng o To, Phng Qun L Khoa Hc, Khoa Sau i Hc v cc

Phng Ban chc nng khc ca Trng i Hc Cn Th to iu kin thun li.

Chn thnh cm n qu Thy C v anh ch ng nghip B mn Sinh hc,

Khoa Khoa Hc T Nhin v Vin Nghin Cu v Pht trin Cng Ngh Sinh Hc

to iu kin thun li, h tr v t vn cho ti trong thi gian hc tp cng nh thc

hin lun n ny.

Cm n ThS. Trn Th Xun Mai t vn thit k cp mi chuyn bit cho

Pseudomonas stutzeri A1501; cm n ng o Vn Sn (nng dn nng trng

Sng Hu) h tr t rung trin khai th nghim chng vi khun cho cy la cao

sn trng ngoi ng; cm n cc hc vin cao hc v cc sinh vin c s cng tc

trong thi gian qua.

Chn thnh cm n Hi ng nh gi lun n tin s cp c s c nhng

kin nhn xt v gp gip cho lun n c hon chnh hn trc khi trnh phn bin

kn v bo v lun n cp Trng; cm n hai nh khoa hc tham gia phn bin kn

cho lun n tin s; xin cm n Hi ng nh gi lun n tin s cp Trng.

Cm n gia nh v nhng ngi thn chia s, ng vin v to iu kin

thun li ti c an tm hc tp v hon thnh lun n tin s.

Xin chn thnh cm n!

Ng Thanh Phong

iii

TM LC

ti Phn lp v tuyn chn vi khun c nh m Pseudomonas spp. bn

cho cy la cao sn vng ng bng sng Cu Long c thc hin t thng 11 nm

2008 n thng 9 nm 2011. Nhng ni dung ca ti c thc hin nhm t n

mc tiu tuyn chn c mt s dng vi khun Pseudomonas spp. c kh nng c nh

m hu hiu vi cy la cao sn. Kt qu nghin cu ca ti t c nh sau:

Phn lp c 150 dng vi khun c nh m t 130 mu t vng r la ca

13 tnh thnh ng bng sng Cu Long trn mi trng Pseudomonas Isolation

Agar (Difco). Tt c 150 dng vi khun u c kh nng tng hp NH4+ trong c

86/150 dng c kh nng tng hp NH4+ cao hn 5 mg/l.

Cc dng tng hp NH4+ cao c phn tch PCR-16S rRNA vi cp mi c

hiu FGPS4-281bis v FGPS1509-153 nhn din Pseudomonas. Kt qu c 55/86

dng vi khun c bng v tr 1500 bp so vi thang chun. Tip tc phn tch PCR-

nifH rRNA vi cp mi c hiu PolF-115 v PolR-476 nhn din Pseudomonas c

on gen nifH , pht hin 32/55 dng vi khun c bng tng ng 361 bp so vi

thang chun.

Chn 20 trong s 32 dng vi khun c on gen nifH kim chng li kh

nng c nh nit v c 20 dng vi khun u biu hin hot tnh ca nitrogenase

thng qua phng php kh acetylene (ARA). nh gi hiu qu ca 20 dng vi

khun ny ln chiu cao v trng lng kh ca cy la cao sn trng trong dung dch

khong (khng s dng m) trong 20 ngy, chn c 6 dng vi khun c hu

hiu cao tin hnh th nghim trn cy la cao sn trng trong chu nh li. Kt

qu th nghim xc nh c 6 dng vi khun c kh nng cung cp 25 75% nhu

cu m sinh hc cho s pht trin ca cy la trng trong chu.

Chn 11 dng vi khun hu hiu nht (bao gm 6 dng hiu qu vi cy la

trng trong chu v 5 dng khc cng c trin vng c nh m cao) tin hnh

nh danh. S dng cp mi PST3422-5 v PST3422-580 (c thit k nhn din gen

nifH ca Pseudomonas stutzeri A1501), pht hin 4/11 dng c bng tng ng

575 bp v tng ng vi bng ca dng vi khun i chng l Pseudomonas stutzeri

A1501 trong ph in di ca cc sn phm PCR. Kt qu gii trnh t DNA v so snh

vi ngn hng d liu NCBI cho thy c 4 dng ny u tng ng di truyn 98-99%

iv

so vi Pseudomonas stutzeri. Trong , hai dng AG9 v BT2 tng ng 99%, hai

dng TG1 v BT1 tng ng 98% vi dng CP000304.1 Pseudomonas stutzeri

A1501 v dng CP002881.1 Pseudomonas stutzeri ATCC 17588. DNA ca 7/11 dng

vi khun cn li c tip tc gii trnh t (theo sn phm PCR da trn cp mi

FGPS4-281bis v FGPS1509-153 tng ng vi on 16S rRNA ca Pseudomonas)

v so snh vi cc dng c trong ngn hng d liu NCBI. Kt qu cho thy c 7 dng

ny u tng ng di truyn 97-100% so vi cc loi thuc ging Burkholderia.

Trong , dng CT1 tng ng 100% vi dng AB568313.1 Burkholderia

vietnamiensis 16S-rRNA, dng VL2 tng ng 99% vi FJ436055.1 Burkholderia

vietnamiensis dng SYe-6586, dng KG14 tng ng 99% vi DQ979871.1

Burkholderia vietnamiensis dng AU0829, dng VL8 tng ng 98% vi

EF158393.1 Burkholderia vietnamiensis dng TVV75, dng KG1 tng ng 98%

vi DQ979872.1 Burkholderia vietnamiensis dng AU0913, dng CM1 v KG8 cng

tng ng 97% vi AY098590.1 Burkholderia kururiensis dng KP23 (dng KG8

cng tng ng 97% vi AY098588.1 Burkholderia brasilensis dng M113) c trong

ngn hng d liu NCBI.

Th nghim ngoi ng c thc hin nh gi kh nng c nh m ca 4

dng vi khun P. stutzeri PS1 (TG1), P. stutzeri PS4 (BT1), B. vietnamiensis BV3

(KG1) v B. vietnamiensis BV5 (CT1) (c chn lc t 6 dng vi khun th

nghim trong nh li) trn cy la cao sn (OM2517) trng trn t ph sa ti nng

trng Sng Hu Cn Th vo v H Thu 2011. Kt qu cho thy tng dng vi

khun P. stutzeri PS4 v B. vietnamiensis BV3 u cung cp n 50% m sinh hc

trong khi dng P. stutzeri PS1 v B. vietnamiensis BV5 ch cung cp c 25% nhu

cu m sinh hc cho s pht trin ca cy la cao sn.

T kha: Burkholderia vietnamiensis, c nh m sinh hc, t ph sa, t vng r,

gen nif, la cao sn, Pseudomonas stutzeri

v

SUMMARY

Thesis "Isolation and selection of nitrogen-fixing bacteria Pseudomonas

spp., its application on high-yielding rice cultivated on the alluvial soil of the

Mekong Delta" was done from November 2008 to September 2011. The aim of this

study was isolation and selection Pseudomonas spp. strains with the high biological

nitrogen fixation (BNF) ability to apply to high-yielding rice cultivated on the soil of

the Mekong Delta. The results achieved as follows:

One hundred and fifty isolates were isolated from 130 soil samples (rice

rhizosphere soils of 13 provinces in Mekong Delta). All of them were able to

synthesize NH4+, 86/150 isolates synthesized NH4

+ higher than 5 mg/l.

The effective isolates (high NH4+ biosynthesis) were analysed by PCR-16S

rRNA technique with primers FGPS4-281bis and FGPS1509'-153 to identify

Pseudomonas spp. The results showed that 55/86 isolates having band at 1500 bp in

comparision to standard ladder; 32/55 isolates were determined nifH gene with PCR

technique with specific primer PolF-115 and PolR-476 which had band at 361 bp in

electrophoresis gel.

Twenty isolates in 32 isolates had high nitrogenase activity through ARA

method. Screening of 20 isolates by evaluation of 20 isolatess effectiveness on rice

cultivated mineral solution free N in 20 days (in-vitro), the results showed that six

isolates having high BNF ability manifested on height and dry weight of high-yielding

rice. With in-pots experiment, these six isolates provided 25 to 75% nitrogen

requirement in the rice growth.

Using primer PST3422-5 and PST3422-580 in PCR technique identified 4/11

isolates having band 575 bp together with reference strain (P. stutzeri A1501) in

electrophoresis. Isolates were sequenced, DNA sequencing were compared with

GenBank database of NCBI by BLAST N software; the results showed that four

isolates were similarity of 98-99% with P. stutzeri, such as AG9 and BT2 strains were

similarity of 99%, TG1 and BT1 strains were similarity of 98% with both strains

CP000304.1 Pseudomonas stutzeri A1501 and CP002881.1 Pseudomonas stutzeri

ATCC 17588. Besides that, seven isolates were further sequenced with DNA from

PCR-16S rRNA products, the results showed that all of them were 97-100% similarity

vi

with species of the genus Burkholderia, such as CT1 strain was similarity of 100%

with AB568313.1 Burkholderia vietnamiensis 16S-rRNA, VL2 strain was similarity of

99% with FJ436055.1 Burkholderia vietnamiensis strain SYe-6586, KG14 strain was

similarity of 99% with DQ979871.1 Burkholderia vietnamiensis strain AU0829, VL8

strain was similarity of 98% with EF158393.1 Burkholderia vietnamiensis strain

TVV75, KG1 strain was similarity of 98% with DQ979872.1 Burkholderia

vietnamiensis strain AU0913, two strains CM1 and KG8 strain were similarity of 97%

with AY098590.1 Burkholderia kururiensis strain KP23 (in which one isolates the

identity 97% with AY098588.1 Burkholderia brasilensis strain M113) in the GenBank

database of NCBI.

A field experiment was conducted to evaluate biological nitrogen fixation

ability of four isolates (P. stutzeri PS1, P. stutzeri PS4, B. vietnamiensis BV3 and B.

vietnamiensis BV5 and they were selected from in-vitro and in-pots experiments) on

high-yielding rice (OM2517) cultivated on alluvial soil of Song Hau farm, Can Tho

city in Summer-Autumn cropping-season 2011. The results showed that PS4 strain

and/or BV3 strain had biological nitrogen fixation ability equivalent to 50% inorganic

fertilizer while two strains (PS1 and/or BV5) only provided 25% nitrogen requirement

for rice growth.

Keywords: alluvial soil, biological nitrogen fixation, Burkholderia vietnamiensis,

high-yielding rice, nif gene, Pseudomonas stutzeri, rhizosphere soil

vii

MC LC

Trang

Li cam oan ............................................................................................................ i

Li cm n ............................................................................................................... ii

TM LC ............................................................................................................ iii

SUMMARY .............................................................................................................. v

MC LC ................................................................................................................ vii

DANH MC CC CH VIT TT ..................................................................... xi

DANH SCH BNG .............................................................................................. xii

DANH SCH HNH ............................................................................................... xiii

CHNG I: M U TNG QUAN TI

.1. Tnh cp thit ca ti ..................................................................................... 1

.2. Mc tiu v nhim v nghin cu ..................................................................... 3

.3. i tng v phm vi nghin cu ..................................................................... 4

.4. Thi gian v a im nghin cu ..................................................................... 4

.5. Nhng ng gp ca lun n ............................................................................ 5

.6. C s l lun v gi thuyt khoa hc ................................................................ 6

CHNG II: TNG QUAN TI LIU

2.1. C nh m sinh hc ................................................................. 7

2.1.1. Khi qut v c nh m sinh hc ...................................................... 7

2.1.2. Chu trnh nit ........................................................................ 8

2.1.3. Vi khun c nh m sng t do ............................. 10

2.2. Tng quan v vi khun Pseudomonas ................................................................ 11

2.2.1. c im ca Pseudomonas ................................................................. 11

2.2.2. Phn loi Pseudomonas ........................................................................ 13

2.2.3. Nhng loi Pseudomonas c kh nng c nh m ............................ 15

2.2.4. Phn lp v xc nh Pseudomonas ..................................................... 16

2.3. C ch c nh m ca Pseudomonas .............................................................. 17

viii

2.3.1. Enzyme nitrogenase ............................................................................. 17

2.3.2. B gen ca Pseudomonas v s iu khin tng hp nitrogenase ..... 20

2.3.3. C ch c nh m .............................................................................. 24

2.3.4. Cc yu t nh hng n qu trnh c nh m ................................ 25

2.4. ng dng c nh m ca Pseudomonas .......................................................... 28

2.4.1. Mt s ng dng .................................................................................. 28

2.4.2. Trin vng ng dng Pseudomonas trong tng lai ............................ 30

2.5. Vai tr ca m i vi cy la .......................................................................... 31

CHNG III: NI DUNG, PHNG TIN V PHNG PHP NGHIN

CU

3.1. Ni dung nghin cu ......................................................................................... 33

3.2. Phng tin nghin cu ..................................................................................... 33

3.2.1. Vt liu ................................................................................................. 33

3.2.2. Dng c ................................................................................................ 34

3.2.3. Thit b ................................................................................................. 34

3.2.4. Cc loi ha cht v mi trng nui cy vi khun ............................. 35

3.3. Phng php nghin cu

3.3.1. Thu mu t vng r la ...................................................................... 38

3.3.2. Phn lp vi khun c nh m t t vng r la .............................. 38

3.3.3. Xc nh kh nng c nh m (in-vitro) ca vi khun ..................... 40

3.3.3.1. Nui cy vi khun trong mi trng Burk khng m ..................... 40

3.3.3.2. o hm lng NH4+ bng phng php Phenol Nitroprusside...... 41

3.3.3.3. Th hot tnh nitrogenase bng phng php kh acetylen (ARA)... 42

3.3.4. Kim tra vi khun bng phng php sinh hc phn t ....................... 43

3.3.4.1. Tch chit DNA (Neumann et al., 1992) .......................................... 43

3.3.4.2. Khuch i DNA .............................................................................. 45

3.3.4.3. in di cc sn phm PCR ............................................................... 46

3.3.4.4. Chp hnh gel in di cha sn phm phn ng PCR ........ .............. 46

3.3.4.5. Gii trnh t DNA v so snh vi ngn hng d liu NCBI ............. 47

ix

3.3.5. nh gi hiu qu c nh m sinh hc ca cc dng vi khun

vi cy la cao sn ............................................................................. 47

3.3.5.1. nh hng ca vi khun ln cy la trng trong ng nghim .......... 47

3.3.5.2. Hiu qu c nh m ca vi khun vi cy la trng trong chu .... 49

3.3.5.3. Hiu qu c nh m ca vi khun vi cy la ngoi ng ............ 50

3.3.6. Kho st mt s vi khun nui cy trong mi trng lng .................. 51

3.3.7. Thng k, x l v phn tch s liu .... 52

CHNG IV: KT QU V THO LUN

4.1. Kt qu thu mu t vng r la ........................................................................ 53

4.2. Kt qu phn lp vi khun c nh m ............................................................. 53

4.2.1. Cc dng vi khun c phn lp 53

4.2.2. c im ca khun lc v t bo vi khun . 54

4.3. Kt qu nh gi kh nng c nh m ca vi khun ....................................... 56

4.3.1. Kh nng tng hp NH4+... 56

4.3.2. Hot tnh nitrogenase ............ 60

4.4. Kim tra vi khun bng phng php sinh hc phn t ..................................... 63

4.4.1. Nhn din cc dng vi khun bng k thut PCR................................. 63

4.4.2. Gii trnh t DNA v so snh vi ngn hng d liu NCBI ................ 65

4.5. Hiu qu c nh m ca cc dng vi khun vi cy la cao sn .................... 72

4.5.1. nh hng ca vi khun vi cy la trng trong ng nghim ..... 72

4.5.2. Hiu qu c nh m ca vi khun vi cy la trong chu ............. 73

4.5.3. Hiu qu c nh m ca vi khun vi cy la ngoi ng ................ 77

CHNG V: KT LUN V NGH

5.1. Kt lun ............................................................................................................... 87

5.2. ngh ................................................................................................................88

DANH MC CC CNG TRNH CNG B ............................................. 89

TI LIU THAM KHO ....................................................................................... 90

x

PH LC

Ph lc 1: Mt s loi mi trng nui cy vi khun .............................................. a

Ph lc 2: Mi trng trch DNA v phn ng PCR ............................................... b

Ph lc 3: Chun b gel agarose v in di ............... ............................................... d

Ph lc 4: Qui trnh gii trnh t DNA ...................................................................... e

Ph lc 5: Phn bn cho cy la th nghim trong chu v ngoi ng .................... h

Ph lc 6: Phn tch m tng s bng phng php micro-Kjeldahl ...................... j

Ph lc 7: ng chun o NH4+ .............................................................................. k

Ph lc 8: Bng kt qu thu mu t vng r la v phn lp vi khun .................... k

Ph lc 9: Bng c im khun lc v hnh dng t bo ca 150 dng vi khun. m

Ph lc 10: Kh nng pht trin v tng hp NH4+ ca 150 dng vi khun.............. p

Ph lc 11: X l s liu ca th nghim trng la in-vitro....................................... u

Ph lc 12: X l s liu ca cc th nghim trong chu (in-pots) ............................ w

Ph lc 13: X l s liu ca cc th nghim ngoi ng ......................................... aa

Phc l 14: Trnh t DNA ca cc dng vi khun BV v BK ....... hh

Ph lc 15: Bng kt qu kh acetylen ca 20 dng vi khun . oo

xi

DANH MC CC CH VIT TT

16S-rRNA/DNA gene 16S-ribosomal RNA/DNA coding gene

ARA Acetylene reduction assay

BNF Biological nitrogen fixation

Blast Basic local alignmeht search tool

BV, BK Burkholderia vietnamiensis, Burkholderia kururiensis

DNA, rRNA Deoxyribonucleic acid, ribosomal ribonucleic acid

dNTP Deoxy nucleoside triphotphate

BSCL ng bng sng Cu Long

EDTA Ethylenediaminetetraacetic acid

EtBr Ethidium Bromide

Gen nif Nitrogen fixing gene

HG, ST, BL, CM Hu Giang, Sc Trng, Bc Liu, C Mau

KG, AG, T, TV Kin Giang, An Giang, ng Thp, Tr Vinh

LA, TG, VL, BT, CT Long An, Tin Giang. Vnh Long, Bn Tre, Cn Th

NCBI National centre for biotechnology information

NT Nghim thc

PCR Polymerase chain reaction

PS Pseudomonas stutzeri

xii

DANH SCH BNG

Bng Tn bng Trang

3.1 Bng s liu xy dng ng chun NH4+

41

3.2 Cc nghim thc trng la trong ng nghim 48

3.3 Cc nghim thc trng la trong chu 49

3.4 Cc nghim thc trng la ngoi ng 50

4.1 Cc dng vi khun c phn lp t t vng r la ng bng sng

Cu Long

53

4.2 T l phn trm v cc c im khun lc v t bo vi khun 55

4.3 Bng tng hp s pht trin v kh nng tng hp NH4+ ca cc dng

vi khun trong mi trng Burk lng khng m

58

4.4 Mt s vi khun (sau 2 ngy nui cy) v hm lng NH4+ c tng

hp 20 dng vi khun

60

4.5 Hm lng NH4+ (mg/l) v hm lng nitrogenase (mM) c tng

hp 10 dng vi khun sau 2 ngy nui cy (t 1)

61

4.6 Hm lng NH4+ (mg/l) v hm lng nitrogenase (mM) c tng

hp 10 dng vi khun sau 2 ngy nui cy (t 2)

62

4.7 Hiu qu ca 20 dng vi khun ln chiu cao v trng lng kh cy

la giai on 20 ngy tui

73

4.8 Hiu qu ca 6 dng vi khun v phn m ha hc trn thnh phn

nng sut v nng sut la cao sn (th nghim trong chu)

75

4.9 Kh nng c nh m sinh hc ca 6 dng vi khun cho nhu cu sinh

trng v pht trin ca cy la cao sn (OM2517) (th nghim trng

la trong chu)

77

4.10 Hiu qu ca 4 dng vi khun v phn m ha hc trn thnh phn

nng sut la cao sn (OM2517) trng trn t ph sa nng trng Sng

Hu, huyn C , Cn Th (v H Thu 2011)

78

xiii

DANH SCH HNH

Hnh Tn hnh Trang

2.1 Chu trnh nit 9

2.2 Quan h tng tc gia vi sinh vt v thc vt nh hng n s tng

trng ca thc vt

10

2.3 Mt s ngun nit cung cp cho cy 11

2.4 Pseudomonas di knh hin vi in t 12

2.5 Khun lc ca Pseudomonas stuzeri v Pseudomonas fluorescens 15

2.6 Cy di truyn (phylogenetic tree) biu din mc tng quan di

truyn gia cc loi Pseudomonas da vo kt qu phn tch di truyn

phn t

16

2.7 Cy di truyn (phylogenetic tree) ca cc loi Burkholderia 17

2.8 Nitrogenase c kh nng xc tc chuyn ha, kh N2 thnh NH3 18

2.9 Cc thnh phn protein cu thnh nitrogenase 19

2.10 Vng c nh m (nitrogen fixation region) ca Pseudomonas

stutzeri A1501

22

2.11 S biu din c ch c nh m sinh hc 25

2.12 Cc phn ng c nh m trong nitrogenase 26

3.1 S phn lp vi khun t mu t vng r la cho n khi tr rng 40

3.2 Cc ng nghim xy dng ng chun o ammonium 42

3.3 Trng la trong dung dch khong c b sung 0,8% agar 48

3.4 S pha long mt s vi khun 51

4.1 Hnh nh mt s khun lc (Theo th t t tri qua phi l cc dng

vi khun TG7, BL4, LA7)

55

4.2 Dng KG7 c chp di knh hin vi in t qut JSM 5500

phng i 8500 ln

56

4.3 Dng VL2 c chp di knh hin vi in t qut JSM 5500

phng i 10000 ln

56

4.4 Mc tng hp NH4+ ca cc dng vi khun 57

4.5 Ph in di sn phm PCR c nhn ln t DNA ca cc dng vi

khun Pseudomonas vi cp mi tng (M: thang chun 100bp plus)

63

xiv

Hnh Tn hnh Trang

4.6 Ph in di sn phm PCR c nhn ln t DNA ca cc dng vi

khun vi cp mi xc nh gen nifH (Thang chun 100bp)

64

4.7 Ph in di ca sn phm PCR c nhn ln t DNA ca 5 dng P.

stutzeri A1501 (PS5), TG1, AG9, BT2 v BT1

65

4.8 So snh trnh t DNA ca dng PS1 vi GenBank 66

4.9 So snh trnh t DNA ca dng PS2 vi GenBank 66

4.10 So snh trnh t DNA ca dng PS3 vi GenBank 67

4.11 So snh trnh t DNA ca dng PS4 vi GenBank 67

4.12 So snh trnh t DNA ca dng PS5 Pseudomonas stutzeri A1501

(dng vi khun i chng) vi GenBank

68

4.13 So snh trnh t ca 5 dng P. stutzeri (PS1, PS2, PS3, PS4 v PS5)

vi nhau cho thy dng PS1 v PS3 ch khc nhau 3 v tr; dng PS4

khc PS2 v PS5 (A1501) 8 v tr.

69

4.14 Cy ph h (phylogenetic tree) trnh by mi quan h v mt di truyn

gia 4 dng vi khun vi dng chun P. stutzeri A1501

70

4.15 Hiu qu ca vi khun PS1 v phn m ha hc trn s pht trin

ca cy la v PS4 ln chiu cao cy la cao sn OM2517

77

4.16 Rung la th nghim ti Nng trng Sng Hu (tri) v cc bi la

cc nghim thc chng P. stutzeri PS4 (phi) c 89 ngy sau khi

gieo s (trc khi thu hoch 1 ngy).

79

4.17 Hiu qu ca 4 chng vi khun v phn m ha hc trn nng sut

la cao sn (tn/ha) trng trn t ph sa Nng trng Sng Hu, Cn

Th (v H Thu 2011).

80

4.18 Tng quan tuyn tnh gia chiu cao cy v s bng/m2 vi nng

sut la mc ngha 5%

81

4.19 Hiu qu ca 4 chng vi khun v phn m ha hc ln hm lng

protein trong go (%)

82

4.20 Tng quan tuyn tnh gia hm lng protein trong go (%) vi

nng sut la (tn/ha) mc ngha 5%

83

4.21 Hiu qu ca 4 chng vi khun v phn m ha hc ln nit tng s

(%) ca t trng la Nng trng Sng Hu, Cn Th

84

xv

1

CHNG I

M U - TNG QUAN V TI

.1. Tnh cp thit ca ti

ng bng sng Cu Long c din tch gn 4 triu ha, trong c 1,7 triu ha t

nng nghip c s dng trng la vi din tch canh tc la hng nm ln n 3,9

triu ha (Nguyn Th Lang v Bi Ch Bu, 2008). Phn bn ni chung v phn m

ho hc ni ring gp phn quan trng trong vic gia tng nng sut cy trng.

m bo nng sut, nng dn s dng rt nhiu phn bn nhng hin nay gi c

phn bn ha hc ngy cng tng cao lm tng gi thnh sn xut v gim hiu qu

kinh t trong nng nghip. S lm dng phn m ha hc s dn n chi ph cao,

ng thi cng s dn n nhng hu qu nh thay i l, ha tnh ca t, gim

ph, mt cn bng sinh thi... gy nh hng tiu cc ln h sinh thi nn khng m bo

thn thin vi mi trng canh tc trong nng nghip (Kannaiyan, 1999). Theo V Minh

Kha (2003), cy la hp thu ch khong 50-60% lng m c bn vo trong t. S

cn li s c lu tn trong t hoc b ra tri gy hng n mi trng nc. Bn

qu nhiu phn m ha hc cho cy trng gp phn dn n chi ph sn xut cao, hiu

qu kinh t thp, ng thi khng m bo cho mt h sinh thi pht trin bn vng.

khc phc nhng tc hi do s dng qu nhiu phn m ha hc th vic s

dng phn m sinh hc c cha cc dng vi khun c kh nng c nh m (BNF:

biological nitrogen fixation) l mt trong nhng bin php c hiu qu m khng gy

nhim mi trng, tit kim chi ph sn xut nhng vn m bo cht lng ng

thi vn tng nng sut nng sn. Vic nghin cu v ng dng vi khun c nh m

sinh hc v ang l nhng ti c nghin cu rng ri khp th gii. Nhng vi

khun c nh m c cc nh khoa hc nghin cu v ng dng nh vi khun

Rhizobium (Keyser v ctv., 1982; Scholla v Elkan, 1984); Bradyrhizobium c nh

m vi cy h u (Elkan, 1992; Dowdl v Bohlool, 1987; Buendia-Clavaria v ctv.,

1994); vi khun Azospirillum c kh nng c nh m, tng hp IAA, ha tan ln v

2

cc cht dinh dng khc (Bilal v ctv., 1990; Seshadri v ctv., 2000; Somers v ctv.,

2005; Lng Ngc Du v ctv., 2007), loi Azospirillum lipoferum cng c phn

lp t cy la ng bng sng Cu Long (Cao Ngc ip v ctv., 2007); vi khun

Azotobacter c kh nng c nh m (Dbereiner, 1974), nh loi Azotobacter

paspali l loi vi khun ni sinh c hiu cho cy c Paspalum notatum v khoai lang

(Dbereiner, 1976); vi khun Gluconacetobacter diazotrophicus c mt s c tnh

nh c nh m, tng hp kch thch t tng trng thc vt (phytohormone) nh

auxin v gibberellin, lm gim pH mi trng ha tan ln kh tan (Bastian v

ctv., 1998; Muthukumarasamy v ctv., 2002a; Tejera v ctv., 2003; Madhaiyan v ctv.,

2004), chng thng hin din t quanh r cy c ph, khm, la, khoai lang

(Jimenaz-Salgalo v ctv., 1997; Hernandez v ctv., 2000; Muthukumarasamy v ctv.,

2002b) ng thi cng xc nh c nhm vi khun ny c kh nng c nh m

mnh m lm tng sn lng ma ng, khm, khoai lang (Prabudoss v Stella,

2009); vi khun Burkholderia c kh nng c nh m v to nt sn vi nhng cy

h u vng nhit i (Mounlin v ctv., 2001), cng sinh vi nhiu loi cy trng gip

c nh m v kch thch s tng trng khi chng hin din t quanh vng r v

trong r ca mt s loi cy trng nh bp, ma, c ph, la (Scarpella v ctv., 2003;

Chen v ctv., 2006), khm, chui (Weber v ctv., 1999) v loi Burkholderia

vietnamiensis c tm thy trong r la trng min Nam Vit Nam (Gillis v

ctv., 1995); Mt s chng Pseudomonas c nh hng quan trng trong s sinh trng

v pht trin thc vt nh tng hp kch t tng trng thc vt nh: auxin, cytokinin,

kch thch s pht trin ca b r cy lm gia tng kh nng hp thu cht dinh dng

trong t mt s loi nh: Pseudomonas putida, Pseudomonas fluorescens,

Pseudomonas syringae (Glickmann et al, 1998; Patten v Glick, 1987; Suzuki v ctv.,

2003; Xie v ctv., 1996), ha tan ln dng kh tan thnh dng d tan gip cy trng

hp th nh: Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas

chlororaphis (Cattenlla v ctv., 1999), c nh m nh Pseudomonas stutzeri

(Krotzky v Werner, 1987).

3

Cc loi thuc ging Pseudomonas c kh nng c nh m c khng nh

t lu (Chan v ctv., 1994), trong c Pseudomonas stutzeri (Krotzky v Werner,

1987; Vermeiren, 1999). Ngoi ra, cc nh khoa hc cng xc nh mt s loi vi

khun c phn lp t t vng r la c kh nng c nh m v gip tng nng

sut la (Gillis v ctv., 1995; Tran Van v ctv., 2000; Nguyn Ngc Dng v ctv.,

2000).

Hin nay, cc nh khoa hc tp trung nghin cu v s dng cc chng vi khun

c nh m sinh hc. Vic nghin cu ng dng cc chng vi khun c kh nng c

nh m hu hiu bn cho cy la ng bng sng Cu Long mang tnh cp thit

nhm gp phn gim s dng phn m ha hc cho cy la nhng vn gi vng nng

sut, bo v mi trng v gp phn m bo cho s pht trin nng nghip bn vng

trong khu vc. Do , ti Phn lp v tuyn chn vi khun c nh m

Pseudomonas spp. bn cho cy la cao sn vng ng bng sng Cu Long c

thc hin nhm nhn din v xc nh c nhng dng Pseudomonas spp. c kh nng

c nh m hu hiu trn cy la cao sn.

.2. Mc tiu v nhim v nghin cu

1.2.1. Mc tiu nghin cu

- Mc tiu chnh: Chn lc c mt s dng vi khun Pseudomonas spp.

mang gen nif c kh nng c nh m hu hiu, cung cp 25-50% nhu cu

m cho qu trnh sinh trng v pht trin ca cy la cao sn.

- Mc tiu c th: (1) Phn lp v tch rng cc dng Pseudomonas bn a

c trong t vng r la ng bng sng Cu Long lm ngun vi khun c

nh m vi cy la cao sn; (2) Kho st c im, kh nng c nh m

sinh hc v nhn din vi khun Pseudomonas trn cc mc hnh thi, ha

sinh v sinh hc phn t (DNA); (3) nh gi hiu qu c nh m ca cc

dng vi khun vi cy la cao sn mc in-vitro cho n nh li v

ngoi ng rung.

4

1.2.2. Nhim v nghin cu

- Phn lp vi khun Pseudomonas spp. t t vng r la ng bng sng

Cu Long da trn mi trng chuyn bit, ng thi kim tra v nhn din

cc dng vi khun bng cc phng php ha sinh v phng php sinh hc

phn t.

- Xc nh s hin din gen nif v nh gi kh nng c nh m ca cc

dng vi khun Pseudomonas spp. phn lp c.

- Chn lc cc dng vi khun Pseudomonas spp. da trn c s cc th

nghim in-vitro v trong nh li tin hnh xc nh hiu qu c nh

m ca cc dng vi khun trn cy la cao sn trng ngoi ng.

.3. i tng v phm vi nghin cu

i tng nghin cu ca ti: cc dng vi khun Pseudomonas spp. c kh

nng c nh m vi cy la cao sn.

Phm vi nghin cu: cc dng vi khun Pseudomonas spp. bn a c phn

lp t t vng r la trng 13 tnh thnh ng bng sng Cu Long, c mang gen

nif v c kh nng c nh m hu hiu vi cy la cao sn trng trn t ph sa

Nng trng Sng Hu, huyn C , Cn Th.

.4. Thi gian v a im nghin cu

1.4.1. Thi gian nghin cu

- Quyt nh cng nhn Nghin cu sinh s 6919/Q-BGDT ngy 15/10/2008

ca B trng B Gio dc v o to.

- Thi gian o to theo h khng tp trung l 4 nm (11/2008 11/2012), theo

Quyt nh giao ti v ngi hng dn Nghin cu sinh s 468/Q-HCT

ngy 13/11/2008 v Quyt nh c cn b, vin chc i o to Nghin cu

5

sinh s 1878/Q-HCT ngy 25/12/2008 ca Hiu Trng trng i Hc

Cn Th.

- Thi gian nghin cu thc t ca nghin cu sinh cho n khi hon thnh cc

ni dung nghin cu theo cng ca ti: 11/2008 9/2011.

1.4.2. a im nghin cu

- Cc rung la c thu mu t vng r 13 tnh thnh ng bng sng Cu

Long.

- Phng th nghim Sinh hc t bo v Nh li thuc Khoa Khoa Hc T

Nhin; Phng th nghim Vi sinh vt t v Phng th nghim Sinh hc phn t

thuc Vin Nghin Cu v Pht Trin Cng Ngh Sinh Hc; Phng th nghim

Chuyn su thuc Phng Qun L Khoa Hc, trng i Hc Cn Th.

- Rung la ca ng o Vn Sn, nng dn p 3, x Thi Hng (Nng trng

Sng Hu), huyn C , TP. Cn Th.

.5. Nhng ng gp ca lun n

- Phn lp c 150 dng vi khun t t vng r la ng bng sng Cu

Long bng mi trng Pseudomonas Isolation Agar (Difco). Dng phng

php Phenol Nitroprusside xc nh c 150 dng vi khun ny u c

kh nng tng hp NH4+, trong c 18 dng tng hp c lng NH4

+ trn

10 mg/l, gp phn tm ra ngun vi sinh vt c nh m sinh hc cung cp cho

cy la cao sn.

- Xc nh c s hin din ca gen nifH 32 dng vi khun, trong c 11

dng vi khun trin vng c gii trnh t DNA, gm c 4 dng vi khun

c xc nh tng ng 98-99% vi Pseudomonas stutzeri, 5 dng vi khun

c xc nh tng ng 98-100% vi Burkholderia vietnamiensis v 2 dng

c xc nh tng ng 97% vi Burkholderia kururiensis, trong c 1

dng cn tng ng 97% vi Burkholderia brasilense [theo Yabuuchi v ctv.

6

(1992), Burkholderia c phn loi t vic xc nh v tch ra t mt s dng

vi khun Pseudomonas ban u].

- S dng 6 dng vi khun c trin vng ng dng c nh m chng cho cy

la trng trong chu, xc nh c 6 dng ny u c kh nng cung cp

25-75% nhu cu m sinh hc cho s sinh trng v pht trin ca cy la cao

sn OM2517 trng trong chu. C 4/6 dng vi khun trin vng trong chu

c th nghim chng cho cy la cao sn trng Nng trng Sng Hu

Cn Th. Kt qu cho thy tng dng vi khun P. stutzeri PS4 (TG1) v B.

vietnamiensis BV3 (KG1) u c kh nng cung cp n 50%N trong khi dng

P. stutzeri PS1 (BT1) v B. vietnamiensis BV5 (CT1) m bo c 25% nhu

cu m cho s pht trin ca cy la cao sn.

.6. C s l lun v gi thuyt khoa hc

- ng bng sng Cu Long hin ang trng nhiu ging la cao sn v nhng

nghin cu gn y cng cho thy c s hin din ca Pseudomonas spp.

vng r ca nhiu loi cy trng trong c cy la cao sn. Tuy nhin, vic

xc nh kh nng c nh m hu hiu ca vi khun Pseudomonas spp. trn

cy la cao sn vn cn hn ch. V vy, nghin cu v vi khun Pseudomonas

spp. ang c quan tm, c bit l tm hiu v kh nng c nh m vi s

hin din ca gen nifH cc dng vi khun ny.

- Gi thnh phn ha hc trn th trng hin ang tng cao l gnh nng cho

nng dn, ng thi vic s dng nhiu phn ha hc s dn n nhim mi

trng, khng m bo s pht trin bn vng, nh hng n s pht trin

kinh t x hi. V vy, hin nay c cc c s sn xut ang ch n phng

n sn xut phn sinh hc. Do , nghin cu v tuyn chn cc dng vi khun

Pseudomonas spp. c kh nng c nh m hu hiu, d nhn nui vi gi

thnh r bn cho cy la cao sn l hng nghin cu cn thit.

7

CHNG II

TNG QUAN TI LIU

2.1. C nh m sinh hc

2.1.1. Khi qut v c nh m sinh hc

C nh m sinh hc l qu trnh kh N2 thnh NH3 di s xc tc ca

enzyme nitrogenase. Sau , NH3 c th kt hp vi cc acid hu c to thnh cc

acid amin v protein. Trong thc t ch c mt s t loi vi sinh vt c kh nng c

nh N2 khng kh, l vi khun lam (cyanobacteria) v mt s vi khun c kh nng

c nh m. Vi khun c nh m c th cng sinh vi nhng cy h u nh ging

vi khun Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium

v Allorhizobium, Methylobacterium (Tan v ctv., 2001; Sy v ctv., 2001); vi khun

c nh m cng c th sng t do nh Azotobacter, Beijerinckia, Klebsiella,

Pseudomonas hoc c th sng t do nhng cng c th ni sinh nh Burkholderia

(Mattos v ctv., 2008).

Qu trnh c nh m xy ra trong t bo ca cc vi sinh vt u ging nhau l

nh chng c h thng gen nif (ni l ch vit tt ca nitrogen nit v f l fixing c

nh) iu khin qu trnh tng hp enzyme nitrogenase. Nitrogenase l h enzyme

xc tc cho phn ng kh N2 thnh NH3. Nh vy, h thng gen nif c xem l h

thng gen iu khin cho qu trnh c nh m sinh hc. Tuy nhin, vi khun lam,

qu trnh c nh m khng phi xy ra bt k t bo no m ch c th xy ra d

bo (heterocyst) (Heldt, 1999).

Trong t nhin c nhng nhm vi sinh vt c kh nng c nh N2 t khng kh

c nh thnh m sinh hc cung cp cho cy. Nhng vi sinh vt ny c mt h

thng sinh ha chuyn bit. Vn ny c ngha to ln v v cng quan trng trong

nng nghip, v vi sinh vt c th c nh nit t khng kh (chim 78,16% theo th

tch v 75,5% theo khi lng bu kh quyn) m cy trng khng trc tip hp thu

8

c (Nester v ctv., 2004). Theo Hardy v ctv., (1973), lng m m cc vi sinh vt

trn tri t c th c nh hng nm ln n 175x106 t tn.

Phn ng c nh m sinh hc xy ra c mt s vi sinh vt l nh s xc

tc ca mt h enzyme chuyn bit l nitrogenase. Enzyme ny xc tc phn ng bin

i m th kh (N2) thnh ammonia (NH3). Nitrogenase c tm thy trong qu

trnh c nh m ca vi khun ho v ym kh l mt phc h bao gm hai protein,

trong protein nh cha Fe v protein ln hn cha Fe v Mo (Bothe v Neuer,

1988).

Theo Evans v Barber (1977) th qu trnh c nh m xy ra nh sau:

Nitrogenase

N2 +6H+ +6e 2NH3

NH3 s kt hp acid hu c to thnh amino acid ri thnh protein:

NH3 + Acid hu c Amino acid Protein

C nh m sinh hc c th c trnh by theo phng trnh sau y, trong

2 phn t ammonium c sn xut t 1 phn t N2 cng vi 16ATP, 8 in t v 8

ion H+: N2 + 8H

+ + 8e

+ 16ATP 2NH3 + H2 + 16ADP + 16Pi

2.1.2. Chu trnh nit

Chu trnh nit l qu trnh bin i ca nit trong sinh quyn, trong nit xut

hin di nhiu dng t do hay kt hp (nit phn t trong kh quyn, cc nitrit, nitrat,

amoni, protein, acid amin...). Khng kh cha 78% nit phn t (N2). Kh nit ny c

th c cc vi sinh vt c nh nh nhng vi sinh vt cng sinh vi thc vt bc cao

hoc sng t do trong t, c bit l t vng r ca thc vt (Nester v ctv., 2004).

Nit l nguyn t thit yu cho cc tin trnh sinh hc, n c trong thnh phn

ca cc acid amin (hp phn bt buc ca protein trong t bo cht c trng cho s

sng); hin din trong DNA, RNA (vt cht di truyn ca t bo cp phn t);

trong cc enzyme; trong mng t bo; mang chc nng cu trc hoc gi chc nng c

9

ng; cung cp nng lng cho c th, tham gia cu to ADP v ATP. Cc sinh vt

u rt cn nit (Nguyn Ngc , 2008)

Vng tun hon nit l vng tun hon c khp kn. S tun hon nit vo v

ra khi cc h sinh thi ph thuc ch yu vo vi khun (Hnh 2.1).

Hnh 2.1. Chu trnh Nit

Trong cc mi trng t nhin, nit tn ti cc dng khc nhau, t nit phn

t dng kh cho n cc hp cht hu c phc tp c trong c th ng, thc vt v

con ngi (Trn Th Cm Vn, 2005). Chu trnh nit bao gm cc qu trnh c bn

sau:

- Qu trnh c nh nit: Nit phn t (N2) c chuyn ha thnh nit hp

cht (Trn c Vin v ctv., 2004). Vic c nh nit bng sinh hc c

gip bi cc vi khun sng t do nh vi khun hiu kh, bn k kh v

ym kh vi nhng thc vt bc cao (ng Kim Chi, 2001).

- Qu trnh nitrate ha: Khi cc c th vi khun c nh nit, cc thc vt v

ng vt cht i, cc acid amin c ng ha thnh ammonia (NH3) hoc

10

ammonium (NH4+). Cc ammonia ny c th c bin i sang dng nitrite

(NO2-) v nitrate (NO3

-) nh cc vi khun nitrite ha (Nitrosomonas v

Nitrosococcus) v vi khun nitrate ha (Nitrobacter) l cc vi khun t

dng hiu kh. Ngoi ra cn c nhm vi sinh vt d dng gm nhiu loi

vi khun v x khun thuc cc ging (chi) Alcaligenes, Anthrobacter,

Corynebacterium, Achromobacter, Pseudomonas (Nguyn Th Kim Thi &

L Hin Tho, 1999)

- Qu trnh phn nitrate ha: Trong iu kin khng c oxy, cc dng nit

hp cht (NO3-, NO2

-) b phn hy to thnh nit phn t bi cc vi

khun kh nitrate (denitrifying bacteria), c gi l qu trnh phn nitrate

ha (qu trnh kh nitrate) lm gim lng nitrate, khp kn v hon thin

vng tun hon nit.

2.1.3. Vi khun c nh m sng t do

Hnh 2.2. Quan h tng tc gia vi sinh vt v thc vt nh hng n s tng trng ca thc vt

11

Vi khun c nh m sng t do vng r la v nhng cy thuc h ha bn

gip cy trng pht trin tt cng nh hn ch n mc thp nht lng m ha

hc trong nn sn xut nng nghip (Kannaiyan, 1999). Chnh v vy m trong nhng

thp nin gn y c nhiu cng trnh nghin cu trn th gii c thc hin nhm

tm ra cc ngun vi sinh vt c kh nng c nh m cho cy la v mt s cy thuc

h ha bn khc, cc dng vi sinh vt c ng dng bn cho cy la v gip tng

nng sut la t 15-54% (Favilli v ctv., 1987; Omar v ctv., 1989). C nh m sinh

hc trn la lm tng m tng s ln 20-25% (Dbereiner, 1992). Theo th nghim

ca Cao Ngc ip (2005), khi ti dch vi khun Pseudomonas spp. ln la cao sn

trng trn t ph sa Cn Th gip tng nng sut la ln 20-37%.

Hnh 2.3. Mt s ngun nit cung cp cho cy

(http://media.gox.vn/edu/document/Thumb/1/01062010035301109_nito.jpg, ngy 10-9-2011)

2.2. Tng quan v vi khun Pseudomonas

2.2.1. c im ca Pseudomonas

Vi khun Pseudomonas thng l vi khun Gram m, hnh que. C chin mao

cc nn c kh nng li tt trong nc, khng c kh nng to bo t. Pseudomonas l

vi khun sng t do, chng hin din khp ni nh trong t, trong nc, thc vt,

ng vt, mt s lm h thc phm. Chng c kh nng h hp hiu kh hay k kh

trong mi trng khng c xi. Nhit thun li chng pht trin l 30 37oc.

12

Tuy nhin mt s chng Pseudomonas li c th sng tt 40C. Pseudomonas c th

c nui trong mi trng n gin v pH trung tnh. Mt vi chng c th to

hunh quang di nh sng tia cc tm bc sng 254nm.

Hnh 2.4. Pseudomonas di knh hin vi in t (http:www.texbookofbacteriology.net/Pseudomonas.ect.html, ngy 19-9-2009)

Mi trng s dng nui cy Pseudomonas trong phng th nghim l mi

trng King B. Mt s chng Pseudomonas c nh hng quan trng trong s sinh

trng v pht trin thc vt nh tng hp kch t tng trng thc vt nh: auxin,

cytokinin, kch thch s pht trin ca b r cy lm gia tng kh nng hp thu cht

dinh dng trong t mt s loi nh: Pseudomonas putida, Pseudomonas

fluorescens, Pseudomonas syringae (Patten v Glick., 1987; Glickmann v ctv., 1998;

Suzuki v ctv., 2003; Xie v ctv., 1996). Mt s loi Pseudomonas c kh nng ha

tan ln dng kh tan thnh dng d tan gip cy trng hp th nh Pseudomonas

fluorescens, Pseudomonas putida, Pseudomonas chlororaphis (Cattenlla v ctv., 1999)

hay khng li mt s vi sinh vt gy hi cho cy trng nh: Pseudomonas fluorescens

HP72 chng nm Rhizoctionia solani (Suzuki v ctv., 2003). c bit, mt s dng

Pseudomonas c kh nng c nh m t ngun nit khng kh nh Pseudomonas

stutzeri (Krotzky v Werner, 1987; Cao Ngc ip, 2005; Yan v ctv., 2008)

13

2.2.2. Phn loi Pseudomonas

Ging (chi) vi khun Pseudomonas c hn 140 loi, hu ht thuc nhm hoi

sinh (Iglewski v Woods, 1983).

Kim tra cc dng Pseudomonas spp. bng phng php sinh hc phn t phn

tch trnh t RNA ribo th (rrs) (k thut PCR-16s-rRNA gen) (Mirza v ctv., 2006) v

gii trnh t gen nif (c nh m nitrogen fixation) da vo cp mi PolF v PolR

xc nh loi ca Pseudomonas thng qua ngn hng d liu NCBI vi phn mm

BLAST N. Mt s nghin cu chng minh nhng loi thuc ging vi khun

Pseudomonas c kh nng c nh m nh Pseudomonas rubrisubalbicans

(Dbereiner v Pedrosa, 1987), Pseudomonas diminuta, P. fluorescens, P.

paucimobilis, P. pseudoflava, P. putida, P. saccharophila, P. stutzeri v P. vesicularis

(Chan v ctv., 1994),. Ngoi ra, cn c mt s dng Pseudomonas sp. cng c

nghin cu nh Pseudomonas aeruginosa, Pseudomonas infection, Pseudomonas

folliculitis, Pseudomonas aeruginosa streatment, Pseudomonas streatment,

Pseudomonas bacteria, Pseudomonas pneumonia, Pseudomonas syringae (Iglewski v

Woods, 1983).

Loi vi khun Alcagenes faecalis A15 l mt loi vi khun c nh m cho cy

la cao sn c nng dn Trung quc s dng nhng sau n c cc nh khoa

hc B phn loi chnh xc da trn b genome ca n l Pseudomonas stutzeri

(Vermeiren, 1999).

Yabuuchi v ctv. (1992) ln u tin tm ra ging Burkholderia da vo vic

phn tch di truyn rRNA nhm II, trn c s phn loi li 2 loi Pseudomonas

pickettii v Pseudomonas solanacearum c chuyn i t ging Ralstonia. Vic

phn loi li mt s loi Pseudomonas v t li tn nhng loi ny l Burkholderia

(Yabuuchi v ctv., 1995). Khi , loi Burkholderia vietnamiensis sng trong r la

trng Vit Nam c pht hin vo nm 1994 (Gillis v ctv., 1995). n nm 1996,

cc nh khoa hc xc nh c Burkholderia vietnamiensis l loi vi khun c kh

14

nng c nh m gip tng nng sut la (Tran Van v ctv., 1996), tng nng sut ma

(Govindanajan v ctv., 2006). Nhng loi Burkholderia c kh nng c nh m nh

Burkholderia brasilensis, Burkholderia tropica cy ma (Baldani, 1996),

Burkholderia kururiensis (Zhang v ctv., 2000), Burkholderia brasilensis,

Burkholderia tropicalis khm v chui (Leonardo v ctv., 2001), Burkholderia

unamae ni sinh trong Bp (Caballero-Mellado v ctv., 2004). Nm 2008, mt s nh

khoa hc ca i hc Cn Th cng tm ra mi trng nhn nui v tn tr hiu

qu i vi dng Burkholderia cepacia TG17 c phn lp t cnh ng la huyn

Chu Thnh thuc tnh Tin Giang (Dng Th Nguyn Quyn v ctv., 2008). Hu

ht cc dng Burkholderia gy bnh u thuc nhm Burkholderia cepacia complex

(Bcc) c lin quan n bnh ly truyn, v d nh bnh vim hch do Burkholderia

pseudomallei complex c pht hin mt bnh nhn thuc huyn C Chi TP. H

Ch Minh vo nm 1999. Cch y vi nm, ngi dn i Loan v Singapore tng

hong s v Burkholderia (Pseudomonas cepacia) khi n gy t vong cho hng chc

ngi, ch yu bnh nhn nm trong bnh vin (http://www.ykhoanet.com/tapchi

yhoc/0012/B3%20%20Duong%204tr305-07.htm, ngy 25-9-2009)

Vic gy bnh do mt s dng Burkholderia lm hn ch tim nng ng

dng. Tuy nhin, Burkholderia c kh nng c nh m rt a dng, c 39 loi

Burkholderia c pht hin. Nhiu loi Burkholderia c tim nng ng dng trong

nng nghip v mi trng nh kh nng c nh m t khng kh, kh nng kch

thch cy trng sinh trng (PGRS Plant Growth-Promoting Rhizobacteria) v pht

trin, kh nng ha tan ln kh tan, kh nng phn hy sinh hc... (Elmerich v

Newton, 2007).

Trc y, c s phn loi vi khun c da vo hnh dng v nhng c tnh

sinh ha l ch yu. Trong nhng nm gn y, khi cng ngh sinh hc pht trin, cc

nh khoa hc xc nh b gen ca vi khun. Sau , ng dng phn tch cu trc

DNA v RNA vo vic phn loi v xc nh mc tng quan di truyn gia cc

dng vi khun (Nester v ctv., 2004).

15

2.2.3. Nhng loi Pseudomonas c kh nng c nh m

Vi khun Pseudomonas sp. phn b rng ri v a dng nhiu chng loi

(Rangarajan v ctv., 2001; 2002). Mt s loi thuc ging Pseudomonas c kh nng

c nh m c khng nh t lu (Krotzky v Werner, 1987; Chan v ctv.,

1994), trong c Pseudomonas stutzeri (Vermeiren, 1999). Ngoi ra, cc nh khoa

hc cng xc nh mt s loi vi khun c phn lp t t vng r la c kh

nng c nh m v gip tng nng sut la (Gillis v ctv., 1995; Tran Van v ctv.,

2000; Nguyn Ngc Dng v ctv., 2000).

Hnh 2.5. Khun lc ca Pseudomonas stuzeri (tri) v Pseudomonas fluorescens (phi)

(Lalucat v ctv., 2006).

Ngoi ra, Burkholderia vietnamiensis c xc nh l loi c kh nng c

nh m cho cy trng nh h thng gen nif c kh nng tng hp enzyme

nitrogennase (Menard v ctv., 2007). Ch c mt loi ca Bcc l Burkholderia

vietnamiensis c kh nng chuyn N2 thnh NH3 (Perin v ctv., 2006).

16

2.2.4. Phn lp v xc nh Pseudomonas

S dng mi trng c chng Pseudomonas Isolation Agar 46,4g/l (Difco).

ng thi, bng k thut di truyn phn tch trnh t rRNA rib th - K thut PCR-

16S-rRNA gen, Mirza v ctv. (2006) xy dng c cy di truyn ca cc nhm

loi Pseudomonas.

Hnh 2.6. Cy di truyn (phylogenetic tree) biu din mc tng quan di truyn gia cc loi Pseudomonas da vo kt qu phn tch di truyn phn t (Menard v ctv., 2007)

Tng t, bng k thut di truyn phn tch 16S-rRNA v phn tch trnh t di

truyn ca gen nif, Menard v ctv. (2007) xy dng c mi tng quan di truyn

ca cc loi Burkholderia c biu din qua cy di truyn (phylogenetic tree).

17

Hnh 2.7. Cy di truyn (phylogenetic tree) ca cc loi Burkholderia (Menard v ctv., 2007)

2.3. C ch c nh m ca Pseudomonas

2.3.1. Enzyme nitrogenase

Nitrogenase l mt a enzyme (phc h enzyme) xc tc cho phn ng c nh

N2, kh N2 thnh NH3. Enzyme nitrogenase c iu khin tng hp bi mt h

thng gen nif c trong b genome ca t bo vi khun. Ngoi ra, enzyme nitrogenase

18

khng ch xc tc cho qu trnh kh N2 thnh NH3, m cn c th xc tc kh cc cht

CH3CN, HCN, NH3, N2O, C2H2 thnh cc sn phm tng ng nh sau:

- N2 + 6H+ + 6e NH3

- NH3 + 2H+ + 2e H2 + NH3

- N2O + 2H+ + 2e N2 + H2O

- C2H2 + 2H+ + 2e C2H4

- HCN + 6H+ + 6e CH4 + NH3

- HCN + 4H+ + 4e CH3NH2

- CH3CN + 6H+ + 6e C3H6 + NH3

- CH3CN + 6H+ + 6e CH3NH2 + NH3 + CH4

- CH2CHCN + 6H+ + 6e C3H6 + NH3

- CH2CCH + 2H+ + 2e CH2CHCH3

Lin on Quc t Sinh ha thng nht phn loi enzyme thnh 6 nhm.

Enzyme nitrogenase thuc nhm EC1 l Oxidoreductases, xc tc cho phn ng oxy

ha kh, nhn in t (e) v nh H2 (hoc nhn in t v nh O2).

Hnh 2.8. Nitrogenase c kh nng xc tc chuyn ha, kh N2 thnh NH3 (Dixon and Kahn, 2004)

Nit t do trng thi rt bn nh lin kt ba (NN) ca n tng ng vi nng

lng t do l 942 kJ/mol. Trong iu kin t nhin, nhng lin kt ba ny ch c th

b b gy nh ngun nng lng nhit rt cao, c th n 4000C nh nhng tia la

Trao i cht

Chuyn in t

Cung cp H

19

in khi c sm st. Trong cng nghip sn xut phn m, NH4+ c to t kh N2

trong phn ng Haber xy ra 400-500C vi p sut vi trm atmostphere. Tuy

nhin, qu trnh c nh m sinh hc, s kh N2 li xy ra trong iu kin sinh l bnh

thng nh nng lng ATP v s xc tc bi enzyme nitrogenase (Dixon and Kahn,

2004).

Nitrogenase l mt phc h enzyme, chng c cu to bi 2 protein. C 2

protein u l phn t nh v u c hot tnh xc tc:

- Mt protein st (protein Fe dinitrogenase reductase): l protein nh hn, c

trng lng phn t khong 60.000 Da, gm 2 tiu n v ging nhau, cha

tm oxy ha - kh Fe4-S4, c 2 v tr gn ATP.

- Mt protein st molibden (protein Mo-Fe dinitrogenase): l protein ln

hn, c trng lng phn t khong 240.000 Da, c cu to t 2 tiu n v

khc nhau cha nhn Fe, Mo v tm oxy ha kh.

Hnh 2.9. Cc thnh phn protein cu thnh nitrogenase (Dixon and Kahn, 2004)

Tng cng, nitrogenase cha 2 nguyn t Mo, 32 Fe v 30 nguyn t S.

Nguyn t Mo l nguyn t vi lng. Nhng nguyn t S rt linh ng.

Protein Mo-Fe Protein Fe Phc h enzyme nitrogenase

20

2.3.2. B gen (genome) ca Pseudomonas v s iu khin tng hp

nitrogenase

Genome v h thng gen nif ca Pseudomonas

B genome ca Pseudomonas stutzeri A1501 c bao gm trong 1 nhim sc

th vng n c 4.567.418 bp, c th m ha 4.146 protein, 59 gen tRNA v 4 operon

rRNA. Thng tin di truyn chuyn bit v s c nh m c xc nh trong b

genome ca Pseudomonas stutzeri A1501. l vng c nh m (nitrogen

fixation region) c kch thc 49kb, gm 59 gen c lin quan. T l (C+G) ca vng

ny cao hn t l (C+G) trung bnh ca b genome (66,8% so vi 63,8%). H thng

gen nif ca Pseudomonas stutzeri A1501 cu thnh nn vng c nh m trong

genome ca n c xc nh tng ng vi vng c nh m ca mt s loi vi

khun khc nh Wolinella succinogenes, Rhizobium leguminosarum, Azotobacter,

Azospirillum, nhng ch khc v t l (C+G).

hai u ca vng gen nif (vng c nh m) Pseudomonas stutzeri

A1501 l nhng gen m ha tng hp Co (cobalamin 5-photphate synthase,

PST1301) mt u v u cn li (PTS1360) l nhng gen m ha cho cc nhn t

gip bo v t bo trnh b tn thng trong qu trnh oxy ha. Nhng gen bo ton

quan trng ni trn khng tn ti vng ny i vi nhng loi Pseudomonas khng

c kh nng c nh m. iu ny cng c th suy lun rng s xut hin vng m

ha cobalamin 5-photphate synthase (PST1301) u ca vng c nh m

Pseudomonas stutzeri A1501 c tip nhn t s chuyn i gen ln cn (Yan v

ctv., 2008).

Th t ca cc gen nif trong cu trc ca vng c nh m Pseudomonas

stutzeri A1501 c khi u l vng PST1301, vng gia ln lt bao gm cc gen

nifQ nifB nifA nifL - nifY2 nifHDKTY nifENX nifUSV nifWZM nifF

v vng PST1360 u cn li (Klipp v ctv., 2004)

21

S iu khin tng hp enzyme nitrogenase

iu ha hot ng gen sinh vt nhn s ch yu din ra giai on phin

m, do s tng tc gia protein iu ha vi trnh t c bit trong vng iu ha ca

gen.

Nhng loi thuc ging (chi) vi khun Pseudomonas c kh nng c nh m

u c h thng gen nif iu khin qu trnh c nh m t nit v c t do trong

khng kh. Do , Pseudomonas thuc nhm vi sinh vt ha t dng

(chemolithotrophs), l nhm vi sinh vt s dng hp cht v c to nng lng

(cht cho in t).

Theo nghin cu ca Yan v ctv. (2008), h thng ca gen nif Pseudomonas

stutzeri A1501 l mt h thng hon chnh gm cc loi gen nif quy nh tng hp cc

thnh phn cu to nn phc nitrogenase.

Phc h enzyme nitrogenase gm 2 protein: protein Mo-Fe c quy nh m

ha bi nifDK v protein Fe c quy nh m ha bi nifH. Cu trc y cc

thnh phn nh trong phc nitrogenase c cu thnh t nhng sn phm ca nhiu

gen nif khc (a dozen other nif genes) c lu gi khi sng t do hay ang cng sinh.

Nhng kim loi v nhng tiu phn xc tc cu thnh nitrogenase c m bo n

nh nh hot ng tng hp ca cc gen nifMZ, nifW v nifUS. Cc gen nifB, nifQ,

nifENX v nifV v nifH chu trch nhim tng hp trung tm Molybden (FeMo-co)

cho n s quy nh cc tiu phn trong protein Mo-Fe. Mc d nhng gen ny l

ging nhau hu ht h thng c nh m cng nh cu to v ni dung, nhng s

iu khin th c khc nhau gia cc ging.

Nh vy, vi khun c kh nng c nh m sinh hc t N2 khng kh l nh h

thng gen nif quy nh tng hp h enzyme nitrogenase. Chnh nitrogenase l h

enzyme s xc tc cho qu trnh chuyn ha N2 thnh m sinh hc. Chc nng ca

22

cc gen nif tham gia quy nh tng hp h enzyme nitrogenase c Curatti v ctv.

(2007) khi qut theo hnh sau y:

Hnh 2.10. Vng c nh m (nitrogen fixation region) ca Pseudomonas stutzeri A1501 (Yan v

ctv., 2008)

Trong vng c nh m ca Pseudomonas stutzeri A1501 tn ti 2 nhm

gen:

- Mt nhm gen u tin (t v tr PST1302 n PST1323) bao gm nifB v

nifQ u lin quan n s iu khin tng hp MoFe-co v iu khin

nifLA. Hai gen nifL v nifA c vai tr m ha protein iu ha (kch thch

hoc c ch) iu khin s hot ng ca operon nif (Martinez-Argudo v

ctv., 2004; Xie, 2006). Operon rnfABCDGEF m ha cho phc h protein

mng chuyn in t n nitrogenase, gn v tr ca nifLA.

- Nhm gen th hai gm mt nhm cc operon nh nifHDKTY, nifNEX,

nifUSV v nifWZMF, mang nhng trnh t m ha b sung, c tm thy

nhng khang nm gia ca cc operon nif hoc nm ngay bn trong ca

nhng operon nif. C rt t nucleotid xut hin cc vng khng m ha

gia cc gen v s chuyn i trng lp gia nhng codon m u v kt

thc c pht hin. Tuy nhin, khng tm thy gen draT v draG l

nhng gen c chc nng iu khin tng hp ADP-nitrogenase gi chc

nng ng v m i vi nitrogenase nh trong bo co v vn ny

Azoarcus v Azospirillum brasilense. V vy, c mt c ch bt hot hoc

kch hot khc Pseudomonas stutzeri A1501 c xut v n do 16

23

gen gia vng nif nh hng s c nh nit (Yan v ctv., 2008;

Elmerich v Newton, 2007; Kraus, 2006).

Nhng loi vi sinh vt c kh nng c nh m sng cng sinh v t do u c

cha nhng gen nif trong h gen. Chnh nh h thng gen nif m nhng vi sinh vt ny

c kh nng tng hp enzyme nitrogenase xc tc cho qu trnh kh N2 thnh NH3.

Nu vng vn hnh khng b protein iu ha c ch th gen cu trc s hot

ng nh ARN-polymerase khi ng tng hp ARN thng tin v ARN thng tin s

quy nh tng hp emzyme. B ba m u 5-AUG-3 m ho cho methionine. vi

khun c hai loi tRNA i vi methionine: mt l tRNA nhn gc methionine a

vo thnh phn ca chui polypeptide Met-tRNA, hai l tRNA nhn gc formyl-

methionine (fMet-tRNA) c vai tr quan trng trong vic khi u tng hp chui

polypeptide. sinh vt nhn s, nhm amine ca methionine c th c formyl ho

bi N10-formyl-tetrahydrofolic acid, nh enzyme transformylase c hiu. Do nhm

amine b gc formyl bao vy nn khng cho php n tham gia vo qu trnh ko di

chui, m ch tham gia vo qu trnh khi u tng hp protein.

sinh vt nhn s, anticodon c nh v gia ca 16S-rRNA. Base b sung

kt hp vi base ca codon mRNA khong cch t 0,3 n 0,4 nm, nh vy s xut

hin lc hp dn Van der Waal. Bng cch ny trc ht tRNA-aminoacyl kt hp vi

tiu phn nh ca ribosome v thc cht l vi mRNA. Phc h ny phn ng vi

nhn t ko di. Nhn t ny c trn tiu phn ln ca ribosome. Ngoi cc yu t

tham gia khi u tng hp protein nh fMet-tRNA, mRNA, cc tiu n v ribosome

30S v 50S, ATP, cn c 3 protein na l cc yu t khi u: IF1 (M 9000), IF2 (M

65000-80000) v IF3 (M 29000). Kt qu ca giai on ny l to thnh phc hp

khi u: fMet-tRNA-mRNA-ribosome 70S (Nester v ctv., 2004).

Qu trnh c bt u bng s kt hp gia yu t IF3 vi tiu n v 30S.

Nh yu t ny m tiu n v 30S kt hp vi mRNA v tr khi u. Trong giai

on hai fMet-tRNA gn vi phc IF2-GTP to thnh IF2-GTP-fMet-tRNA (t hp

24

3). giai on ba v bn phc hp IF3-30S kt hp vi t hp 3. Nh yu t IF2 m

fMet-tRNA c a vo khu vc P. Ngi ta cha bit chnh xc vai tr ca IF1, c

th n tham gia vo vic i mi chu trnh bng cch gp phn gii phng yu t IF2

ra khi phc hp. Cui cng tiu n v 50S kt hp vi tiu n v 30S to ra

phc hp ribosome 70S, ng thi gii phng c 3 yu t khi u cng nh gii

phng c GDP v Pi (Nester v ctv., 2004).

2.3.3. C ch c nh m

Trong thnh phn cu to nitrogenase, s nguyn t Fe v nguyn t S c th

khng n nh vi acid. Phn t protein nh hn c chc nng vn chuyn e, trong

e ca ferredoxin hoc flavodoxin vn chuyn ln phc h Mo-Fe.

Nitrogenase c cu trc FeMo-co c khi gia l Fe7S8 kt hp vi mt phn t

homocitrat vi phn ui l Mo. C 2 phn t FeMo-co hin din trong mt enzyme

nitrogenase. S c nh nit hnh thnh khi cc cht cho in t flavodoxin chuyn

in t n dinitrogenase reductase v chuyn tip n ditrogenase, cung cp cc in

t di dng H2 n cc N2 to thnh NH3. Sau , NH3 kt hp vi cc acid hu c

to ra cc acid amin hu dng cho cy trng (Cao Ngc ip v Nguyn Hu Hip,

2009).

Phn ng c nh m N2 + 8H+ + 8e

+ 16ATP 2NH3 + H2 + 16ADP + 16Pi

c thc hin trn cc nhm sinh vt s hch c kh nng sn xut enzyme

nitrogenase. Ni mt cc n gin, enzyme nitrogenase c cu to bi 2 protein: 1

protein Fe v 1 protein Mo-Fe. Phn ng c nh m xy ra theo trnh t protein Fe

s kh v cung cp in t trc cung cp in t trong Ferredoxin. K n, protein

Mo-Fe kh v cung cp in t n N2 to thnh HN=NH. Trong 2 chu k tip theo

cng i hi cung cp in t to ra H2N=NH2 t HN=NH, v cui cng to NH3 t

H2N=NH2. Ty theo nhm vi sinh vt m Ferredoxin kh s c cung cp in t t

qu trnh quang hp hoc h hp hay ln men. Qu trnh ny c iu khin bi cc

gen nif (Cao Ngc ip v Nguyn Hu Hip, 2009).

25

Qu trnh c nh thnh NH3, NH3 s kt hp vi cc hp cht khc to

thnh m sinh hc theo hnh sau y:

Hnh 2.11. S biu din c ch c nh m sinh hc (Moore v ctv., 1998)

Ngoi ra, trong qu trnh vn chuyn e cn nng lng do ATP cung cp.

ATP kt hp vi protein cha Fe qua 1 cu Mg v sau c thu phn thnh ADP

v Pi. S thu phn lm thay i hnh dng ca protein enzyme, e c vn

chuyn ln phc h FeMo. vn chuyn 1e cn 2 ATP. Phc h FeMo thc cht

l 1 N2-reductase. Phc h nh hn l 1 protein cha Fe, phc ln hn l protein cha

Fe/Mo, phc ln ny thc cht l dinitrogen-reductase.

S thay i ho tr Mo c vai tr quan trng vn chuyn e t phc Fe/Mo

n N2.

1. 2e v 2H+ c kt hp vi nguyn t Mo

2. Nguyn t H2 ang gn vi enzyme b y ra bi N2

3. N2 c kh thnh N=N do tip nhn H+ + e

4. N=N c kh thnh =NNH2 do tip nhn H+ + e

26

5. =NNH2 c kh thnh NH3 do tip nhn H+ + e

, NH3 c tch ra khi

phc h enzyme

6. Nguyn t N ang gn vi enzyme c kh thnh NH3 do tip nhn 3e v

3H+ v sau NH3 c tch ra khi phc h.

Hnh 2.12. Cc phn ng c nh m trong nitrogenase (Charpy-Roubaud v Charpy, 2001)

S gn N2 vi phc h FeMo cn 1 lin kt trc ca phc h ny vi H2.

V vy, kt qu l bn cnh to ra NH3 cn xut hin H2, phn t ny c th c tch

ra thnh H+ v e v e ny c a tr li h thng.

2.3.4. Cc yu t nh hng n qu trnh c nh m

NH3 c to nn bi nitrogenase c a n t bo cht ca t bo ch v

y c s dng tng hp nn amino acid. S tng hp enzyme nitrogenase c

iu khin bi enzyme glutamate synthetase, xc tc cho tng hp glutamin t NH3.

Nu trong h thng c t NH3 th glutamate synthetase kch thch tng hp nitrogenase,

nng NH3 cao th c ch s tng hp nitrogenase. C ch iu khin ny rt c

ngha, tuy nhin khi cung cp NH3 d tha th nitrogenase khng c s dng, v

c NH3 (Van v Sloger, 1981). vi khun sng t do, t l c nh nit b nh

hng do s tch lu NH3 trong t bo vi khun v s cung cp khng y carbon

hu c. Loi vi khun c nh nit sng t do cn cho s trao i cht ca chng

27

nhng hp cht hu c d phn gii, nh ng v cc acid hu c, thng c trong

t vi lng khng . V vy kh nng c nh m ca vi khun sng t do c bn

l thp. Kh nng c nh N2 b nh hng rt nhiu khi nng O2 cao.

Phc h enzyme nitrogenase khng bn khi c mt oxy. iu ny trong thc t

c khc phc khc nhau. Vi khun t do c nh m ch th hin hot tnh iu

kin ym kh nh s dng in t xut hin trong qu trnh tng hp ATP ngn

nga oxy xm nhp. Vi khun lam c mc thch nghi hon thin hn, qu trnh c

nh m ca n xy ra trong d bo (heterocyst) c vch dy ngn khng cho oxy xm

nhp. Hon thin hn c l vi khun Rhizobium sng cng sinh trong r u: vi khun

nhn nng lng t r cy v c bo v i vi tc ng ca oxy nh protein

leghemoglobin ca cy ch.

Nitrogenase 0oC s d mt hot tnh hn nhit thng. Nhng iu

kin k kh v lnh su (bo qun trong nit lng) th nitrogenase li c th gi hot

tnh kh lu. Ngoi ra, vic b sung etanol c th bo v hot tnh ca nitrogenase.

Ngoi ra, hiu qu cng sinh ca vi khun v cy trng, s nh hng n qu

trnh pht trin ca cy trng, hiu qu c nh m ph thuc vo nhiu yu t khc

nhau nh chng vi sinh vt, loi thc vt, loi t, mt s vi sinh vt chng vo cng

nh vi sinh vt trong t vng r v iu kin mi trng (Starkey, 1958). Thm vo

l c ch ng m trong qu trnh iu khin hot ng ca operon nif (Xie, 2006).

Nh vo dng kt hp ca hot tnh nitrogenase trong sinh vt sng, iu ny s

rt l th nu bit c s bn phn m c nh hng n s c nh m trong r

la hay khng. Nhiu nghin cu chng minh hu nh c s c ch ca bn phn

m trn hot tnh c nh m ca to lam sng t do (Roger v ctv., 1980). Ngc

li nhng nghin cu c tnh cht h thng v nh hng ca s c nh m kt hp

th vn cn t.

Ngi ta bit rng s c nh m xy ra trong t khi lng C hu c c y

v hm lng m khong trong t thp. Trong nhng th nghim trong ng nghim

28

cch y lu cho thy rng s c nh m b ngng tr khi c s hin din ca m

khong. Tuy nhin t c nghin cu liu n c nh hng n c nh m trong t

khi c trng cy.

Vng r la l ni l tng cho tin trnh kh c li. S kh vi sinh ca phn t

m thnh amoniac (Ishizawa v ctv., 1987; Yoshida v Ancajas, 1973). Vi khun d

dng kt hp vi r cho s c nh N2 pht trin hot tnh nitrogenase trong trng

hp m kt hp thp trong mi trng (Van v Sloger, 1981).

nh hng c ch ca m kt hp c bit l m nitrate c quan st trn

lng ht ca r, s khi u ca nt r, s pht trin (Munnus, 1977) v trn hot tnh

nitrogenase ca cy h u (Mengel, 1994; Cherney v Duxbury, 1994).

Th nghim ca Trolldeneir (1977) chng minh rng nh hng ca s c

nh m trn vng r nng m 10 ppm dng phn ur. Ngc li, ng ta cng

bo co rng, khng c nh hng c ch ca phn m i vi ph ca t la

ngp nc bi v hm lng m gim nhanh chng trong dung dch t.

Nng mui trong t nh hng n mt s Pseudomonas vng r, nng

mui cao s lm gim mt s Pseudomonas. t c nhiu hu c c kh nng lm

gim s tn hi do nng mui v phc hi mt s Pseudomonas (Rangarajan v

ctv., 2002).

Ngoi ra, hiu qu c nh m ca vi khun v cy trng, s nh hng n

qu trnh pht trin ca cy trng ph thuc vo nhiu yu t khc nhau nh chng vi

sinh vt, loi thc vt, loi t, mt s vi sinh vt chng vo cng nh vi sinh vt trong

t vng r v iu kin mi trng (Starkey, 1958).

2.4. ng dng c nh m ca Pseudomonas

2.4.1. Mt s ng dng

Nhng nghin cu gn y cho bit tng s nit c nh c ng dng bi vi

sinh vt trn ton th gii khong 175 triu tn trong mt nm. Nhiu loi vi sinh vt

29

c kh nng s dng N2 t khng kh chuyn thnh ngum m sinh hc nh h

thng sinh ha chuyn bit. Theo Hardy v ctv. (1973), lng m m cc vi sinh vt

trn Tri t c th c nh hng nm ln n 175x106 t tn. C nh m sinh hc

trn la lm tng m tng s ln 20-25% (Dbereiner, 1992). Theo th nghim ca

Cao Ngc ip (2005), khi ti dch vi khun Pseudomonas spp. ln la cao sn trng

trn t ph sa Cn Th gip tng nng sut la ln 20-37%. V vy, vic nghin

cu ng dng vi khun c nh m sinh hc mang hiu qu. Do , vic trin khai

tm kim v ng dng vi khun c nh m l vn cp thit c cc nh khoa

hc trin khai nhm lm tng tnh hiu qu ng dng vi sinh vt c nh m, c bit

l nhm vi khun Pseudomonas c ng dng c nh m vi cy trng, trong c

cy la nc l mt loi cy trng ph bin v chim s lng rt ln vng ng

bng sng Cu Long.

Hin nay, gi c phn bn ha hc tng cao nn nh khoa hc v sn xut

chuyn sang nghin cu v ng dng nhiu vi khun c nh m sng t do v ni

sinh trong cy la lm phn bn sinh hc (Cao Ngc ip v ctv., 2007; Lng Ngc

Du v ctv., 2007).

Nhiu cng trnh nghin cu trn th gii pht hin cc nhm vi khun c

kh nng c nh m cho cy la gip tng nng sut cy trng t 15-54% (Favilli v

ctv., 1987; Omar v ctv., 1989). Hy Lp (ma h 1990) s dng phn m sinh

hc lm cho nng sut la tng 15%-20%, sn lng ca bp, la m, la mch tng

3%-54% so vi nhng v i chng khng bn phn. Vi nhng nghin cu chng vi

khun c nh m trn la m Mexico lm cho nng sut tng t 23%-63% v 24%-

43% (Okon v Labandera-Gonzalez, 1994).

Theo Nguyn Tin Huy (1999) s dng phn m vi sinh (Azospirilum,

Pseudomonas, Enterobacter) cho thy la c th tng nng sut 10-15%, tit kim

30-40 kg m/ha.

30

2.4.2. Trin vng ng dng Pseudomonas trong tng lai

Nm 2009 tng sn lng la go c nc l 38,9 triu tn; ring ng bng

sng Cu Long (BSCL) l 22,5 triu tn, sn lng v din tch canh tc la ca

BSCL khng ngng gia tng. T cho thy, BSCL l va la ca c nc v

ng vai tr quan trng trong vic m bo an ninh lng thc v sn lng go xut

khu, c nng sut cao v c tim nng ln.

(http://dddn.com.vn/20090928023411384cat119/san-xuat-lua-xk-gao-nam-2009-deu-

duoc-mua.htm, ngy 17/9/2010)

Vi dn s khng ngng gia tng trong tng lai, la go phi c sn xut

nhiu hn trn din tch ngy cng thu hp (Shenoy v ctv., 2001). p ng nhu

cu ngy cng cao c v cht lng v sn lng la, khng ch Vit Nam m c cc

nc khc trn th gii, bin php m nh nng hay s dng l khng ngng thm

canh tng v v s dng phn ha hc nhiu, c bit l m ha hc v m l ngun

dinh dng chnh ca cy trng, cng ng ngha vi lng m thot ra mi trng

gia tng. Do bn qu nhiu m, nht l m ha hc khng nhng dn n chi ph

cao, m cn dn n nhim mi trng, gy hiu ng nh knh, tn hi sc khe v

h sinh thi

Trong tng lai, nhu cu s dng phn bn hu c vi sinh ngy cng tng v

nhng l do sau y:

- S dng phn bn hu c vi sinh s thay th dn vic bn phn ho hc trn

ng rung, t trng trt m vn m bo c nng cao nng sut thu

hoch;

- S dng phn bn hu c vi sinh v lu di s dn dn tr li ph nhiu

cho t nh lm tng lng phospho v kali d tan trong t canh tc, ci

to, gi bn ca t i vi cy trng nh kh nng cung cp hng lot

cc chuyn ho cht khc nhau lin tc do nhiu qun th vi sinh vt khc

nhau to ra;

31

- Vic s dng phn bn hu c vi sinh cn c ngha rt ln l tng cng

bo v mi trng sng, gim tnh c hi do ho cht trong cc loi nng

sn thc phm do lm dng phn bn ha hc;

- Gi thnh h, nng dn d chp nhn, c th sn xut c ti a phng

v gii quyt c vic lm cho mt s lao ng, ngoi ra cng gim c

mt phn chi ph ngoi t nhp khu phn ho hc.

2.5. Vai tr ca m i vi cy la

Cy la cn nhiu loi dng cht khc nhau. C nhng cht cy la cn vi s

lng ln, gi l cht a lng nh N, P, K, Si, Ca, Mg, c cht cy trng cn vi

s lng rt t, gi l cht vi lng nh Fe, Zn, Cu, S, thiu hoc tha mt trong s

cc cht ny, cy la s pht trin khng bnh thng. Trong m l nguyn t quan

trng nht i vi cy la, v m l cht to hnh cho cy la, l thnh phn ch yu

ca protein v cht dip lc lm cho l xanh tt, gia tng chiu cao, tng s chi, kch

thc thn l. Do , c th da vo mu sc v kch thc l, chiu cao v kh nng

n bi ca cy la chn on tnh trng dinh dng m trong cy (Nguyn Ngc

, 2008).

Theo De Datta (1981), cy la trong t ngp nc hp thu m t phn bn, t

s c nh m do to v cc vi khun d dng hay m c khong ha t cht

hu c v xc b thc vt.

Cy trng ni chung v cy la ni ring u s dng m di hai dng ch

yu l ammonium (NH4+) v nitrat (NO3

-) nhng ph bin l dng ammonium (NH4+),

cy la hp thu m ammonium nhanh hn m nitrat. D vy cy la vn khng tch

ly c ammonium trong t bo l, lng ammonium d tha s kt hp thnh

asparagin trong l. Ngc li khi nng NO3- trong mi trng cao th cy la tch

ly nhiu NO3- trong t bo (Nguyn Ngc , 2008).

Ty theo giai on sinh trng ca cy la m cy c nhu cu m khc nhau.

cc giai on tng trng th m gip cy tng trng v chiu cao, to chi,

32

nhnh, tng s l trn cy v tng din tch l. Nu thiu m cy la t n bi, chi

nh, l ngn hp v tr nn vng v ri sm, cy la ci cc khng pht trin. Ngc

li khi tha m cy sinh trng qu mc, tn l xum xu to iu kin cho su bnh

pht trin v nh hng rt ln n vic ng cc giai on sau. trong cy m

ch yu c tch ly trong thn, l, khi la tr c khong 48 71% m c a ln

bng. Trong giai on sinh sn, m c vai tr trong vic to ra mm hoa, tng s ht

trn gi, tng s gi trn bng v cn gip tng s chi hu hiu. Nu thiu m cy

la s thnh lp bng ngn, t ht, ht nh v c nhiu ht thoi ha. Cn khi tha

m, cy la s pht trin thn l qu mc, m non, mm d ng, tn l rm rp, d

nhim su bnh nn lm gim nng sut la rt ln. Trong giai on chn th qu trnh

chuyn ha cht hu c trong l v thn din ra rt mnh, l cn gia tng tc quang

hp nhng khi thiu m th qu trnh tng hp dip lc t khng xy ra dn n tc

quang hp gim. Khi tinh bt khng chuyn vo bn trong ht lm cho ht b

lp (Nguyn Ngc , 2008)

33

CHNG III

NI DUNG, PHNG TIN V PHNG PHP NGHIN CU

3.1. Ni dung nghin cu

t c mc tiu Phn lp v xc nh vi khun Pseudomonas spp. c

mang gen nif c nh m hin din trong t vng r la ng bng sng Cu Long

v chn lc cc dng Pseudomonas spp. c hu hiu cao trn la cao sn, cc vn

c xy dng cho qu trnh thc hin ti bao gm nhng ni dung nghin

cu nh sau:

- Phn lp vi khun Pseudomonas spp. t t quanh vng r la bng mi

trng nui cy c hiu v nhn din chng bng cp mi chuyn bit da

theo PCR-16S-rRNA.

- nh gi kh nng c nh m ca cc dng vi khun lm c s chn

lc cc dng vi khun hu hiu, ng thi xc nh gen nif ca cc dng vi

khun Pseudomonas spp. phn lp c.

- Chn lc cc dng vi khun hu hiu da trn cc kt qu th nghim v kh

nng c nh m ca cc dng vi khun trong phng th nghim v nh

li, tin hnh kim tra v nh gi hiu qu c nh m cc dng vi khun

Pseudomonas spp. trn cy la cao sn trng ngoi ng.

3.2. Phng tin nghin cu

3.2.1. Vt liu

Mu t vng r la c thu 13 tnh, thnh thuc ng bng sng Cu Long.

Ging la OM2517 c ngun gc t t hp lai OM1325 v OMCS94, c

cng nhn ging Quc gia nm 2004 theo Quyt nh s 2182 Q/BNN-KHCN ngy

29/7/2004. Ging la OM2517 c thi gian sinh trng ngn (90-95 ngy), t nng

34

sut trung bnh 5-6 tn/ha v c th t n 8 tn/ha, thch nghi c hai v ng Xun

v H Thu, ph hp t ai vng BSCL (Nguyn Th Lang v Bi Ch Bu, 2008).

3.2.2 Dng c

- a Petri, kim cy, n cn

- ng nghim c np cao su, ng ong

- Bnh tam gic

- Ci nghin mu

- Micropipette (1 - 100l, 100 - 1000l)

- Lame, lamelle, v mt s dng c khc

3.2.3. Thit b

- T cy vi sinh vt Laminar Flow (Vit Nam)

- T sy Memmert (c)

- T lnh tr mu Sanyo (Nht Bn)

- T vi sinh vt Memmer (c)

- Knh hin vi Olympus CH -20 (Nht Bn)

- My lc mu Heidolph MR 3001 (c)

- Ni kh trng nhit t HVE- 50 (Nht Bn)

- My o OD Beckman Coulter DU 640B (c)

- My chp hnh k thut s Sony Cyber- shot 7.2 (Nht Bn)

- Cn in t Mettler Toler (Switzerland)

- pH k Thermo election corporation (M)

- My trn mu Vortex

- L viba

- My ly tm MIKRO 12- 24 (Trung Quc)

35

- B in di mt chiu BioRad 2000 (Hoa K)

- My o nng DNA Beckman DU 600 (c)

- n UV dng kh trng v pht hin DNA bc sng 260nm

- My c v chp gel

- My PCR Perkin Elmer 9700 (Hoa K)

- My vi tnh phn tch v lu tr s liu

3.2.4. Cc loi ha cht v mi trng nui cy vi khun

3.2.4.1. Ha cht dng phn lp vi khun

- Cn

- Nc ct mt ln

- Glycerol

- Mi trng Pseudomonas Isolation Agar 46.4g/l (Difco) (Mirza v ctv., 2006)

3.2.4.2. Mi trng nui cy vi khun v ha cht dng xc nh amonium

- Mi trng Burk lng khng m (Park v ctv., 2005)

Thnh phn Nng (g/l)

Glucoz 10

KH2PO4 0,41

K2HPO4 0,52

Na2SO4 0,05

CaCl2 0,2

MgSO4.7H2O 0,1

FeSO4 .7H2O 0,005

NaMoO4.2H2O 0,025

- Phenol

36

- Phenol - Nitroprusside: + 5g phenol /0,5lit

+ 0,025g Nitroprusside / 0,5lit

- Sodium - Hypochloride: + 5g NaOH / 0,5lit

+ 15ml hypochloride / 0,5lit

- NH4 chun(1ppm)

3.2.4.3. Ha cht trch DNA

- Mi trng LB (Bennasar v ctv., 1998)

Thnh phn Nng (g/l)

Bacto yeast extract 5

Bacto tryptone 10

NaCl 10

- TE pH 8,0 ha tan DNA

- Sodium dodecyl sulfate (SDS) 10% ha tan DNA v gip proteinase K hot ng

tch protein ra khi DNA hiu qu hn.

- Isopropanol nhm ta DNA

- Ethanol 70% dng ra DNA

- Hexadecyltrimethylamemonium bromide (CATB) 10% NaCl 0,75M to mi

trng n nh DNA.

- Proteinase K (20 mg/l) loi b protein hoc polysaccharide khi DNA

- Chloroform: Isoamyl alcohol (24:1) nhm ta protein v to mng ngn gia

DNA v protein.

- Bi nc (nc ct 2 ln)

3.2.4.4. Cc ha cht trong phn ng PCR

- Bi nc

37

- MgCl2 (25 mM)

- PCR buffer (10X Tab buffer (NH4) SO4 (Fermentas)

- BSA (Bovine Serum Albumin Acetylated 10 mg/ml)

- Deoxyribonucleoside triphophate (dNTPs -200 M)

- Primer FGPS4-281bis (2,50 ng/l) (5 3) (Mirza v ctv., 2006)

AGAGTTTGATCCTGGCTCAG

- Primer FGPS1509-153 (2,50 ng/l) (5 3)

AA GAGGTGATCCAGCCGCA

- Taq polymerase (5 U/l, Gibco)

- Template DNA vi khun

- H2O tinh khit

- Cp mi xc nh gen nifH l ca Pseudomonas:

PolF: TGCGAYCCSAARGCBGACTC

PolR:ATCGCCATCATYTCRCCGGA

PCR on 360 bp t v tr 115 476 ca gen nif (Mirza v ctv., 2006)

- Thit k cp mi nhn din on gen nifH ca Pseudomonas stutzeri A1501

3.2.4.5. Ha cht dng in di sn phm PCR

- Agarose 1%

- TAE buffer

- Loading Buffer (cht ch th mu LB)

- Ethidium bromide (Cht ch th hunh quang- EtBr)

- Thang chun DNA 100bp plus, 100bp

38

3.3. Phng php nghin cu

3.3.1. Thu mu t vng r la

Thu mu t vng r la 13 tnh v thnh ph thuc ng bng sng Cu Long,

(thu 10 mu t 10 rung la khc nhau mi tnh hoc thnh ph). Cc bc tin hnh

nh sau:

- Xc nh cc a im thu mu: Chn 3 huyn (hoc qun) trong mi tnh (hoc

thnh ph); trong mi huyn (hoc qun), chn 3 x (hoc phng); trong mi x

(hoc phng), chn 1 2 rung la ang canh tc.

- Nh la ly phn t bm st b mt r (20-50g) ca 3 5 bi la cho vo bc

nilon ( lu tr trong qu trnh phn lp), trn u v ly khong 10g t cho vo

bc nilon nh hn ( s dng trong qu trnh phn lp). Ghi k hiu trn bc

nilon v ghi ch v trng thi cy la, rung la, a im thu mu... vo s tay.

- Thi im thu mu t vng r la c thc hin sau khi s hoc sau khi cy

khong 20 ngy tr i cho n trc khi thu hoch, m bo thi gian v c s

tng tc gia vi khun v vng quanh r la.

- t t vng r trong bc nilon nh (10g) c tch ring ra cho vo cc nh c

nh s v kh t nhin nhit phng th nghim (25-32C).

3.3.2. Phn lp vi khun c nh m t t vng r la

3.3.2.1. Tri mu

- Dng ci s nghin t vng r cho mn, trn u v cn 1g cho vo bnh tam gic

cha 100ml nc ct ngui sau khi kh trng.

- Dng my lc (Heidolph MR3001 - c) lc mu t trong 12 gi vi tc

200 vng/pht (cho cc ht t ri ra v vi khun phn tn trong nc). Sau

lng khong 3 gi.

- Dng micropipette ht 10 l phn dung dch trong, nh ln 5 vin bi cy (ng

knh 2-3 mm, c kh trng) c trong tng a Petri c mi trng

39

Pseudomonas isolation Agar Difco (Ph lc 1) (Mirza v ctv., 2006). Lc nh v

xoay u cc hng cho cc vin bi tri vi sinh vt u trn b mt mi trng c,

m np a cho kh. Sau y np li, lc u mt ln na v bi ra ngoi.

Bc ny c thc hin trong bung cy v trng (Laminar Flow - Vit Nam).

- t cc a cy vo t (Memmer - c), gi nhit 30C t 1 n 2 ngy.

3.3.2.2. Lm thun, quan st, m t c im khun lc v t bo vi khun

- T a Petri ban u, tin hnh chn lc cc khun lc ri rc c hnh dng, kch

thc khc nhau. Sau , t mi khun lc tin hnh cy chuyn nhiu ln trn cc

a Petri khc cha mi trng Pseudomonas isolation Agar theo phng php cy

ria phn lp tng dng vi khun. Tin hnh phn lp n khi cc khun lc ng

nht v khi quan st ch c mt dng vi khun di knh hin vi quang hc

(Olympus CH -20, Nht Bn) phng i 400 ln (c trc vi th knh), ghi nhn

hnh dng, kh nng chuyn ng v o kch thc ca t bo vi khun.

- Cy cc mu rng tr li trn mi trng Pseudomonas isolation Agar quan st

c im khun lc (hnh dng, mu sc, ni, dng ba, kch thc).

3.3.2.3. Tr mu rng

- Chn ly cc khun lc ri rc tr trong ng nghim hoc chai c np. Cc

vi khun sau khi lm rng (thun) v tr trong mt ng nghim th c xem

nh l mt dng (chng), tr 4C (t lnh Sanyo Nht Bn) v dng s

dng trong thi hn 30 ngy.

- Khi cn lu tr lu di (6-12 thng) th cc dng vi khun rng s c nui

cy mi trng nh trn nhng c b sung glycerol 10%, 2 ngy, sau

nh thm glycerol trn b mt khun lc trc khi tr ngn ng ca t lnh

(Sikorski v ctv., 2002).

40

Lc mu t Chn khun lc khc nhau v cy chuyn nhiu ln

Tr mu rng Chn khun lc ri

Hnh 3.1. S phn lp vi khun t mu t vng r la cho n khi tr rng

3.3.3. Xc nh kh nng c nh m (in-vitro) ca vi khun

3.3.3.1. Nui cy vi khun trong mi trng Burk khng m

Cc dng vi khun sau khi phn lp c nui cy trong mi trng Burk

khng m (Park v ctv., 2005), c th lng hay c (Ph lc 1). Nhng dng vi khun

no c kh nng sng st v pht trin trn mi trng ny l du hiu ban u cho

thy chng c kh nng c nh m.

Tri trn mi trng c hiu

30oC, 2 ngy

Kim tra rng di knh hin vi

v lu tr mu

41

Mi trng Burk c chnh pH 7,0 bng dung dch NaOH chun. Sau

cho 10ml mi trng Burk vo tng ng nghim c np cao su, kh trng nhit t

(121C, 20 pht), ngui. Sau cy vi khun t nhng dng tr vo ng nghim

bng que cy trong t cy v trng. nhit 30C trong 2-8 ngy (mi ngy lc

ng nghim 1-2 ln) v o hm lng NH4+ sau 2, 4, 6 v 8 ngy nui cy. Nhng

mu ny c tin hnh m s t bo vi khun bng bung m sau 2 ngy nui cy

(bung m Neubauer, c, s t bo/ml = S t bo trong bung m x 104 x pha

long), dch vi khun t 107 t bo/ml s c dng lm chun so snh c v

xc nh s pht trin ca cc dng vi khun sau 2 ngy nui cy.

3.3.3.2. o hm lng NH4+ bng phng php Phenol Nitroprusside

Mi dng vi khun c nhn nui trong 3 ng nghim c np cha mi trng

Burk lng khng m (Park v ctv., 2005) t 2 n 8 ngy. Tin hnh o lng NH4+ sinh

ra t cc dng vi khun trong mi ng nghim trn bng phng php Phenol

Nitroprusside qua my o OD (Beckman Coulter DU 640B, c) xc nh kh nng

tng hp NH4+ ca tng dng vi khun 8 sau 2, 4, 6 v 8 ngy nui.

Xy dng ng chun vi cc ng nghim chun sau y (bng 3.1):

Bng 3.1 Bng s liu xy dng ng chun NH4+

ng 0 1 2 3 4 5

H2O (ml) 5 4 3 2 1 0

NH4 chun (ml) 0 1 2 3 4 5

Nitroprusside (ml) 5 5 5 5 5 5

Sodium Hypochloride (ml) 5 5 5 5 5 5

Phng php o:

- Lc u dch nui vi khun sau 2 (hoc 4, 6, 8) ngy, ly 1,5ml mu

cn o cho vo tube eppendorf, li tm 12000 vng/pht trong 5 pht.

- Pha dung dch Nitroprusside:

42

+ 5g phenol /0,5 lt

+ 0.025g Nitroprusside / 0,5 lt

- Pha dung dch sodium Hypochloride

+ 5g NaOH / 0,5 lt

+ 15ml hypochloride / 0,5 lt

- Ly 2,5ml nc khong cho vo mi ng nghim ng chun v mu

- Chun b mu o: H2O (4 ml) + mu (1ml) + Nitroprusside (5ml) +

sodium Hypochloride (5ml). Mi mu lp li 2 ln.

- Vortex, sau yn khong 15 pht; ri tin hnh o OD bc

sng 640nm.

Hnh 3.2 Cc ng nghim xy dng ng chun o ammonium

Tnh ton nng NH4+ v thng k nh gi s b v kh nng c nh m

thng qua hm lng NH4+ m vi khun tng hp c.

Da vo phng trnh ng chun: y = a*x + b, trong : x l nng ca

mu (mg/l), y l hp th quang. Sau , o hp th quang ca mu cn phn tch

tnh hm lng N-NH4 c trong mu theo cng thc: x = (y b)/a.

3.3.3.3. Th hot tnh nitrogenase bng phng php kh acetylen (ARA)

Phng php ny dng o hot lc ca enzyme nitrogenase. S dng

phng php kh acetylen (C2H2) thnh etylen (C2H4) xc nh hot tnh ca

0 1 2 3 4 5

43

nitrogenase trong phng th nghim. kh mt phn t N2 th cn 6 in t, cn kh

mt phn t C2H2 ch cn 2 in t. Nh vy, phi kh 3 phn t C2H2 mi tng

ng vi vic kh mt phn t N2. Cn c lng C2H4 sinh ra (hoc lng C2H2 b

kh) m xc nh lng nit c nh c ca tng loi vi sinh vt. Cng c th dng

lng C2H4 sinh ra biu th hot tnh ca enzyme nitrogenase. Thnh phn I

(protein Mo-Fe) v thnh phn II (protein Fe) rt mn cm vi oxy. V vy, trong qu

trnh chit xut v phn tch nitrogenase u cn tin hnh trong iu kin k kh

(anaerobic).

C th, t phn ng N2 + 3H2 2NH3 l phn ng pht nhit, trong

nitrogenase hot ng cung cp hydrogen cho phn ng ny. Nh vy, t lng

hydrogen s suy ra c lng nitrogenase. T , th nghim kh acetylen cho thy

rng: nitrogenase to ra hydrogen cng nhiu th acetylen b kh cng nhiu v s to

c nhiu etylen.

Nui vi khun trong mi trng Burk lng khng m trong ng nghim kn

trn my lc, sau 2 ngy bm 0,5 ml kh acetylene vo tng ng nghim, sau khi bm

acetylene 30 pht ht 1 ml kh trong mi ng nghim o trn my sc k kh

SHIMADZU GC 1415. Trong qu trnh c tin hnh pp li 3 ln. T hm

lng acetylene b kh thnh ethylene suy ra lng nitrogenase.

3.3.4. Kim tra vi khun bng phng php sinh hc phn t

3.3.4.1. Tch chit DNA (Neumann v ctv., 1992)

Cy chuyn khun lc kim tra rng vo ng nghim c cha mi trng LB

(Luria Bertani broth) bao gm Bacto yeast extract 5g/l, Bacto tryptone 10g/l v NaCl

10g/l (Bennasar v ctv., 1998), lc 200 vng/pht trong 12-18 gi thu sinh khi vi

khun. Sau , tin hnh trch DNA ca vi khun vi cc bc nh sau:

- Chuyn 4ml dch vi khun t ng nghim nui cy sang 2 tup Eppendorf

2ml.

44

- Ly tm tup Eppendorf trn vi tc 13.000 vng/pht trong 5 pht

nhit phng. Loi b phn nc.

- Ht 250l dung dch TE pH = 8,0 cho vo tup trn. Dng tay nh tan

sinh khi.

- Thm vo 50l SDS 10% ha tan DNA.

- Tip theo thm 5l proteinase K (10mg/ml) phn hy cc protein lin kt

vi DNA.

- em 65C trong 20 pht, 5 pht o ngc tup 1 ln.

- Thm vo 400l CTAB 10% 0,7M NaCl.

- em 65C trong 20 pht.

- Thm vo 600l Chloroform: Isoamyl alcohol loi b cc thnh phn

khng mong mun trong mu, ch yu l cc protein bng tc dng lm

bin tnh protein ng thi khng ha tan acid nucleic, mang khu trang

cn thn v l ha cht c, tin hnh trong t ht.

- Lc tup tht mnh ri em ly tm vi tc 12.000 vng/pht trong 10

pht nhit phng. Protein khi b bin tnh s khng cn ha tan

trong pha nc c cha acid nucleic v sau khi ly tm s hnh thnh mt

mng ngn gia DNA v protein. Protein ta thnh mt lp nm bn di

mng ngn, ta thu c pha nc pha trn c cha acid nucleic.

- Chuyn phn dch trong pha trn sang tup eppendorf 1,5ml mi, thao

tc cn thn trnh lm v mng ngn...

- Thm vo 1ml Isopropanol ta DNA v thu c sn phm dng c

c ( bo v DNA khi s phn hy ca cc enzyme, c th ha tan

DNA li trong dung dch theo nng mong mun), trn u. Gi tup

-20C t nht 30 pht.

- em ly tm vi tc 13.000 vng/pht trong 10 pht nhit phng

th nghim c my iu ha nhit dao ng trong khong 22 2C.

Loi b phn nc. Cc DNA c trng lng phn t thp (b gy do thao

tc hoc cc enzyme ni bo gii phng ra mi trng khi ph v t bo)

45

khng b ta, v th c th loi chng ra khi sn phm khi ta bng

isopropanol.

- Phn DNA ta c ra loi b cc du vt ca isopropanol cn dnh li

trn mu bng cch cho vo 1ml ethanol 70% (2 ln). Mi ln ra s em

ly tm 12.000 vng/pht trong 5 pht nhit phng.

- em ly tm chn khng trong 15-20 pht 45C loi b ethanol.

- Ha tan trong 30l nc ct hai ln v trng (Bi nc) v tr - 20C

chun b chy PCR.

3.3.4.2. Khuch i DNA

DNA ca cc dng vi khun sau khi trch, tin hnh chy phn ng PCR bng

my PCR Perkin Elmer PE 9700 (M) nhn din cc dng vi khun c nh m

phn lp.

u tin, cp mi FGPS c s dng khuch i on 1500 bp ca

Pseudomonas theo phng php sinh hc phn t phn tch trnh t RNA ribo th - K

thut PCR -16S rRNA (Mirza v ctv., 2006) (Ph lc 2). Cp mi FGPS c trnh t v

chu k phn ng PCR nh sau:

Trnh t cp mi:

- Mi xui FGPS4-281bis (5 3): AGA GTT TGA TCC TGG CTC AG

- Mi ngc FGPS1509-153 (5 3): AAG GAG GTG ATC CAG CCG CA

Phn ng PCR: 95C/5; 35 chu k: (95C/45, 60C/45, 72C/130); 72C/10.

Tip theo, chn cc dng vi khun cho bng v tr 1500 bp tip tc thc hin

phn ng PCR vi cp mi c hiu (specific primers) xc nh gen nifH 16S rDNA

ca vi khun c m thuc nhm Pseudomonas (Poly v ctv. 2001) (Ph lc 2). Cp mi

Pol (khuch i on 361 bp ca nifH) c trnh t v chu k phn ng PCR nh sau:

Trnh t cp mi:

46

- Primer PolF-115: 5-TGCGAYCCSAARGCBGACTC

- Primer PolR-476: 5-ATCGCCATCATYTCRCCGGA

Phn ng PCR: 95C/5; 35 chu k: (95C/45, 58C/45, 72C/2); 72C/10.

Sau , s dng cp mi PST3422 s khuch i phn on 575 bp ca gen

nifH P. stutzeri A1501 (Yan v ctv., 2010) (Ph lc 2). Cp mi PST3422 c thit

k bng chng trnh DNAMAN da trn gen nifH ca vi khun P. stutzeri A1501

c cng b trn ngn hng d liu gen (NCBI database). Cp mi PST3422 c trnh

t v chu k phn ng PCR nh sau:

Trnh t cp mi PST3422:

- Mi xui PST3422-5: 5-AGGATCGAGAGCGGGTACT-3

- Mi ngc PST3422-580: 5-CACCTATCCACTTTCCGATG-3

Phn ng PCR: 95C/5; 35 chu k: (95C/45, 53C/45, 72C/1); 72C/10.

Thc hin cc bc tip theo ca chu k phn ng PCR. Cui cng em tr sn

phm khuch i 4C cho n khi dng.

3.3.4.3. in di cc sn phm PCR

Cc sn phm DNA vi khun sau khi khuch i bng phn ng PCR s tip tc

c phn tch bng phng php in di trn gel agarose 1% vi 6X loading buffer (LB)

c nhum vi ethidium bromide (0,5mg/l). Sau khi in di trn gel agarose, cc mu

DNA ca vi khun c quan st kt qu di tia UV (bc sng 260 nm) ca my chp

hnh gel Bio-Rad Universal HoodII (M) xc nh cht lng ca DNA, cng nh

pht hin vi khun c nh m. S dng thang chun DNA 100bp plus (vi cp mi

tng) v 100bp (vi cp mi c hiu) c lng kch thc ca bng DNA vi khun

trn hnh gel chp c (Ph lc 3).

3.3.4.4. Chp hnh gel in di cha sn phm phn ng PCR