Research Article Molecular Cloning and Characterization of Novel …downloads.hindawi.com/journals/bmri/2014/973790.pdf · 2019. 7. 31. · Research Article Molecular Cloning and

Research ArticleMolecular Cloning and Characterization of Novel PhytocystatinGene from Turmeric Curcuma longa

Seow-Neng Chan1 Norliza Abu Bakar2 Maziah Mahmood1

Chai-Ling Ho3 and Noor Azmi Shaharuddin1

1 Department of Biochemistry Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (UPM)43400 Serdang Malaysia

2 Biotechnology Research Center Malaysian Agricultural Research and Development Institute (MARDI) 43400 Serdang Malaysia3 Department of Cell and Molecular Biology Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (UPM)43400 Serdang Malaysia

Correspondence should be addressed to Noor Azmi Shaharuddin noorazmiupmedumy

Received 3 April 2014 Accepted 8 June 2014 Published 14 July 2014

Academic Editor Marco Bazzicalupo

Copyright copy 2014 Seow-Neng Chan et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Phytocystatin a type of protease inhibitor (PI) plays major roles in plant defense mechanisms and has been reported to showantipathogenic properties and plant stress tolerance Recombinant plant PIs are gaining popularity as potential candidates inengineering of crop protection and in synthesizing medicine It is therefore crucial to identify PI from novel sources like Curcumalonga as it is more effective in combating against pathogens due to its novelty In this study a novel cDNA fragment encodingphytocystatin was isolated using degenerate PCR primers designed from consensus regions of phytocystatin from other plantspecies A full-length cDNA of the phytocystatin gene designated CypCl was acquired using 5101584031015840 rapid amplification of cDNAends method and it has been deposited in NCBI database (accession number KF5459541) It has a 687 bp long open reading frame(ORF) which encodes 228 amino acids BLAST result indicated that CypCl is similar to cystatin protease inhibitor from Cucumissativus with 74 max identity Sequence analysis showed that CypCl contains most of the motifs found in a cystatin including a Gresidue LARFAV- QxVxG sequence PWdipeptide and SNSL sequence at C-terminal extension Phylogenetic studies also showedthat CypCl is related to phytocystatin from Elaeis guineensis

1 Introduction

In an effort to increase food security and provide protec-tion to plants that are constantly in combat with pest andpathogens various genes coding for defense related proteinshave been incorporated into crops An example that gainspopularity as a suitable candidate gene for crop protection isprotease inhibitors (PIs) A great number of PIs have beencharacterized from different plant species and engineeredinto crop plants for protections against pest and pathogens[1ndash4] PIs are commonly found in plants and play a role inregulating digestive proteases by forming a complex with thetarget protein either on the active site or on the allostericsite [4] In a nonhost plant pathogenesis PIs are released asone of the defense responses by plants to protect themselves

against the attacks of their predators [3ndash5] However as pestand pathogens are slowly adapting and acquiring resistanceagainst the currently utilised PIs it is crucial to continuouslydiscover PIs from novel sources PIs from novel sources havenot been exposed to the crop pest and pathogens before andthis helps in overcoming the resistance adaptation by pest andpathogens [3 4]

Cysteine protease inhibitor or phytocystatin is a type of PIthat has long been identified and reported to have importantroles in plants [6] It acts as an endogenous proteolysisregulator during seed maturation and development involvesin programmed cell death and inhibits exogenous proteasesreleased by plant predators like insects nematode bacteriaand pathogens during the attack [6ndash12] In recent yearshowever phytocystatin has also been suggested to be involved

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 973790 9 pageshttpdxdoiorg1011552014973790

2 BioMed Research International

Table 1 Efficiency of different methods of total RNA extraction from turmeric (Curcuma sp) plant

Method 119860260280

ratio Yield concentration (ng120583L) Time durationModified CTAB Method 200ndash220 sim300 SlowRNAzol RT 180ndash200 800ndash1000 FastRNeasy Plant Mini kit 170ndash200 300ndash500 Fast

1 2 3 4 5 6

Figure 1 Total RNA integrity test on 10 agarose gel Intact 28S and18S total RNA bands can be observed on the agarose gel indicatinga good integrity of the total RNA after being extracted by differentmethods (lanes 1 and 2 modified CTAB method lane 3 4 RNAzolRT and lane 5 6 RNeasy Plant Mini kit)

in response to biotic and abiotic stresses as they are highlyexpressed during harsh condition such as cold droughtsalt stress oxidant stress and wounds [8 9 12] All theseproperties encourage more research to be done on PIsespecially on phytocystatin Ayurvedic [2ndash4]

Generally phytocystatins contain several conservedmotifs such as QxVxG (the active site) a PW dipeptidein the region towards the C-terminal and one or tworesidues of G towards the N-terminal Another notablemotif is the conserved [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-X-[EDQV]-[HYFQ]-N sequence which is found in theupstream of the active site The sequence formed an 120572-helixstructure but lacks disulphide bonds and glycosylation sites[6ndash9 13 14] Phytocystatins identified so far have differentsizes the majority are small proteins that range from 12 kDato 16 kDa and contain no disulphide bonds [6 7 10]Several phytocystatins have slightly higher molecularmasses around 23 kDa due to the C-terminal extensioncontaining the motif SNSL and they are reported to inhibitlegumain [15] In potato and tomato multicystatins havebeen identified with multiple cystatin domains and have amolecular mass of 85 kDa [6 7 10]

Turmeric (Curcuma sp) a medicinal plant from Zingib-eraceae family is one of such novel sources and up till nowthere are no PIs that are being characterized and reportedTurmeric is commonly used as traditional medicines andspices for culinary arts in country like India Malaysia andother Asian countries Turmeric as stated in Ayurvedicmedicine has the characteristic of antifungal and anti-inflammatory properties [16 17] Recent studies have alsoproven that turmeric does exhibit antitumour antimicro-bial anti-HIV nematocidal and antioxidant properties [18ndash20] Sookkongwaree et al [21] had extracted secondary

compounds from Zingiberaceae family and it was proven toexhibit antiviral and antiprotease properties It is suggestedthat PIs could have possibly contributed to the antiviral andantiprotease properties of the secondary compounds

In this study we have identified and characterized a novelfull-length sequence of cysteine protease inhibitor gene fromCurcuma longa by using RACE-method (rapid amplificationof cDNA ends) The phylogenetic relationship of the genewith other plant species and predictions of the putativefunctions of the identified gene was also conducted

2 Results

21 Comparison of Total RNA Extraction with DifferentMethods Total RNA of turmeric (Curcuma longa) was suc-cessfully extracted from mature leaves of turmeric plant bythree different methods (i) modified CTAB (cetyltrimethylammonium bromide) method (ii) RNAzol RT (MolecularResearch Center Inc USA) and (iii) RNeasy Plant Mini kit(Qiagen USA) Intact bands of the 28S and 18S RNA can beclearly observed in the agarose gel for all the three methods(Figure 1) As for the quantitative test of the total RNA withthe raw tissue weight of 200mg our results showed thattotal RNA extraction by RNAzol RT produced the highestyield with concentration of sim800 ng120583L while extraction byRNeasy PlantMini kit and by amodifiedCTABmethod yieldssim300 ng120583L of total RNA Both RNAzol RT and RNeasy PlantMini kit producedA

260280ratio at the range of 170ndash180while

a modified CTAB method produced the ratio at the range of200ndash220 Table 1 summarised the comparison of the threeextraction methods

Therefore RNAzol RT was chosen as the preferredmethod to extract the total RNA from different parts ofturmeric plant (leaf flower and rhizome) because of thehighest extraction yield and faster time duration as comparedto the other methods However based on the agarose gelelectrophoresis result (Figure 2) it shows that RNAzol RT isonly able to extract total RNA from leaves and is not suitablefor flowers and rhizomes

22 Cloning and Sequence Analysis of Curcuma longa Phyto-cystatin cDNA Degenerate polymerase chain reaction (PCR)was conducted to isolate the novel target gene phytocystatinA 495 bp cDNA fragment was successfully amplified fromtotal cDNA reverse transcribed from the total RNA ofleaves sample of Curcuma longa 5101584031015840 RACE (rapid ampli-fication of cDNA ends) was conducted and the full-lengthcDNA designated CypCl was then deposited to the NCBI(National Center for Biotechnology Information) databasewith accession number KF5459541 The cDNA (Figure 3(a))

BioMed Research International 3

1 2 3

Figure 2 Total RNA extracted by using RNAzol RT on differenttissues samples (1) leaves (2) flowers and (3) rhizomes The resultsshowed that only the total RNA from the leaves samples wassuccessfully extracted as compared to the other two samples

is 870 bp long and contains a 687 bp long open reading frame(ORF) encoding 228 amino acids

Sequence analysis of the full-length CypCl gene(Figure 3(b)) showed that the deduced amino acid sequenceof CypCl contained motifs that are generally found inphytocystatin The detected motifs are (i) a single G residueand [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-X-[EDQV]-[HYFQ]-N sequence towards the N-terminal (ii) QxVxGsequence and (iii) PW dipeptide towards the C-terminalFurthermore CypCl is shown to contain C-terminalextension as multiple alignments of the deduced amino acidsequence of CypCl along with phytocystatins of other plantspecies show sequence homology (Figure 4) Additionalevidence includes the detection of the motif SNSL at C-terminal proximity of the gene The predicted pI of theputative protein is 572 and a calculated molecular mass is258 kDa A putative start codon is positioned at 22ndash24 andthe stop codon is at the position of 724ndash726 (Figure 3(a))When the deduced amino acid sequence was subjectedto protein BLAST using BLASTP (Basic Local AlignmentSearch Tool) [22] several conserved domains belongingto cystatin superfamily were detected on the sequence(Figure 5) BLASTP also showed that the sequence showedthe highest similarity (74 max identity) to the predictedcysteine protease inhibitors from Cucumis sativus (accessionno XP 0041655171) with query coverage of 88 and 119864 valueof 4119890 minus 102

23 Sequence Analysis and Phylogenetic Tree ConstructionIn order to study the evolution relationship and predictedfunction of the novel phytocystatin from turmeric a phy-logenetic tree was generated with deduced amino acidof CypCl The unrooted phylogenetic tree (Figure 6) con-structed with phytocystatin containing C-terminal extensionby using neighbour-joining method showed that the novelCypCl from turmeric is related to phytocystatin from Elaeisguineensis (African oil palm) CypCl is grouped together withphytocystatins from other monocot plants forming one clade

while the majority of eudicot plants formed another cladeexcept for the Brassicaceae family

With the presence of the motifs on the sequence BLASTresults and the expected molecular mass (approximately23 kDa) it is suggested that full-length cDNA CypCl isidentified as phytocystatin and belongs to the group with theC-terminal extension It is also suggested to be a novel phy-tocystatin and sequence is expected to exhibit the commonfunctions of a phytocystatin with C-terminal extension

3 Discussion

31 Total RNA Extraction with Different Methods Curcumalonga plant is amedicinal plant that contains a high amount ofsecondary metabolites and phenolic compounds In relationto that three different extraction methods including oneconventionalmethod (CTABmethod) and two commerciallyavailable kits (RNAzol RT and Qiagen RNeasy Plant Minikits) were employed The extraction result was comparedto identify the most suitable method to extract total RNAfromCurcuma longa for downstream applications andwe hadchosen RNAzol RT as the extraction method

Based on the result in Figure 1 andTable 1 all of themeth-ods successfully extracted total RNA from Curcuma longarsquosleaves However in terms of the speed of extraction RNAzolRT and Qiagen RNeasy Plant Mini kits were fast and able toextract total RNA in less than an hour whereas CTABmethodrequired 3 days to achieve the same result Qiagen RNeasyPlant Mini kit utilises the silica-based membrane column tobind total RNA while RNAzol RT is a single-step total RNAextractionmethodwhich excludes the addition of chloroformto phase out the pure total RNA and the separation isdone based on the interaction of phenol and guanidine withother cellular components [23] CTABmethod in comparisoncontained much more steps including a phenol chloroformextraction ethanol and LiCl precipitation that lengthen theextraction period While Qiagen RNeasy Plant Mini kit andRNAzol RT kit had similar speed in total RNA extractionRNAzol produced a higher total RNA yield about twice asmuch as yields of the other two methods

However the total RNA extracted by RNAzol RT andQiagen RNeasy Plant Mini kits had a lower A

260280ratio

from 170 to 200 compared to CTAB method with A260280

ratio from 200 to 220 A pure and good quality totalRNA will have A

260280ratio at 200 and the ratio below

this value normally is caused by phenol or protein contam-ination CTAB-based extraction method is a recommendedmethod to extract total RNA from difficult samples includingwoody tissues and tissues rich in secondary metabolitesCTAB-based extraction buffer contained PVPP (polyvinylpolypyrrolidone) that helps in eliminating polyphenolicscompounds from the homogenate by forming a complexthrough hydrogen bonding [24 25]Thebuffer also contained120573-mercaptoethanol to prevent the production of quinonesfrom phenolics thus preserving RNA during the extraction[25] However the amount of secondarymetabolites is higherin rhizome and flower samples than in leaves samples Thismay explain why RNAzol RT method was unable to extracttotal RNA from the rhizome and flower samples as RNAzol


800 bp

M 1

600bp

(a)

1 atgctcatcccactcagccgagccctcttctttcttgtcgccctcattctgatctccgat

M L I P L S R A L F F L V A L I L I S D

61 tccctgttcttctcgcttgcgcttgcatccatggttggagatgtgagagaatcggatggg

S L F F S L A L A S M V G D V R E S D G

121 gcgcagaacagtgccgagctcgaggagctcgctcgcttcgctgtcgaggagcacaacaaa

A Q N S A E L E E L A R F A V E E H N K

181 aaagagaatagacttctggaatttactcgagtggtgaaggcaaaagagcaagttgtggca

K E N R L L E F T R V V K A K E Q V V A

241 ggaacacttcattacttgaccttggaggcaatcgacgcagggaagaagaagctgtatgaa

G T L H Y L T L E A I D A G K K K L Y E

301 gccaaggtatgggtgaagccgtggctccactttaaagagcttcaagagtttgagcatata

A K V W V K P W L H F K E L Q E F E H I

361 ggagactctccctcttttaccactgctgacctcggtgctaaacgagaggagcttggtcct

G D S P S F T T A D L G A K R E E L G P

421 ggtttgcgcactgttcctgtaaatgatcctctggtcagagatgcagcagaccatgctttg

G L R T V P V N D P L V R D A A D H A L

481 aagaccatccaacaaaggtccaattccctgtttccttacgagctgctggaggtcctccat

K T I Q Q R S N S L F P Y E L L E V L H

541 gcaaaggcagaggtgattgaagaaactgcaaagtttgacatgctccttaaagtaaagaga

A K A E V I E E T A K F D M L L K V K R

601 ggaagcaaggaggagaaatttaaggccgaggtccacaagaacctggaaggtaactttctt

G S K E E K F K A E V H K N L E G N F L

661 ttgaatcagatgcaacaggagaattag

L N Q M Q Q E N -

(b)

Figure 3 (a) cDNA of CypC with 870 bp long was amplified using primers covering from 51015840 end to 31015840 end designed from the informationobtained in 5101584031015840 RACE-method (lane M 100 bp DNA ladder lane 1 CypCl) (b) Nucleotide and deduced amino acid sequences of theidentified full-length cDNA of CypCl A signal peptide (from amino acid position 1ndash29th) was predicted by using SignalP30 program Thecleavage site is between amino acids 29 and 30 (as indicated with black arrow) Several motifs of phytocystatin were detected in the full-lengthsequence such as (i) one G residues towards N-terminal (ii) LARFAVEEHN (iii) QVVAG (iv) PW dipeptide and (v) SNSL The respectivemotifs had been boxed

RT lacked PVPP and 120573-mercaptoethanol which are crucialfor total RNA extraction in samples with rich secondarymetabolites [25] Since only total RNA from leaves sampleswas needed for the isolation of CypCl RNAzol RT methodwas chosen as the extraction method

32 Analysis of Full-Length Phytocystatin mRNA in Curcumalonga The full-length CypCl that was amplified from thecDNA of Curcuma longa was homology-analysed throughBLAST against the existing phytocystatin genes in the Gen-Bank and it was found to contain all the motifs that arecommon in phytocystatin family The discovery of thesemotifs on the full-length sequence is crucial to identifyand provide more information on the gene The QVVAGmotif and the PW dipeptide motif (Figure 3(b)) found onthe full-length gene belong to the region of the protein thatform hairpin loops respectively and are responsible for theinhibitory activity of cystatin [26] By studying the structuralmodel of oryzacystatin a well-characterised phytocystatinfrom rice genome both of the hairpin loops form a tripartite

wedge with the conserved G residues (found on the N-terminal)which then slots into the active site cleft of the targetenzyme and inhibits it The role of the tripartite structuralelements was confirmed when mutagenesis including aminoacid substitution partial deletions random mutations andmutant variants on these hairpin loops had showed reductionor termination of the inhibitory effect of cystatin towardspapain [10] The presence of these motifs on the aminoacid sequence of the full-length CypCl suggested that thefull length of gene is probably functionally active in papaininhibition

In addition the analysis of the full-length CypCl againstother phytocystatins in the database also showed that it con-tained a conserved [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-X-[EDQV]-[HYFQ]-Nmotif where the sequence in the cDNAis LARFAVEEHN and the SNSL motif in C-terminal exten-sion The [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-X-[EDQV]-[HYFQ]-N conserved motif is found within the 120572-helixstructure of phytocystatin but the function of this structureis still unknown [10 26] However on the evolutionary point


1 ABG898561 (1)

(1) (1)

(1) (1)

(1) (1)

(1) (1) (1) (1) (1) (1) (1)

(1)

(1)

(1)

(1)

(1) (1) (1) (1) (1) (1)

(78) (72) (39) (90) (72) (88) (34) (38) (65) (81) (37) (41) (69) (74) (85) (37) (37) (77) (43) (37) (70) (37) (66) (81)

EOY195841 CAA896971 AAD138121 AAF231261 AAU815971 AAK150901

CypCl Os01g0270100

ACF065481 AAL308301 CAA606341

NP0011052951 ADP507601 ABG810971 ADB028941 AAQ072591 NP1967751

AAM883971 ADD511911 ABK786891 CAH601631

NP0012377341 91 180

ABG898561EOY195841CAA896971AAD138121AAF231261AAU815971AAK150901

CypClOs01g0270100

ACF065481AAL308301CAA606341

NP0011052951ADP507601ABG810971ADB028941AAQ072591NP1967751

AAM883971ADD511911ABK786891CAH601631

NP0012377341181 268

(161) (158) (129) (173) (155) (171) (117) (121) (148) (164)(120)(103)(131)(136)(148)

(99)(107)(136)(102)(120)(153)(121)(155)(164)

ABG898561EOY195841CAA896971AAD138121AAF231261

CypCl

AAK150901

Os01g0270100

AAU815971

ACF065481AAL308301CAA606341


AAM883971ADD511911ABK786891CAH601631

NP0012377341

--MLIKFSFLLPHSSTILLLFSLIFFFSPSSQGSCSDFESEP----------SMATLGGLRES-QGAANDAEIESLARFAVDEHNKKENA

--------MRKPNCLLFSFFSVAIQFLFFDKFSAESILCSEP----------MATTLGGVHPSHG-SQNSAELENLARFAVDEHNKKENA

----------------------------------------------------MATVQGGVHDSPQGTANNAEIDGIARFAVDEHNKKENA

MRVRFFFYFAYFLITLVFFPSVTLQSYSGGHRQEATGFCGEEGEREDNLIRMATTTLGGISDSAS-AENSVEIESLARFAVEEHNKKENA

--------MRVIRSRAILIVLFLVSAFGLSEQGKSGGFCSEE-----------MATLGGVHDSHGSSQNSDEIHSLAKFAVDEHNKKENA

-MRVNRNATVFIALNLILFLVFVFTAFALSGQENTGGFCGEEEEK--GNNYIEMALLGGIRDSHPESQNSDEIHSLAKFAVDEHNKKENA

-----------------------------------------------------MATLGGVHDSNS----NPDTHSLARFAVDQHNTKENG

----------------------------------------------------MASTLGDVRDSQGA-SNSADVDELARFAVEEHNKKENS

--------------MLIPLSRALFFLVALILISDSLFFS-----------LALASMVGDVRESDGA-QNSAELEELARFAVEEHNKKENR

---MRVAATTRPASSSAAAPLPLFLLLAVAAAAAALFLVGSAS------LAMAGHVLGGAHDAPSA-ANSVETDALARFAVDEHNKRENA

-----------------------------------------------------MATVGDVRDSQIN-ENSVEIEELGRFAVQEHNKKENA

------------------------------------------------MSSDGGPVLGGVEP--VGNENDLHLVDLARFAVTEHNKKANS

-------------ARVVPLVAALLVLLALAVSSTRNRNAQE--------GEESMALDGGIKDV-PANENDLHLQELARFAVDEHNKKANA

----------MRKHRIVSLVAALLVLLALAAVSSTRSTQKESV------ADNAGMLAGGIKDV-PANENDLQLQELARFAVNEHNQKANA

-----MRRFHRALGSVAAPALLLLLLLALVLPSIQTHSQKQSAGEKAAMAEPARRLAGGIVES-PGREKDPYIVGLARFAVSEHNKEANT

-----------------------------------------------------MTTVGGISET-PGTQNSLQIDALARFAVDEHNKKQNT

-----------------------------------------------------MATVGGIKEV-EGFANSVEIDGLARFAVDDYNKKQNA

-------MADEQPKKPVPAAEEAPAAEAEAEEEEGLHLEDDQEPR-------EHPIMGGIYDAPLNNENGFDKEDLARFAVREYNNKNNA

-------------------------------------MADQQAG----------TIVGGVRDIDAN-ANDLQVESLARFAVDEHNKNENL

-----------------------------------------------------MALMGGIVDVEG-AQNSAEVEELARFAVDEHNKKENA

-----------MEFNRLVLLSLSFVLVSQIGFCREDSFIRMP---------KTMNLLGGVRDCGG-IQNSAEIESIARFAVQEHNKNQNA

-----------------------------------------------------MALLGGVRDVPAY-QNSDEVESLARFAVDEHNKKENV

---MKLRSQSNSSPSFVFFFVLFFSFIALS----------------------EMATLGGIRDSPAGSENSLETEALGRFAVDDHNQKQNG

MRALTSSSSTFIPKRYSFFFFLSILFALRSSSGGCSEYHHHHA---------PMATIGGLRDSQGS-QNSVQTEALARFAVDEHNKKQNS

LLEFARVVKAKEQVVAGTLHHFTIEAIDAGKKKLYDAKVWVKPWMNFKELQEFKHTEDS-------PSFTSSDLGAIREGHAPGWKEVPV

MVEFVRVVKATEQVVAGTLHHLTVEAIDAGKKKLYEAKVWVKPWMNFKELQEFKHAGDADVS----PSFTASDLGVKKDVHGPGLQALPT

MVEFGRVLKAKEQVVAGTLHHLTIEAIEAGKKKIYEAKVWVKPWLNFKELQEFKHATDVADTTASHPSFTSSDLGVKREGHGAEWKEVAA

MIELVRVVKAEEQVVAGKLHHLTLEVIDAGKRKLYEAKVWLKPWMNFKELQGFNHIEDI-------PTLTSSDLGAKRDWPNTGLKSVPV

MIELARVVKAQEQTVAGKLHHLTLEVMDAGKKKLYEAKVWVKPWLNFKELQEFKHVEDV-------PTFTSSDLGVKQVEQNSGLKSVPV

MFELARVVKAKEQVVAGTLHHLTLEVVDAGKKKLYEAKVWVKPWLNFKELQEFTHVEDA-------PAITSSDLGVKKEEQCSGFKSVPV

LLELVRVVEAREQVVAGTLHHLVLEVLDAGKKKLYEAKIWVKPWMDFKQLQEFKHVRDV-------PSFTSSDLGAKTDDQVSGWRPVPV

LLEFVRVVKAKEQVVAGTLHHLTVEVVEAGKKKLYEAKVWVKSWMNFKELQEFKHAGDV-------PSITPSDLGAKKGDHPQGWREVAP

LLEFTRVVKAKEQVVAGTLHYLTLEAIDAGKKKLYEAKVWVKPWLHFKELQEFEHIGDS-------PSFTTADLGAKREELGPGLRTVPV

LLEFVRVVEAKEQVVAGTLHHLTLEALEAGRKKVYEAKVWVKPWLDFKELQEFRNTGDA-------TTFTNADLGAKKGGHEPGWRDVPV

LLEFARVVKAKEQVVAGTLHHLTLEVMEAGKKKLFEAKVWVKPWLNFKELQEFTHLGDS-------PSVTAADLGAKHGGHEPGWRTVPA

LLEFEKLVSVKQQVVAGTLYYFTIEVKEGDAKKLYEAKVWEKPWMDFKELQEFKPVDASANA----------------------------

LLGYEKLVKAKTQVVAGTMYYLTVEVKDGEVKKLYEAKVWEKPWENFKELQEFKPVEEGASA----------------------------

LLGFEKLVKAKTQVVAGTMYYLTIEVKDGEVKKLYEAKVWEKPWENFKQLQEFKPVEEGASA----------------------------

RLEFDKVVKVKEQAVAGRLYYITIQVDEGGAKKLYEAEVLEQLWLDVKKLLEFKPAEAASPNV---------------------------

LLQFGGVVSVKQQVVAGTMYYITLEAMDGDKKKLYEAKVWEKPWMNFKELQEFKPVGDASSA----------------------------

LLEYKRVVNAKQQVVAGTIYYITLEVEDGGQKKVYEAKIWEKPWLNFKDVQEFKLIGDAPTQSTPTESTA--------------------

LLEFVRVVKAKEQVVSGMMHYLTVEVNDAGKKKLYEAKVWEQVWMNFRQLQEFTYLGDA-------------------------------

TLEYKRLLGAKTQVVAGTMHHLTVEVADGETNKVYEAKVLEKAWENLKQLESFNHLHDV-------------------------------

LLQFSRLVKAKQQVVSGIMHHLTVEVIEGGKKKVYEAKVWVQAWLNSKKLHEFSPIGDS-------SSVTPADLGVKRDAHEAEWLEIPT

LLEFAKVLKAKEQVVASKIYYLTLEAIDAGKKKIYEAKVWVKPWMNFKELQEFKHAQET-------KSFTSSDLGVKQDGHGSEWQVVPT

MLEFVRVVKAKEQVVAGTLHHLVVEAIDGGKKKLYEAKVWVKPWLNFKQVQEFKHAGEPETV-SGTPSFTSSDLGVKQGGHAPGWQDVHP

LLEFSRVVRTQEQVVAGTLHHLTLEAIEAGEKKLYEAKVWVKPWLNFKELQEFKPAG-------DVPSFTSADLGVKKDGHQPGWQSVPT

HDPEVQNAAEHAVKTIQQRSNSLFPYELQEIAHAKAEVVEDTAKFNLHLKVKRGNKDEIFNVEVHKSSD-GNYNLNKMGNIQPEIENQ

HDPVVQDAANHAVKTIQQRSNSLVPYELKEIVHAKAEVLEDFAKLDMLLKVKRGDKEEKFKVEVHHKSE-GTFHLNRMEPDHS-----

HDPVVQDAATHAVNTIQQRSNSLFPYQLQEIVHAKAQVVDDFAKFDMILKVKRGTSEEKFKVEVHKNNE-GTFLLNQMEPHT------

NDPVVQEAAQHAVKTIQQRSNSLLPYELQEIVHANAEVIDDSAKVHMLIKTKRGEKEEKFSVEVPKNNE-GAFFLNHMAPANS-----

HDPVVEEAAEHAIKTIQQRSNSIHPYKLQEIVHANAEMADDSTKLHLVIKTSRGGKEEKFKVQVQHNNE-GAFHLNRMEPDN------

HDPVVQEAAEHAIKTIQQRSNSLLPYELQEIVHANAEMIEDSTKLHMLIKTSRGGKEEKFKVQVHHSNE-GAFHLNHMEPDR------

HDPVVQDAAHHAIKTIQERSNSLFPYELSEVVHANAEVVDTSAKFDMLLKVKRGGKEEKYKVEVHKSTEEGGFNLKKVDLDHS-----

HDPHVQDAAQHALRTIQQRSNSLVPYELLEIIHAKAEVIEDAAKFDLLLKLKRGSKEEKFKVEVHKNNE-GNFLLNQMVQDHS-----

NDPLVRDAADHALKTIQQRSNSLFPYELLEVLHAKAEVIEETAKFDMLLKVKRGSKEEKFKAEVHKNLE-GNFLLNQMQQEN------

HDPVVKDAADHAVKSIQQRSNSLFPYELLEIVRAKAEVVEDFAKFDILMKLKRGNKEEKFKAEVHKNLE-GAFVLNQMQQEHDESSSQ

HDPVVQEAANHAVKTIQQRSNSLAPYELLEILLAKAEVIEDSAKFDLLLKLKRGSKEEKLKVEVHKNLE-GTFHLNKMQEHSDSGC--

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NDPEVQDAANHVVKSIQMRSNSIFRYELLEILLAKAKVIEGSAKFDLLLKLKWGSKDVKFKAEVNKNIE-GKFCSTQWKEDHL-----

DDPEVQHVADHAVKTIQQRSNSLFPYELQEVVHANAEVTGEAAKYNMVLKLKRGDKEEKFKVEVHKNHQ-GVLHLNHMEQHHD-----

QDPQVQDAANHAVKSLQQKSNSLFPYELQEVVHAKSEVMEEHAKFNMLLKLKRGDKEEKYKVEVHKNNE-GAYNLNQMEVEH------

HDPQVQDAANHAIKTIQQRSNSLVPYELHEVADAKAEVIDDFAKFNLLLKVKRGQKEEKFKVEVHKNNQ-GGFHLNQMEQDHS-----

HDPVVQDAANHAVKSIQQRSNTLFPYELLEILHAKAKVLEDLAKIHLLLKLKRGSREEKFKVEVHKNIE-GTFHLNQMEQDHSDSGN-

90

XP 0041655171

XP 0041655171

XP 0041655171

LLEFARVVKAKEQVVAGTMHHLTLEIVEAGKKKLYEAKVWVKPWLNFKELQEFKPAS------DGAPSITPSDLGCKKGEHESGWREVPG

Figure 4 Multiple alignments of the deduced amino acids of the full-length sequence of CypCl (indicated with black arrow) withother phytocystatin sequences from other plants obtained from the GenBank database in the NCBI website They were Amaranthushypochondiacus (ABG898561) Theobroma cacao (EOY195841) Ricinus communis (CAA896971) Ipomoea batatas (AAD138121) Solanumlycopersium (AAF231261) Petunia x hybrida (AAU815971) Sesamum indicum (AAK150901) Cucumis sativus (XP 0041655171)Oryza sativajaponica group (Os01g0270100) Elaeis guineensis (ACF065481) Oryza sativa (AAL308301) Sorghum bicolour (CAA606341) Zea mays(NP0011052951) Triticum aestivum x Secale cereal (ADP507601) Pelargonium x hortorum (ABG810971) Jatropha curcas (ADB028941)Ananas comosus (AAQ072591) Arabidopsis thaliana 1 (NP196751) Colocasia esculenta (AAM883971) Vitis cinerea var helleri x Vitis riparia(ADD511911) Brassica rapa (ABK786891) Fragaria x ananassa (CAH601631) and Glycine max (NP0012377341)

of view this sequence could possibly shed some light onthe evolutionary scheme of cystatins among the animals andplantsThis sequence is generally found in phytocystatins butseldom to be found in animal cystatin which supports thetheory of a commonancestor before the split between animals

and plants [10] While the SNSL sequence in C-terminalextension of the full-length CypCl is only found in somephytocystatins phytocystatins with C-terminal extensioncontaining this sequence are predicted to inhibit legumainactivity Martinez et al [15] had proved that the SNSL site


Query sequence1 25 50 75 100 125 150 175 200 228

Specific hits

Nonspecifichits

Superfamilies

Putative proteinase inhibition site Putative proteinase inhibition siteCY CY

CY CYCystatin

Atha cystat relCY superfamily CY superfamily

Atha cystat rel superfamily

Figure 5 Graphical summary of BLAST results of the deduced amino acids of CypCl on conserved domains It shows that several conservedcystatin superfamily domains are detected on the sequence suggesting a novel putative phytocystatin isolated from Curcuma longa

gi|52546928 Petunia x hybrida

gi|76160986 Solanum tuberosum

gi|7595872 Ipomoea batatas

gi|13183179 Sesamum indicum

gi|110748606 Amaranthus hypochondriacus

gi|1638842 Ricinus communis

gi|224073041 Populus trichocarpa

gi|225464047 Vitis vinifera

gi|508727687 Theobroma cacao

gi|27752377 Malus domestica

gi|70907497 Fragaria x ananassa

gi|351721816 Glycine max

gi|357515449 Medicago truncatula

gi|502158281 Cicer arietinum

gi|44945869 Cucumis sativus

gi|118197452 Brassica rapa

gi|18158608 Brassica oleracea

gi|79596870 Arabidopsis thaliana

gi|22001329 Colocasia esculenta

CypCl

gi|192910880 Elaeis guineensis

gi|195622972 Zea mays

gi|514773386 Setaria italica

gi|115435888 Oryza sativa japonica group

gi|109238653 Hordeum vulgare subsp vulgare

gi|194338899 Triticum aestivum

100

100

100

100

71

100

100

98

100

100

41

40

52

71

86

63

88

41

30

14

46 13

18

005

Figure 6 Phylogenetic tree generated with phytocystatins containing C-terminal extensions by using neighbour-joining method with 100bootstrapping CypCl is shown to be related to phytocystatin from Elaeis guineensis (African oil palm)


of the phytocystatins contained in the C-terminal extensionis essential when phytocystatin with and without C-terminalextension was tested for legumain-inhibition activity onlythose with C-terminal extension are able to inhibit legumainactivity Martinez et al [15] also showed the importance ofamino acid N (ASN asparagine) in SNSL sequence as whenit is replaced by a K (Lys lysine) residue the phytocystatinis unable to inhibit legumain With the presences of the con-served sequences coding for the tripartite wedge responsiblefor papain inhibition and the C-terminal extension which isresponsible for legumain inhibition the novelCypCl could beexpressed as a bifunctional protease inhibitor

4 Materials and Methods

41 Plant Treatment and Sampling Before sampling Cur-cuma longa plant (around 6 months old) was treated with500120583L of methyl jasmonate diluted in 4500 120583L of ethanol(in 1 9 ratios) and placed inside a sealed growth chamberfor 24 hours The mixture was pipetted to a cotton swab andkept at the bottom of the growth chamber without havingany direct contact with the plant [27ndash29] After 24 hoursthe leaves flower and rhizome samples were collected withsterilized blade washed with distilled water to remove dirtand grounded immediately in liquid nitrogen About 200mgof the ground samples was aliquoted in 2mLmicrocentrifugetubes labeled and proceeded with total RNA extraction

42 Total RNA Extraction Total RNA was extracted witha conventional method CTAB method [30] and two com-mercially available kits RNAzol RT (Molecular ResearchCenter Inc USA) and RNeasy Plant Mini kit (Qiagen USA)according to the manufacturerrsquos protocol respectively Thequality and integrity of the total RNA extractedwere analysedin agarose gel electrophoresis and were quantified withNanoDrop ND 1000 spectrophotometer (Thermo ScientificUSA)

43 Data Mining and Degenerate Primer Design Transcrip-tomic data of phytocystatins from different plants specieswere gathered from the GenBank of NCBI website The fullmRNA sequences in nucleotide and amino acids were alignedusing AlignX Vector NTI Advance 11 (Invitrogen USA)From the alignment conserved regions were detected anda pair of degenerate primers was designed with forwarddegenerate primer Cys F (51015840-CTCGCTCGHTTCGCCGTC-GAYGAG-31015840) and reverse degenerate primer Cys R (51015840-GTTCTTGTGHACYTCDACCTTGAA-31015840)

44 cDNA Construction and the First Round PolymeraseChain Reaction (PCR) cDNA was constructed with 1120583gof extracted total RNA using iScript Reverse TranscriptionSupermix for RT-qPCR (BioRad CA USA) according to themanufacturerrsquos protocol The first round polymerase chainreaction (PCR) was carried out in a 20120583L reaction usingGoTaqFlexi DNA polymerase kit (Promega USA) with thedesigned degenerate primers according to themanufacturerrsquosinstructions PCR was performed on a thermocycler with the

following parameters an initial heating of PCR activationat 94∘C for 3 minutes followed by 30 cycles of 94∘C for45 seconds 42∘C for 45 seconds and 72∘C at 45 secondsand a final extension at 72∘C for 7 minutes The 495 bpamplified PCR product was purified from gel usingQIAquickPCR Purification kit (Qiagen USA) and ligated into pGEM-T easy vector (Promega USA) Then it was transformedinto top 10 E coli chemically competent cells and culturedovernight on an ampicillin-containing Luria-Bertani (LB)agar spread with 40 120583g120583L X-gal and 40 120583L of 500mMIPTG at 37∘C Bluewhite colony screening was carried outand selected colonies were cultured overnight in ampicillin-containing LB broth Plasmids were extracted using Nucle-oSpin Plasmid (Macherey-Nagel Germany) according tothe manufacturerrsquos instructions and the plasmid was sentfor sequencing at 1st base (Malaysia) The identity of thePCR product was determined by subjecting it to the BasicLocal Alignment Search Tool (BLAST) [22] at NCBI website(httpblastncbinlmnihgov)

45 Molecular Cloning of CypCl Gene and Sequence AnalysisOnce the PCR product was proven to show similarity toother phytocystatins gene-specific 51015840 primer Cyp5 R (51015840-CTTGCTCTTTTGCCTTCACCACTC-31015840) and 31015840 primerCyp3 F (51015840-TACGAGCTGCTGGAGGTCCTCCATGC-31015840)were designed based on the sequence of the PCR productTo amplify 51015840 cDNA end and 31015840 cDNA end RACE specificcDNA was generated using SMARTer RACE kit (ClontechCA USA) according to the manufacturerrsquos protocol and5101584031015840 RACE was performed From the obtained informationon 51015840 and 31015840 cDNA end RACE the full-length cDNA ofCypCl covering the start codon until the stop codon wasamplified using forward primer CypF F (51015840-GCTATCGAA-GCGTGGCATCAT-31015840) and reverse primer CypF R (51015840-GAGGTCACCCAAAGTCGTTACACA-31015840) PCR conditionsinclude initial activation at 94∘C for 3 minutes followedby 35 cycles of 94∘C for 45 seconds 60∘C for 45 secondsand 72∘C at 45 seconds and a final extension at 72∘C for10 minutes The resulted PCR product was analysed underultraviolet light on a 10 agarose gel (Vivantis Malaysia)stained with ethidium bromide and a 100 bp DNA marker(Vivantis Malaysia) was used The band was purified ligatedin vector and transformed into Top10 E coli competent cellsas described in the previous stepThe sequencing results werethen subjected to BLAST and signal peptide analysis usingSignalP 30 program

46 Phylogenetic Tree Construction Phytocystatins fromother plants were gathered from NCBI website and alignedusing MEGA 52 software [31] The resulting alignmentwas trimmed and the alignment was subjected to phyloge-netic tree construction by MEGA 52 software [31] usingneighbour-joining method with 100 bootstrapping

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

The research was funded by eScience MOSTI Malaysia(Ministry of Science Technology and Innovation) andRUGSgrants The authors would like to thank Universiti PutraMalaysia (UPM) and Malaysia Agricultural Research andDevelopment Institute (MARDI) for providing facilities tocarry out the research They would also like to thank AssocProf Dr Parameswari UPM for her insightful commentsthroughout the research works

References

[1] M A Jongsma and C Bolter ldquoThe adaptation of insects to plantprotease inhibitorsrdquo Journal of Insect Physiology vol 43 no 10pp 885ndash895 1997

[2] P K Lawrence and K R Koundal ldquoPlant protease inhibitors incontrol of phytophagous insectsrdquo Electronic Journal of Biotech-nology vol 5 no 1 pp 93ndash109 2002

[3] S K Haq S M Atif and R H Khan ldquoProtein proteinaseinhibitor genes in combat against insects pests and pathogensnatural and engineered phytoprotectionrdquo Archives of Biochem-istry and Biophysics vol 431 no 1 pp 145ndash159 2004

[4] H Habib and K M Fazili ldquoPlant protease inhibitor a defensestrategy in plantsrdquo Biotechnology andMolecular Biology Reviewvol 2 pp 68ndash85 2007

[5] M C Heath ldquoNon-host resistance and nonspecific plantdefensesrdquoCurrentOpinion in Plant Biology vol 3 no 4 pp 315ndash319 2000

[6] Y Wang Y Zhan C Wu et al ldquoCloning of a cystatin gene fromsugar beet M14 that can enhance plant salt tolerancerdquo PlantScience vol 191-192 pp 93ndash99 2012

[7] M Martinez Z Abraham M Gambardella M Echaide PCarbonero and I Diaz ldquoThe strawberry gene Cyf1 encodesa phytocystatin with antifungal propertiesrdquo Journal of Experi-mental Botany vol 56 no 417 pp 1821ndash1829 2005

[8] M Benchabane U Schluter J Vorster M Goulet and DMichaud ldquoPlant cystatinsrdquo Biochimie vol 92 no 11 pp 1657ndash1666 2010

[9] P Bangrak andWChotigeat ldquoMolecular cloning and biochemi-cal characterization of a novel cystatin fromHevea rubber latexrdquoPlant Physiology and Biochemistry vol 49 no 3 pp 244ndash2502011

[10] MM Popovic A Bulajic D Ristic B Krstic R M Jankov andM Gavrovic-Jankulovic ldquoIn vitro and in vivo antifungal prop-erties of cysteine proteinase inhibitor from green kiwifruitrdquoJournal of the Science of Food and Agriculture vol 92 no 15pp 3072ndash3078 2012

[11] M Pernas E Lopez-Solanilla R Sanchez-Monge G Salcedoand P Rodrıguez-Palenzuela ldquoAntifungal activity of a plantcystatinrdquoMolecular Plant-Microbe Interactions vol 12 no 7 pp624ndash627 1999

[12] K R B Pereira S Botelho-Junior D P Domingues et alldquoPassion fruit flowers kunitz trypsin inhibitors and cystatindifferentially accumulate in developing buds and floral tissuesrdquoPhytochemistry vol 72 no 16 pp 1955ndash1961 2011

[13] R Margis E M Reis and V Villeret ldquoStructural and phyloge-netic relationships among plant and animal cystatinsrdquo Archivesof Biochemistry and Biophysics vol 359 no 1 pp 24ndash30 1998

[14] W Megdiche C Passaquet W Zourrig Y Zuily Fodil andC Abdelly ldquoMolecular cloning and characterization of novel

cystatin gene in leaves Cakile maritima halophyterdquo Journal ofPlant Physiology vol 166 no 7 pp 739ndash749 2009

[15] M Martinez M Diaz-Mendoza L Carrillo and I DiazldquoCarboxy terminal extended phytocystatins are bifunctionalinhibitors of papain and legumain cysteine proteinasesrdquo TheFEBS Letters vol 581 no 16 pp 2914ndash2918 2007

[16] A Apisariyakul N Vanittanakom and D Buddhasukh ldquoAnti-fungal activity of turmeric oil extracted from Curcuma longa(Zingiberaceae)rdquo Journal of Ethnopharmacology vol 49 no 3pp 163ndash169 1995

[17] R C Lantz G J Chen A M Solyom S D Jolad and B NTimmermann ldquoThe effect of turmeric extracts on inflammatorymediator productionrdquo Phytomedicine vol 12 no 6-7 pp 445ndash452 2005

[18] R Selvam L Subramanian R Gayathri and N AngayarkannildquoThe anti-oxidant activity of turmeric (Curcuma longa)rdquo Jour-nal of Ethnopharmacology vol 47 no 2 pp 59ndash67 1995

[19] C A C Araujo and L L Leon ldquoBiological activities ofCurcumalonga LrdquoMemorias do Instituto Oswaldo Cruz vol 96 no 5 pp723ndash728 2001

[20] G K Jayaprakasha L J M Rao and K K Sakariah ldquoChemistryand biological activities of Clongardquo Trends in Food Science andTechnology vol 16 no 12 pp 533ndash548 2005

[21] K Sookkongwaree M Geitmann S Roengsumran A Pet-som and U H Danielson ldquoInhibition of viral proteases byZingiberaceae extracts and flavones isolated from Kaempferiaparviflorardquo Pharmazie vol 61 no 8 pp 717ndash721 2006

[22] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[23] P Chomcyznski W Wilfinger A Kennedy M Rymaszewskiand K Mackey ldquoRNAzol RT a new single-step method forisolation of RNArdquo Nature Methods Application Notes vol 72010

[24] M E John ldquoAn efficient method for isolation of RNA and DNAfrom plants containing polyphenolicsrdquo Nucleic Acids Researchvol 20 no 9 article 2381 1992

[25] R Ghangal S Raghuvanshi and P C Sharma ldquoIsolation ofgood quality RNA from a medicinal plant seabuckthorn richin secondary metabolitesrdquo Plant Physiology and Biochemistryvol 47 no 11-12 pp 1113ndash1115 2009

[26] W Bode R Engh D Musil et al ldquoThe 20 A X-ray crystalstructure of chicken egg white cystatin and its possible modeof interaction with cysteine proteinasesrdquo EMBO Journal vol 7no 8 pp 2593ndash2599 1988

[27] E E Farmer R R Johnson and C A Ryan ldquoRegulation ofexpression of proteinase inhibitor genes by methyl jasmonateand jasmonic acidrdquoPlant Physiology vol 98 no 3 pp 995ndash10021992

[28] D S Moura and C A Ryan ldquoWound-inducible proteinaseinhibitors in pepper Differential regulation upon woundingsystemin and methyl jasmonaterdquo Plant Physiology vol 126 no1 pp 289ndash298 2001

[29] P R Lomate and V K Hivrale ldquoWound and methyl jasmonateinduced pigeon pea defensive proteinase inhibitor has potencyto inhibit insect digestive proteinasesrdquo Plant Physiology andBiochemistry vol 57 pp 193ndash199 2012

[30] C J Tsai L J Cseke and S A Harding ldquoIsolation andpurification of RNArdquo in Handbook of Molecular and Cellular


Methods in Biology and Medicine p 31 CRC Press 2nd edition2004

[31] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Efficiency of different methods of total RNA extraction from turmeric (Curcuma sp) plant

Method 119860260280

ratio Yield concentration (ng120583L) Time durationModified CTAB Method 200ndash220 sim300 SlowRNAzol RT 180ndash200 800ndash1000 FastRNeasy Plant Mini kit 170ndash200 300ndash500 Fast

1 2 3 4 5 6

Figure 1 Total RNA integrity test on 10 agarose gel Intact 28S and18S total RNA bands can be observed on the agarose gel indicatinga good integrity of the total RNA after being extracted by differentmethods (lanes 1 and 2 modified CTAB method lane 3 4 RNAzolRT and lane 5 6 RNeasy Plant Mini kit)

in response to biotic and abiotic stresses as they are highlyexpressed during harsh condition such as cold droughtsalt stress oxidant stress and wounds [8 9 12] All theseproperties encourage more research to be done on PIsespecially on phytocystatin Ayurvedic [2ndash4]

Generally phytocystatins contain several conservedmotifs such as QxVxG (the active site) a PW dipeptidein the region towards the C-terminal and one or tworesidues of G towards the N-terminal Another notablemotif is the conserved [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-X-[EDQV]-[HYFQ]-N sequence which is found in theupstream of the active site The sequence formed an 120572-helixstructure but lacks disulphide bonds and glycosylation sites[6ndash9 13 14] Phytocystatins identified so far have differentsizes the majority are small proteins that range from 12 kDato 16 kDa and contain no disulphide bonds [6 7 10]Several phytocystatins have slightly higher molecularmasses around 23 kDa due to the C-terminal extensioncontaining the motif SNSL and they are reported to inhibitlegumain [15] In potato and tomato multicystatins havebeen identified with multiple cystatin domains and have amolecular mass of 85 kDa [6 7 10]

Turmeric (Curcuma sp) a medicinal plant from Zingib-eraceae family is one of such novel sources and up till nowthere are no PIs that are being characterized and reportedTurmeric is commonly used as traditional medicines andspices for culinary arts in country like India Malaysia andother Asian countries Turmeric as stated in Ayurvedicmedicine has the characteristic of antifungal and anti-inflammatory properties [16 17] Recent studies have alsoproven that turmeric does exhibit antitumour antimicro-bial anti-HIV nematocidal and antioxidant properties [18ndash20] Sookkongwaree et al [21] had extracted secondary

compounds from Zingiberaceae family and it was proven toexhibit antiviral and antiprotease properties It is suggestedthat PIs could have possibly contributed to the antiviral andantiprotease properties of the secondary compounds

In this study we have identified and characterized a novelfull-length sequence of cysteine protease inhibitor gene fromCurcuma longa by using RACE-method (rapid amplificationof cDNA ends) The phylogenetic relationship of the genewith other plant species and predictions of the putativefunctions of the identified gene was also conducted

2 Results

21 Comparison of Total RNA Extraction with DifferentMethods Total RNA of turmeric (Curcuma longa) was suc-cessfully extracted from mature leaves of turmeric plant bythree different methods (i) modified CTAB (cetyltrimethylammonium bromide) method (ii) RNAzol RT (MolecularResearch Center Inc USA) and (iii) RNeasy Plant Mini kit(Qiagen USA) Intact bands of the 28S and 18S RNA can beclearly observed in the agarose gel for all the three methods(Figure 1) As for the quantitative test of the total RNA withthe raw tissue weight of 200mg our results showed thattotal RNA extraction by RNAzol RT produced the highestyield with concentration of sim800 ng120583L while extraction byRNeasy PlantMini kit and by amodifiedCTABmethod yieldssim300 ng120583L of total RNA Both RNAzol RT and RNeasy PlantMini kit producedA

260280ratio at the range of 170ndash180while

a modified CTAB method produced the ratio at the range of200ndash220 Table 1 summarised the comparison of the threeextraction methods

Therefore RNAzol RT was chosen as the preferredmethod to extract the total RNA from different parts ofturmeric plant (leaf flower and rhizome) because of thehighest extraction yield and faster time duration as comparedto the other methods However based on the agarose gelelectrophoresis result (Figure 2) it shows that RNAzol RT isonly able to extract total RNA from leaves and is not suitablefor flowers and rhizomes

22 Cloning and Sequence Analysis of Curcuma longa Phyto-cystatin cDNA Degenerate polymerase chain reaction (PCR)was conducted to isolate the novel target gene phytocystatinA 495 bp cDNA fragment was successfully amplified fromtotal cDNA reverse transcribed from the total RNA ofleaves sample of Curcuma longa 5101584031015840 RACE (rapid ampli-fication of cDNA ends) was conducted and the full-lengthcDNA designated CypCl was then deposited to the NCBI(National Center for Biotechnology Information) databasewith accession number KF5459541 The cDNA (Figure 3(a))


1 2 3







3 Discussion




260280ratio






800 bp

M 1

600bp

(a)
























L N Q M Q Q E N -

(b)







1 ABG898561 (1)

(1) (1)

(1) (1)

(1) (1)

(1) (1) (1) (1) (1) (1) (1)

(1)

(1)

(1)

(1)

(1) (1) (1) (1) (1) (1)

(78) (72) (39) (90) (72) (88) (34) (38) (65) (81) (37) (41) (69) (74) (85) (37) (37) (77) (43) (37) (70) (37) (66) (81)


CypCl Os01g0270100

ACF065481 AAL308301 CAA606341


AAM883971 ADD511911 ABK786891 CAH601631

NP0012377341 91 180


CypClOs01g0270100

ACF065481AAL308301CAA606341


AAM883971ADD511911ABK786891CAH601631

NP0012377341181 268

(161) (158) (129) (173) (155) (171) (117) (121) (148) (164)(120)(103)(131)(136)(148)

(99)(107)(136)(102)(120)(153)(121)(155)(164)


CypCl

AAK150901

Os01g0270100

AAU815971

ACF065481AAL308301CAA606341


AAM883971ADD511911ABK786891CAH601631

NP0012377341



























































----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------






90

XP 0041655171

XP 0041655171

XP 0041655171






Query sequence1 25 50 75 100 125 150 175 200 228

Specific hits

Nonspecifichits

Superfamilies


CY CYCystatin























CypCl







100

100

100

100

71

100

100

98

100

100

41

40

52

71

86

63

88

41

30

14

46 13

18

005















Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3







3 Discussion




260280ratio






800 bp

M 1

600bp

(a)
























L N Q M Q Q E N -

(b)







1 ABG898561 (1)

(1) (1)

(1) (1)

(1) (1)

(1) (1) (1) (1) (1) (1) (1)

(1)

(1)

(1)

(1)

(1) (1) (1) (1) (1) (1)

(78) (72) (39) (90) (72) (88) (34) (38) (65) (81) (37) (41) (69) (74) (85) (37) (37) (77) (43) (37) (70) (37) (66) (81)


CypCl Os01g0270100

ACF065481 AAL308301 CAA606341


AAM883971 ADD511911 ABK786891 CAH601631

NP0012377341 91 180


CypClOs01g0270100

ACF065481AAL308301CAA606341


AAM883971ADD511911ABK786891CAH601631

NP0012377341181 268

(161) (158) (129) (173) (155) (171) (117) (121) (148) (164)(120)(103)(131)(136)(148)

(99)(107)(136)(102)(120)(153)(121)(155)(164)


CypCl

AAK150901

Os01g0270100

AAU815971

ACF065481AAL308301CAA606341


AAM883971ADD511911ABK786891CAH601631

NP0012377341



























































----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------






90

XP 0041655171

XP 0041655171

XP 0041655171






Query sequence1 25 50 75 100 125 150 175 200 228

Specific hits

Nonspecifichits

Superfamilies


CY CYCystatin























CypCl







100

100

100

100

71

100

100

98

100

100

41

40

52

71

86

63

88

41

30

14

46 13

18

005















Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


800 bp

M 1

600bp

(a)
























L N Q M Q Q E N -

(b)







1 ABG898561 (1)

(1) (1)

(1) (1)

(1) (1)

(1) (1) (1) (1) (1) (1) (1)

(1)

(1)

(1)

(1)

(1) (1) (1) (1) (1) (1)

(78) (72) (39) (90) (72) (88) (34) (38) (65) (81) (37) (41) (69) (74) (85) (37) (37) (77) (43) (37) (70) (37) (66) (81)


CypCl Os01g0270100

ACF065481 AAL308301 CAA606341


AAM883971 ADD511911 ABK786891 CAH601631

NP0012377341 91 180


CypClOs01g0270100

ACF065481AAL308301CAA606341


AAM883971ADD511911ABK786891CAH601631

NP0012377341181 268

(161) (158) (129) (173) (155) (171) (117) (121) (148) (164)(120)(103)(131)(136)(148)

(99)(107)(136)(102)(120)(153)(121)(155)(164)


CypCl

AAK150901

Os01g0270100

AAU815971

ACF065481AAL308301CAA606341


AAM883971ADD511911ABK786891CAH601631

NP0012377341



























































----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------






90

XP 0041655171

XP 0041655171

XP 0041655171






Query sequence1 25 50 75 100 125 150 175 200 228

Specific hits

Nonspecifichits

Superfamilies


CY CYCystatin























CypCl







100

100

100

100

71

100

100

98

100

100

41

40

52

71

86

63

88

41

30

14

46 13

18

005















Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 ABG898561 (1)

(1) (1)

(1) (1)

(1) (1)

(1) (1) (1) (1) (1) (1) (1)

(1)

(1)

(1)

(1)

(1) (1) (1) (1) (1) (1)

(78) (72) (39) (90) (72) (88) (34) (38) (65) (81) (37) (41) (69) (74) (85) (37) (37) (77) (43) (37) (70) (37) (66) (81)


CypCl Os01g0270100

ACF065481 AAL308301 CAA606341


AAM883971 ADD511911 ABK786891 CAH601631

NP0012377341 91 180


CypClOs01g0270100

ACF065481AAL308301CAA606341


AAM883971ADD511911ABK786891CAH601631

NP0012377341181 268

(161) (158) (129) (173) (155) (171) (117) (121) (148) (164)(120)(103)(131)(136)(148)

(99)(107)(136)(102)(120)(153)(121)(155)(164)


CypCl

AAK150901

Os01g0270100

AAU815971

ACF065481AAL308301CAA606341


AAM883971ADD511911ABK786891CAH601631

NP0012377341



























































----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------






90

XP 0041655171

XP 0041655171

XP 0041655171






Query sequence1 25 50 75 100 125 150 175 200 228

Specific hits

Nonspecifichits

Superfamilies


CY CYCystatin























CypCl







100

100

100

100

71

100

100

98

100

100

41

40

52

71

86

63

88

41

30

14

46 13

18

005















Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Query sequence1 25 50 75 100 125 150 175 200 228

Specific hits

Nonspecifichits

Superfamilies


CY CYCystatin























CypCl







100

100

100

100

71

100

100

98

100

100

41

40

52

71

86

63

88

41

30

14

46 13

18

005















Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Molecular Cloning and Characterization of Novel …downloads.hindawi.com/journals/bmri/2014/973790.pdf · 2019. 7. 31. · Research Article Molecular Cloning and