CHAPTER IV Binary vector construction with bkt H. pluvialisshodhganga.inflibnet.ac.in/bitstream/10603/39819/9... · 2018. 7. 2. · H. pluvialis was shown to possess three different

CHAPTER IV

Binary vector construction with bkt(β-carotene ketolase) from

H. pluvialis and Rubisco promoter (RBCS2) from D. bardawil

Binary vector construction with bkt and rubisco promoter |CHAPTER IV

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Summary

This chapter deals with the construction of a binary vector harbouring bkt from

H. pluvialis which is driven by Rubisco smaller subunit promoter (RBCS2) with its

transit peptide. Genomic DNA clone of bkt (1.8 kb size- NCBI accession No.

GQ214765) from H. pluvialis was used in vector construction. The sequence analysis

of the cloned bkt revealed that there was some nucleotide polymorphism when aligned

and compared with the known bkt (D45881). The presence of six exons and five

introns in the cloned bkt gene was observed. Driving the gene with a chloroplast

specific promoter may increase the expression of BKT in Dunaliella where β-carotene

accumulation specifically occurs in inter-thylakoid membrane of chloroplast. Rubisco

smaller subunit promoter (RBCS2) is reported as a highly efficient promoter for

expression of chloroplast proteins. As bkt does not contain a signal peptide sequence

for targeting the enzyme to chloroplast, the construct required fusion of bkt with

transit peptide for chloroplast targeting. The RBCS2 promoter region along with the

transit peptide was amplified from D. bardawil using adaptor ligated genomic

walking method. The 1.4 kb amplicon was confirmed by sequencing and was shown

to have 98% similarity with other Rubisco sequences. Promoter analysis through

SIGNAL SCAN revealed the presence of several cis elements like TATA box,

CCAAT motif, I box and G box across the promoter and was shown to possess

several light responsive elements. The presence of chloroplast targeted signal peptide

was also analysed. The analysis revealed the presence of a cleavage site between 62 &

63 bp. Two promoter constructs viz, pTZ-RBB (1.2 kb promoter region) and pTZ-

RBS (400 bp promoter region) were initially constructed. Binary vector construct

p1304-CaMV-BKT was digested initially with XhoI and XbaI to obtain bkt fragment

which was later ligated to pTZ-RBB and pTZ-RBS. The construct so obtained (pTZ-

RBB-BKT and pTZ-RBS-BKT) were digested with XbaI and KpnI enzymes to

release the BKT-RBB/RBS fragment which were finally ligated to p1304-CaMV-

BKT by replacing its CaMV-BKT region and the final constructs obtained were

named as p1304-RBB-BKT and p1304-RBS-BKT. All the constructs were confirmed

by restriction digestion.


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4.1 Introduction

The tools of metabolic and enzyme engineering have been well developed in

recent times and are being used for strain improvement for the sustainable increase in

value added products from the available biological sources, both in terms of quality

and quantity. Metabolic engineering, using a variety of carotenoid biosynthesis genes

is one of the most powerful methods used to produce keto-carotenoids in heterologous

systems because; organisms which are capable of synthesizing such keto-carotenoids

are not common in nature. A considerable number of microbial and plant carotenoid

genes can be used in metabolic engineering for the construction of novel pathways,

re-engineering of pre-existing ones, and tailoring of enzymatic activity to a specific

need. Metabolic engineering thus leads to the development of productive, economical,

safe, and environmentally sound manufacturing processes for the production of keto-

carotenoids for industrial and nutritional purposes. Different approaches have been

followed to modify the carotenoid content in plants to enhance the nutritional value:

1. Modification of the carotenoid pathway by introduction of new gene(s) into

other species for production of new compound or conversion of existing

compound into other valuable compounds.

2. Increasing the amounts of pre-existing carotenoids.

3. Engineering a carotenogenic pathway in tissue that is completely devoid of

carotenoids.

There is growing interest worldwide in manipulating carotenoid biosynthesis in

carotenoid producing organisms. Exhibiting high activity of carotenoid hydroxylase

but lacking β-carotene ketolase (bkt/crtW) activity, plants are capable of forming 3-

hydroxy carotenoids (e.g. lutein and zeaxanthin) but are unable to synthesize 4-

ketocarotenoids (e.g. canthaxanthin and astaxanthin). Cloning of most of the

astaxanthin biosynthesis genes in H. pluvialis has now opened the door to genetically

manipulating this pathway not only in algae, but also in other organisms.

Haematococcus pluvialis, the green micro alga produces a high value keto-

carotenoid, astaxanthin which has been shown to have higher antioxidant activity than

β-carotene and α-tocopherol (Kobayashi and Sakamoto, 1999) along with several

other beneficial effects. The genes responsible for the synthesis of astaxanthin from

the β-carotene in Haematococcus pluvialis have been well studied. Cloning of genes

like β-carotene ketolase (bkt), β-carotene hydroxylase (chy), phytoene synthase (psy),


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phytoene desaturase (pds), lycopene cyclase (lcy) etc have been well characterized

and also expressed in vitro in H. pluvialis. In vitro studies of BKT and CHY were

attempted by several workers (Lotan and Hirschberg, 1995; Kajiwara et al, 1995; Tao

et al, 2006; Vidhyavathi et al, 2008).

Two enzymatic activities convert β-carotene into astaxanthin through several

alternative biosynthetic intermediates: a ketolase which incorporates two keto groups

at C-4 and C-4' in the molecule of β-carotene, and a hydroxylase which introduces

two hydroxyl groups at C3 and C-3'. β-carotene ketolase genes have been isolated

from astaxanthin-producing non-photosynthetic bacteria (Proteobacteriai) such as

Paracoccus sp. and Brevundimonas sp. (Lee and Kim, 2006; Misawa et al, 1995),

Brevundimonas sp. (Choi et al, 2005), soil bacteria Bradyrhizobium sp. (Hannibal

et al, 2000) and cyanobacteria. Their homologous genes (encoded by BKT) have

also been isolated from H. pluvialis (Kajiwara et al, 1995; Lotan and Hirschberg,

1995) and also from other green algae C. reinhardtii and Chlorella zofingiensis

(Huang et al, 2006). H. pluvialis was shown to possess three different β-carotene

ketolase genes, which were named bkt1, bkt2 and bkt3 (Huang et al, 2006). These β-

carotene ketolases are varied on their catalysis functions. β-carotene ketolase (BKT)

from H. pluvialis has notable substrate preference for β-carotene and echinenone.

Screening of several green algal bkt cDNAs for their catalytic function with

respect to astaxanthin synthesis was carried out by Yu-Juan Zhong et al (2011). The

bkt cDNA from C. reinhardtii (Lohr et al, 2005), a green alga with no astaxanthin

accumulation in vegetative cells, was found to encode a bi-functional ketolase that

can convert β-carotene to canthaxanthin and zeaxanthin to astaxanthin efficiently. In a

study by Yu-Juan Zhong et al (2011), bkt from C. reinhardtii and the corresponding

cDNAs from H. pluvialis and Chlorella zofingiensis, which encode ketolases with

different efficiencies, were used to add keto groups to zeaxanthin in A. thaliana to

reveal the limiting step for astaxanthin biosynthesis in plants. They suggested from

their results that the selection of a ketolase with increased substrate specificity for 3-

hydroxy β-carotene derivatives leads to higher expression of ketolase over

hydroxylase, thereby increasing the production of astaxanthin in transgenic plants.

CrtW and BKT2 enzymes accept zeaxanthin as well as β-carotene as the substrates,

i.e., they introduce a keto group not only into the β-ionone rings but also into the

3(3’)-hydroxylated β-ionone rings at the 4 (4’) positions (Choi et al, 2007). Besides


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CrtW and BKT, another group of β-carotene ketolases is represented by CrtO of

Synechosystis sp. (Gonzalez et al, 1997). These enzymes catalyze the direct

conversion of a methylene group (in the 4 and 4' positions of the β-ionone ring of β-

carotene) producing echinenone and canthaxanthin (Breitenbach, 1996). CrtO is

structurally different from CrtW but similar in sequence to bacterial phytoene

desaturase enzymes. It is unusual in that its catalysis product formed from the

symmetrical substrate β-carotene is the monoketo echinenone rather than the

diketo canthaxanthin (Tao and Cheng, 2004).

The activity of the endogenous plant carotene hydroxylase(s) to act either on

β-carotene or heterologous ketolated carotenoids in order to form astaxanthin and its

intermediates has been reported by several workers. The Haematococcus β-carotene

ketolase has been efficiently expressed in tobacco resulting in keto-carotenoid

production in the nectary tissue (Mann et al, 2000). The gene has also been expressed

in a seed specific manner in Arabidopsis thaliana (Stalberg et al, 2003). Both these

approaches depend on the activity of the endogenous plant carotene hydroxylase(s) to

act either on β-carotene or heterologous ketolated carotenoids in order to form

astaxanthin and its intermediates. β-carotene ketolase can function in conjunction with

the intrinsic β-carotene hydroxylase, which adds hydroxyls at C3 and C3', to produce

astaxanthin (Mann et al, 2000). Thus addition of a single enzyme, β-carotene ketolase,

is sufficient to obtain high levels of keto-carotenoids in a plant species that normally

does not produce these compounds.

An important aspect in designing transgenic plants is how to obtain significant

levels of trans-gene expression in desired plant tissues at desired plant development

phases. The role of promoters is essentially important in this aspect. To ensure proper

transcription of the trans-gene, a promoter region that is adequately recognized by the

RNA polymerase of the host must precede the trans-gene. Lack of highly active

promoters has been a major hurdle in the development of microalgal transformation

systems (Lumbreras et al, 1998). Similarity between codon usage of the transcript and

that of the host organism is another important aspect that should be considered. One

of the most widely used universal constitutive promoter in plant molecular biology is

the cauliflower mosaic virus 35S (CaMV 35S) promoter (Odell et al, 1985). Although

CaMV 35S promoter drives strong and constitutive expression in most dicotyledonous

plants (Benfey et al, 1990), it has not been shown to be useful promoter in most algal


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species. CaMV 35S promoter has been used to drive transient expression in Ulva

lactuca (Huang et al, 1996). Lumbreras et al (1998) reported that expression of

foreign genes under the control of this promoter in C. reinhardtii have been

unsuccessful.

Chloroplasts are plant-specific organelles that are considered to be valuable

sites of protein accumulation due to their abundance in the plant cell and large size

(Staub et al, 2000). Majority of chloroplast proteins are encoded in the nucleus and

synthesized in the cytosol as precursors with N-terminal extensions called transit

peptides which are later cleaved off by stromal processing peptidase in stroma (Gavel

and von Heijine, 1990). The N-terminal transit peptide generally possesses necessary

and sufficient information for the correct targeting of proteins to chloroplasts (Lee et

al, 2002). In Dunaliella the production and accumulation of β-carotene is localized in

inter-thylakoid membranes of the chloroplast. Inorder to express BKT in Dunaliella,

proper targeting of bkt with the help of a highly active homologous promoter to

chloroplast is thus necessary.

Algal transformation has been most successful using promoters derived from

highly expressed algal genes. A widely used promoter for C. reinhardtii

transformation is derived from the 5’ untranslated region of the C. reinhardtii ribulose

biphospahte carboxylase/oxygenase small subunit (RBCS2) (Stevens et al, 1996). The

D. tertiolecta RBCS2 promoter and 3’ untranslated regions were shown to drive

expression of the bleomycin resistance gene (ble) in C. reinhardtii (Walker et al,

2005a). However, transformation efficiency was low compared to a homologous gene

promoter, and the phenotypes of the transgenic clones were unstable. High levels of

expression have made the promoter of the RBCS2 gene a widely used choice to drive

expression of heterologous proteins in transformed C. reinhardtii (Stevens et al,

1996).

In C. reinhardtii the expression of bkt1 was driven by the Chlamydomonas

constitutive promoter of the Rubisco small subunit (RBCS2) and the resulting protein

was directed to the chloroplast by the transit peptide sequences of Rubisco small

subunit (RBCS2) (Rosa et al, 2007). In carrot, the pea Rubisco small subunit transit

peptide along with double CaMV 35S or Arabidopsis-ubiquitin, or RolD promoters

were used to target the BKT enzyme to plastids in leaf/roots for the production of

keto-carotenoids (Jayaraj et al, 2008). The crtW gene with transit peptide sequence of


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the pea Rubisco small subunit under the regulation of the CaMV 35S promoter was

introduced to Lotus japonicas for astaxanthin production in flower petals (Suzuki et

al, 2007). In tobacco the cDNA of the bkt was transferred under the regulation of the

tomato pds (phytoene desaturase) promoter. The transit peptide of pds from tomato

was used to target the CRTO polypeptide to the plastids (Mann et al, 2000). All these

studies prove the high efficiency of Rubisco peptide sequence to target the enzyme to

chloroplast.

Understanding the conversion of β-carotene into astaxanthin thus paved the

way for possibility of introducing the last steps of xanthophylls/keto-carotenoid

pathway, in alternative microbial systems like Dunaliella which is rich in β-carotene

production but lacks the enzymes for its conversion into xanthophylls/keto-

carotenoids. Essentially the metabolic engineering discussed in the current study is an

attempt to extend the existing carotenoid pathway in Dunaliella through a single gene

(bkt) for production of keto-carotenoids including astaxanthin using β-carotene as the

substrate. In the present chapter the construction of a suitable binary vector

harbouring cloned bkt from H. pluvialis under the control of RBCS2 promoter with a

chloroplast transit peptide for proper expression of BKT in Dunaliella is described.

4.2 Materials and Methods

4.2.1 D. bardawil culture and maintenance

D. bardawil culture, maintenance of the culture and growth conditions were followed

as in the section 2.3.1, 2.3.1 and 2.3.2 of chapter II. Glass wares and plastic wares

used in the experiments are same as described in section 2.2.2, 2.2.3 and 2.2.4 of

chapter II.

4.2.2 Bacterial strain, plasmid isolation and restriction digestion

E. coli strain DH5α was used for transformation and plasmid maintenance. The clones

of pTZ-R/T and pRT100 was maintained in LB medium containing 100 mgL-1

ampicillin and clones of pCAMBIA 1304 was maintained in LB medium containing

50 mgL-1 kanamycin. All the restriction enzymes were obtained from MBI Fermentas,

Germany. Restriction digestion, ligation, transformation and plasmid isolation

experiments were carried out following the procedure of Sambrook et al (1989) and

are provided in the appendix.


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4.2.3 Amplification of β-carotene ketoalse (bkt) from pCAMBIA1304-CaMV-

BKT

The binary vector constructs pCAMBIA 1304 harbouring bkt driven by CaMV

promoter (p1304-CaMV-BKT) and cloning vector construct pRT-BKT with CaMV-

bkt-polyA cassette were obtained (Kathiresan, 2009) and used in the experiments

(Figure 4.1). Genomic DNA clone of bkt (1.8 kb size- NCBI accession No.

GQ214765) from H. pluvialis was used in the above vector construction. In the

construct bkt was introduced in the MCS region at HindIII site, with CaMV-bkt-Poly

A cassette derived from pRT100 clone carrying bkt.

Figure 4.1: T-DNA region of binary vector construct pCAMBIA1304-CaMV-BKT

The vector construct was checked initially using full gene amplification of bkt

and by restriction digestion. PCR amplification for bkt was carried out using p1304-

CaMV-BKT as the template and BKTF (5’-3’) – TGACTCGAGTGGGCGACAC

AGTATCACAT and BKTR (5’-3’) – ACTCTAGAACCAGGTCATGCCAAG as the

forward and reverse primers in a Biorad thermocycler (Biorad, Germany). The

program consisted of an initial denaturation of four minutes at 940C and step of 35

cycles consisting the denaturation of one minute at 940C, annealing of one minute at

600C and final extension of one minute at 720C followed by a final extension of 10

minutes at 720C. Amplified products were resolved based on their molecular weight

by running the products on a 1.0% agarose gel and was documented using a Herolab

documentation unit (Herolab 442K, E.A.S.Y., and Germany) (Figure 4.2 A). Binary

vector p1304-CaMV-BKT was digested with HindIII enzyme to check the presence of

bkt. p1304-CaMV-BKT is having an internal site for HindIII so as release fragments

of 1.5 kb and 1.0 kb. Control p1304 was also digested with the same enzyme (Figure

4.2 B).


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Figure 4.2: (A) Amplification of bkt from p1304-CaMV-BKT using BKTF and

BKTR primers (B) Restriction digestion of p1304-CaMV-BKT using

HindIII

4.2.4. Amplification of Rubisco smaller subunit (RBCS2) promoter along with

transit peptide from D. bardawil

RBCS2 promoter was amplified from genomic DNA of D. bardawil using modified

PCR based adaptor ligated genome walking method which is described section 4.3.14

of chapter I. Primer sequences were designed based on D. teritiolecta promoter

sequence with NCBI accession no. AY530156. Genomic DNA of D. bardawil was

carried out using Genelute Plant Genomic DNA isolation kit (Sigma–Aldrich, USA).

This was digested using previously mentioned blunt cutting restriction enzymes

(Refer section 2.3.14 of Chapter I1), followed by adaptor ligation to create the library.

Primer sequences ASP1 and ASP2 are same as that used in section 2.3.14. Initial

primers TR1 and TR2 lies in the signal peptide region of RBCS2 gene and TR3 in the

promoter region. The PCR program included initial denaturation of five minutes at

940C and step of 35 cycles consisting the denaturation of one minute at 940C,

annealing of one minute at 580C and final extension of one minute at 720C followed

by a final extension of 10 minutes at 720C.The primary PCR product was separated on

1% agarose gel stained with ethidium bromide. The PCR product (700 bp) after

tertiary PCR was purified, ligated to T-Tail cloning vector pTZ R/T and sequenced.

The primer TR4 was designed based on the obtained sequence and the procedure was

followed for getting a 750 bp fragment after the tertiary PCR which was purified and

sequenced. The final primer TF1 was designed based on the second sequence result

for the full amplification of the promoter along with transit peptide portion with a

total size of 1.4 kb. The primers used for amplification is listed in table 4.1.

AB


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Table 4.1 Primer sequences for RBCS2 promoter amplification.

Primer Sequence(5’-3’)

TR4 GGTCAAGGAGAAGAGAGGTCTGAGG

TR3 GTTGGGGCAATGACAACAGC

TR2 CGTATAAAGTAAAGTGGTAA

TR1 CTCGAG CACCATCATCTGGTTGGCGCG

TF1 GTCAAC GGTACC GAGCTGGGACTTGACCACCAAG

TF2 GTCAAC GGTACC TTACTTTGCATGATGCAGCA

The promoter was amplified in full length using primers TF1 and TR1 and

was sequenced. The primers TF1 and TF2 were tagged with HincII and KpnI and TR1

with XhoI sites. The amplification using TF1 and TR1 was used for complete

promoter construct (1.4 kb) and TF2 and TR1 were used for smaller deletion construct

(600 bp).

Amplified fragment of promoter using TF1 and TR1 were cloned in a pTZ

R/T T-tailed vector (MBI Fermentas, Germany). The cloned construct pTZ-RB was

sequenced. The sequence analyses for the promoter were performed using Signal P

(http://www.cbs.dtu. dk/services/SignalP/) for the presence of signal peptide.

Nucleotide BLAST searches of sequence was carried out using NCBI-BLAST and the

sequence was aligned using Dialign software (http://bibiserv.techfak.uni-

bielefeld.de/cgi-bin/dialign). The sequence obtained was analyzed against the PLACE

database that identifies transcription factor binding sites or cis-acting sequences in

plant promoters. PLACE (http://www.dna.affrc.go.jp/htdocs/PLACE/) is a database of

319 cis-acting regulatory DNA elements that were collected from previously

published reports (http://www.dna.affrc.go.jp/PLACE/signalscan.html) (Higo et al,

1999; Prestrige, 1991).

4.2.5 Construction of binary vector with bkt and Rubisco (RBCS2) promoter

Rubisco promoter regions were used to substitute CaMV in the vector construct

p1304-CaMV-BKT. The vector construction consisted of three step procedure.

Rubisco promoter regions were amplified from the pTZ-RB using the primers

TF1&TR1 and TF2&TR1. T- tail ligation in pTZ R/T was carried out as described


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above. The clones were confirmed using restriction digestion using EcoRI and BamHI

enzymes which release full insert from the clone. pTZ-RBB contained 1.4 kb

promoter fragment and pTZ-RBS contained 0.6 kb promoter fragment (Figure 4.3).

From the positive clones, only those containing insert in reverse orientation was

selected for further cloning. pTZ-RBB and pTZ-RBS was digested with enzymes

KpnI and XbaI so that only those clones which contain insert in reverse direction

released the full insert.

Figure 4.3: Linear vector map of pTZ-RBB and pTZ-RBS in reverse orientation.

pTZ-RBB and pTZ-RBS were digested with XhoI and XbaI restriction

enzymes to obtain the linearised vector backbone. The vector construct pRT-BKT has

bkt inserted in the MCS region of the vector with XhoI-XbaI restriction sites (Figure

4.4). pRT-BKT was also digested using the same set of enzymes to obtain the bkt

insert.

Figure 4.4: Linearised vector map of the construct pRT-BKT

The digested fragments (bkt gene fragment -1.8 kb) and linearised vector

backbone of pTZ-RBB (4.4 kb) and pTZ-RBS (3.7 kb) were gel run, eluted and was

further used for ligation. The ligated clones was digested with restriction enzymes

KpnI and XbaI for the presence of insert with size 3.2 kb (1.8 kb BKT+ 1.4 kb RBB)

and 2.4 kb (1.8 kb BKT+ 0.6 kb RBS) from pTZ-RBB-BKT and pTZ-RBS-BKT

respectively.

The binary vector construct p1304-CaMV-BKT was double digested with

KpnI and XbaI to release the CaMV-BKT cassette. The linearised vector after

removal of CaMV-BKT was used as the vector backbone for ligation reaction. The


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linearised vector thus contains part of the MCS and the poly A region of the CaMV-

BKT-polyA cassette. The vector constructs pTZ-RBB-BKT and pTZ-RBS-BKT was

also digested with the same set of enzymes to obtain the 3.2 kb and 2.5 kb inserts to

be used in the ligation mix. The digestion mix was run on 1% agarose gel and was gel

extracted using Qiagen gelelute kit (Qiagen, GmbH, Germany) was further used for

ligation. Positive plasmids were checked with enzymes KpnI and XbaI to release the

inserts. The plasmid DNA was also confirmed using PCR by amplification of Rubisco

promoter region using primers (TF1 & TR1; TF2 & TR1).

The schematic representation of the cloning procedure followed for binary

vector construction p1304-RBB-BKT and p1304-RBS-BKT is represented in figure

4.5


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Figure 4.5: Schematic representation of the cloning strategy followed for vector construction p1304-RBB-BKT and p1304-RBS-BKT


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4.3 Results

4.3.1 Amplification of RBCS2 promoter region with transit peptide

RBCS2 promoter region was amplified using PCR based adaptor ligated walking

method (Figure 4.6).

Figure 4.6: Gel images of RBCS2 promoter amplification (A) Genomic DNA digestion of D. bardawil with various blunt cutting enzymes (Lanes 1-8) and lane M is the 3 kb marker (B) primary PCR with TR2 and AP1 primer (Lane 1-11) and Lane M: 3 kb marker (C) Tertiary PCR giving the amplicon of 700 bp (Lane 1-2) and Lane M:3 kb marker (D) Full length promoter amplicon of 1.4 kb (Lane 1) and Lane M:3 kb marker (E) Insert release from pTZ-RBB Lane(1-2) and Lane M: 3 kb marker

The sequence of RBCS2 along with transit peptide with positions of primers,

Start codon and transit peptide is given in figure 4.7. The sequence of 1.4 kb was

analysed in NCBI-BLAST which showed 98% similarity with the D. teritiolecta

sequence and was aligned (Figure 4.8). The peptide cleavage site was identified

between 62-63 bp (21 amino acids) (Figure 4.9). Further analysis using the same

software revealed a cleavage site also between 135-136 bp (45 amino acids) after start

codon in the sequence. The presence of TATA boxes and other transcription factors

across the promoter which was analysed using PLACE database is given in figure

4.10.


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CCATTAACCAGTTTAAAGAGCTGGGACTTGACCACCAAGGTGCCAATAAAC

TCGCTCGCAAACTTCATGCCCATTCTGTCATGTATGCCAACAAGCTTGTTAC

CACTAGGTATGCTATTGAAAACAAGAATGCTTCTCGTACCCAGGTTATGGAG

CCAGGTGCTTCCAATAACCCTCCAGATCCAAATTCTTTCCAGATTTTTTACTA

CACAGCTTTGTGGTGGAGGGCCCCCCCGGGGATTCAAGGCTCTTTTGAGCC

AATGTGTCCCCTTTTCCTTAATTGATGTAGGGAGGTTTTCCTCTGCCTATGTA

GTCTTTTCTTTTCTTGTCTATTGAAGGGACCGTTCCTGCTGCCAATTGTATAT

CATATGATATAAACTATATATATTTAGTAGAGAGAAATTAGTCTTCCTTTCCAT

AAAGGGAATGCACCTTGCATTGGCCTCAGACCTCTCTTCTCCTTGACCAAAG

TGTGAAGTGATTCTGCTGCCTCAGAACTTTTTTCCTTTGTAACAGCAATCAAT

TGCAGCACAAATACCTTCACAAACACCGCCACAGTGGCCTGCTTGGCCCTTT

TCTCTCACTGCCATATCTGTACAGTGAAGTGGACGCATGTGGGCCCTTTTGC

TTGTCTTCCTTCCACAGACAAATGATCCTCACAATTACCCTTGCGTTGTGTGC

CCCGAAATATCATCTCAATGTTGTCACATGACAAATCCACAAACATAGCAGA

CCGCACAACACCATTAATGCAACACTTCATTGCCCAGTGGGGTGTCTGCGC

GTGTGCATGTGTGTGTGTTTGCGCGCACATGCGTGTGTGTGCGTGCGTGCA

TGCGTGCATGCGTGCGTGGGTGTGTTGTTTACTTTGCATGATGCAGCAAGC

CCCAAGCTTTTGATGGGTGAGGCTGTTGTCATTGCCCCAACCAAATCTGCCT

GCTTTATCCGCCAGCATCTCACAGGGCCTGATTGATTAGAGAACAAGAAGTT

CACTAGCCTGCTCTTAGGGGGGGGTGCAGAAGGTTTTGGAGAAATGGTGGA

TGCACTCCTTGCTCACCCTTGGCTGGACTTTCACGCGTCTCTGCACCTAAAC

ATCTTCACTTGAATGAACTGGATGAGGAATTTCCTAAAGGAGCAACAACATT

GTCACCCGAAGAGCATGGGTTCACACTTATCACTCAAAATCACGCCTTTTAA

GTTTGTGAGATTGCTCGCATTACCACTTTACTTTATACGTCCATTCCTGCACA

CACCCACTCACACACCACCCCAGTCAGACACAATGGCCTCTCTCATTGCAAA

GTCCGCCTCCGTGGCCCCTGTTGCCAGCCGCACCTCCACCA^AGGTGCAG

GCCTCTTTGAAGCCTGCCGTGCGCGCCGTGCCCAAGGCCCAGGCTCCCGC

TGTGCGCGCCAACCAG^ATGATGGTG

Figure 4.7: Sequence analysis of RBCS2 promoter region along with transit peptide amplified from D. bardawil. Blue (TR1), yellow (TR2), pink(TR3) and red (TR4) highlighted portions are used as primers for walking. Sequence in blue font is TF1 and in red font is TR1. TATA box position is underlined with bold font (-52). The region in green font is used as TF2. Grey highlighted portion indicated start of the RBCS2 gene(postion-1286 bp) and subsequent transit peptide sequence. ^ indicates the cleavage region of transit peptide.


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Sequences producing significant alignments (Details from NCBI nucleotide blast)

AY530156.1 Dunaliella tertiolecta ribulose- 98%

1,5-bisphosphate carboxylase/oxygenase

small subuni(rbcS2) gene, complete cds

Length=3321

Score = 2523 bits (1366); Expect = 0.0; Identities = 1429/1456 (99%);

Gaps = 18/1456 (1%); Strand=Plus/Plus

RBCS 1 CCATTAACCA GTTTAAAGAG CTGGGACTTG ACCACCAAGG TGCCAATAAA

D.ter 1 CCATTAACCA GTTTAAAGAG CTGGGACTTG ACCACCAAGG TGCCAATAAA

RBCS 51 CTCGCTCGCA AACTTCATGC CCATTCTGTC ATGTATGCCA ACAAGCTTGT

D.tert 51 CTCGCTCGCA AACTTCATGC CCATTCTGTC ATGTATGCCA ACAAGCTTGT

RBCS 01 TACCACTAGG TATGCTATTG AAAACAAGAA TGCTTCTCGT ACCCAGGTTA

D.tert 101 TACCACTAGG TATGCTATTG AAAACAAGAA TGCTTCTCGT ACCCAGGTTA

RBCS 151 TGGAGCCAGG TGCTTCCAAT AACCCTCCAG ATCCAAATTC TTTCCAGATT

D.tert 151 TGGAGCCAGG TGCTTCCAAT AACCCTCCAG ATCCAAATTC TTTCCAGATT

RBCS 201 TTTTACTACA CAGCTTTGTG GTGGAGGGCC CCCCCGGGGA TTCAAGGCTC

D.tert 201 TTTTACTACA CAGCTTTGTG GTGGAGGGCC CCCCCGGGGA TTCAAGGCTC

RBCS 251 TTTTGAGCCA ATGTGTCCCC TTTTCCTTAA TTGATGTAGG GAGGTTTTCC

D.tert 251 TTTTGAGCCA ATGTGTCCCC TTTTCCTTAA TTGATGTAGG GAGGTTTTCC

RBCS 301 TCTGCCTATG TAGTCTTTTC TTTTCTTGTC TATTGAAGGG ACCGTTCCTG

D.tert 301 TCTGCCTATG TAGTCTTTTC TTTTCTTGTC TATTGAAGGG ACCGTTCCTG

RBCS 351 CTGCCAATTG TATATCATAT GATATAAACT ATATATATTT AGTAGAGAGA

D.tert 351 CTGCCAATTG TATATCATAT GATATAAACT ATATATATTT AGTAGAGAGA

RBCS 401 AATTAGTCTT CCTTTCCATA AAGGGAATGC ACCTTGCATT GGCCTCAGAC

D.tert 401 AATTAGTCTT CCTTTCCATA AAGGGAATGC ACCTTGCATT GGCCTCAGAC

RBCS 451 CTCTCTTCTC CTTGACCAAA GTGTGAAGTG ATTCTGCTGC CTCAGAACTT

D.tert 451 CTCTCTTCTC CTTGACCAAA GTGTGAAGTG ATTCTGCTGC CTCAGAACTT

RBCS 501 TTTTCCTTTG TAACAGCAAT CAATTGCAGC ACAAATACCT TCACAAACAC

D.tert 501 TTTTCCTTTG TAACAGCAAT CAATTGCAGC ACAAATACCT TCACAAACAC

RBCS 551 CGCCACAGTG GCCTGCTTGG CCCTTTTCTC TCACTGCCAT ATCTGTACAG

D.tert 551 CGCCACAGTG GCCTGCTTGG CCCTTTTCTC TCACTGCCAT ATCTGTACAG

RBCS 601 TGAAGTGGAA CGCCATGTGG GCCTTTTGCT TGTCTTCTCC ACCAGACCAA

D.tert 601 TGAAGTGGAA CGCCATGTGG GCCTTTTGCT TGTCTTCTCC ACCAGACCAA

RBCS 651 TGATCTCACC AATTACCCTT GCGGTTGTGT GCCCCGACAT ATCATCTCAA

D.tert 651 TGATCTCACC AATTACCCTT GCGGTTGTGT GCCCCGACAT ATCATCTCAA

RBCS 701 TGTTGGGTCA CATTGACAAA TCACAAAACA TAGCAGACCG CACAACACCA

D.tert 701 TGTTGGGTCA CATTGACAAA TCACAAAACA TAGCAGACCG CACAACACCA

RBCS 751 TTAATGCAAC ACTTCATTGC CCAGTGGGGT GTCTGCGCGT GTGCATGTGT

D.tert 751 TTAATGCAAC ACTTCATTGC CCAGTGGGGT GTCTGCGCGT GTGCATGTGT


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RBCS 801 GTGTGTTTGC GCGCACATGC GTGTGTGTGC GTGCGTGCAT GCGTGCATGC

D.tert 801 GTGTGTTTGC GCGCACATGC GTGTGTGTGC GTGCGTGCAT GCGTGCATGC

RBS 851 GTGCGTGGGT GTGTTGTTTA CTTTGCATGA TGCAGCAAGC CCCAAGCTTT

D.tert 851 GTGCGTGGGT GTGTTGTTTA CTTTGCATGA TGCAGCAAGC CCCAAGCTTT

RBCS 901 TGATGGGTGA GGCTGTTGTC ATTGCCCCAA CCAAATCTGC CTGCTTTATC

D.tert 901 TGATGGGTGA GGCTGTTGTC ATTGCCCCAA CCAAATCTGC CTGCTTTATC

RBCS 951 CGCCAGCATC TCACAGGGCC TGATTGATTA GAGAACAAGA AGTTCACTAG

D.tert 951 CGCCAGCATC TCACAGGGCC TGATTGATTA GAGAACAAGA AGTTCACTAG

RBCS 1001 CCTGCTCTTA GGGGGGGGTG CAGAAGGTTT TGGAGAAATG GTGGATGCAC

D.tert 100 CCTGCTCTTA GGGGGGGGTG CAGAAGGTTT TGGAGAAATG GTGGATGCAC

RBCS 1051 TCCTTGCTCA CCCTTGGCTG GACTTTCACG CGTCTCTGCA CCTAAACATC

D.tert1051 TCCTTGCTCA CCCTTGGCTG GACTTTCACG CGTCTCTGCA CCTAAACATC

RBCS 1101 TTCACTTGAA TGAACTGGAT GAGGAATTTC CTAAAGGAGC AACAACATTG

D.tert1101 TTCACTTGAA TGAACTGGAT GAGGAATTTC CTAAAGGAGC AACAACATTG

RBCS 1151 TCACCCGAAG AGCATGGGTT CACACTTATC ACTCAAAATC ACGCCTTTTA

D.tert 115 TCACCCGAAG AGCATGGGTT CACACTTATC ACTCAAAATC ACGCCTTTTA

RBCS 1201 AGTTTGTGAG ATTGCTCGCA TTACCACTTT ACTTTATACG TCCATTCCTG

D.tert1201 AGTTTGTGAG ATTGCTCGCA TTACCACTTT ACTTTATACG TCCATTCCTG

RBCS 1251 CACACACCCA CTCACACACC ACCCCAGTCA GACACAATGG CCTCTCTCAT

D.tert1251 CACACACCCA CTCACACACC ACCCCAGTCA GACACAATGG CCTCTCTCAT

RBCS 1301 TGCAAAGTCC GCCTCCGTGG CCCCTGTTGC CAGCCGCACC TCCACCAAGG

D.tert1301 TGCAAAGTCC GCCTCCGTGG CCCCTGTTGC CAGCCGCACC TCCACCAAGG

RBCS 1351 TGCAGGCCTC TTTGAAGCCT GCCGTGCGCG CCGTGCCCAA GGCCCAGGCT

D.tert1351 TGCAGGCCTC TTTGAAGCCT GCCGTGCGCG CCGTGCCCAA GGCCCAGGCT

RBCS 1401 CCCGCTGTGC GCGCCAACCA GATGATGGTG tg-------- ----------

D.tert1401 CCCGCTGTGC GCGCCAACCA GATGATGGTG ctcgagtggg cgacacagta

Figure 4.8: Result from NCBI nucleotide blast and alignment of the sequence with

D. tertiolecta (AY530156.1) sequence using Dialign program (RBCS-

cloned RBCS2 promoter region; D.tert- Dunaliella tertiolecta sequence)


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SignalP-HMM result:

Prediction: Signal peptideSignal peptide probability: 0.838Signal anchor probability: 0.159Max cleavage site probability: 0.195 between pos. 62 and 63

5'3' Frame 1

Met A S L I A K S A S V A P V A S R T S T K ^ V Q A S L K P A V R A V P K A

Q A P A V R A N Q ^ Met Met V L E W A T Q Y H Met

Figure 4.9: Result for presence of chloroplast signal peptide in the cloned sequence using Signal P software for the first cleavage site. Translated sequence of coding region is also presented with possible cleavage sites indicated by ^(between amino acids 21 & 22 and 45 & 46).

(+) = Current Strand(-) = Opposite Strand

1 CCATTAACCAGTTTAAAGAGCTGGGACTTGACCACCAAGGTGCCAATAAA (-)WUSATAg S000433 (-)GT1CORE S000125 (+)MYB1AT S000408 (+)TAAAGSTKST1 S000387 (+)DOFCOREZM S000265 (-)NODCON2GM S000462 (-)OSE2ROOTNODULE S000468 (+)ELRECOREPCRP1 S000142 (+)WBOXATNPR1 S000390 (+)WBOXNTERF3 S000457 (+)WRKY71OS S000447 (+)CCAATBOX1 S000030 (+)CAATBOX1 S000028 (+)POLASIG1 S000080

51 CTCGCTCGCAAACTTCATGCCCATTCTGTCATGTATGCCAACAAGCTTGT (+)BIHD1OS S000498 (-)WRKY71OS S000447


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(+)RAV1AAT S000314 (-)GAREAT S000439

101 TACCACTAGGTATGCTATTGAAAACAAGAATGCTTCTCGTACCCAGGTTA (+)CACTFTPPCA1 S000449 (-)CAATBOX1 S000028 (+)HSELIKENTACIDICPR1 S000056 (-)HSELIKENTACIDICPR1 S000056 (+)CURECORECR S000493 (-)CURECORECR S000493

151 TGGAGCCAGGTGCTTCCAATAACCCTCCAGATCCAAATTCTTTCCAGATT (+)EBOXBNNAPA S000144 (+)MYCCONSENSUSAT S000407 (-)EBOXBNNAPA S000144 (-)MYCCONSENSUSAT S000407 (-)RAV1BAT S000315 (+)CCAATBOX1 S000030 (+)CAATBOX1 S000028 (+)AGMOTIFNTMYB2 S000444 (-)DOFCOREZM S000265 (+)ARR1AT S000454 (-)CCA1ATLHCB1 S000149

201 TTTTACTACACAGCTTTGTGGTGGAGGGCCCCCCCGGGGATTCAAGGCTC (+)CACTFTPPCA1 S000449 (-)DOFCOREZM S000265 (+)SORLIP2AT S000483 (-)SORLIP2AT S000483 (+)ARR1AT S000454 (+)NODCON2GM S000462 (+)OSE2ROOTNODULE S000468 (-)DOFCOREZM S000265

251 TTTTGAGCCAATGTGTCCCCTTTTCCTTAATTGATGTAGGGAGGTTTTCC (+)CCAATBOX1 S000030 (+)LEAFYATAG S000432 (+)CAATBOX1 S000028 (+)PYRIMIDINEBOXOSRAMY1A S000259 (-)DOFCOREZM S000265 (-)GT1CONSENSUS S000198 (-)CAATBOX1 S000028 (-)GT1CONSENSUS S000198

301 TCTGCCTATGTAGTCTTTTCTTTTCTTGTCTATTGAAGGGACCGTTCCTG (-)DOFCOREZM S000265 (-)POLLEN1LELAT52 S000245 (-)DOFCOREZM S000265 (-)POLLEN1LELAT52 S000245 (-)CAATBOX1 S000028 (-)MYBCOREATCYCB1 S000502

351 CTGCCAATTGTATATCATATGATATAAACTATATATATTTAGTAGAGAGA (+)CCAATBOX1 S000030 (+)CAATBOX1 S000028 (+)EBOXBNNAPA S000144 (+)MYCCONSENSUSAT S000407 (-)EBOXBNNAPA S000144 (-)MYCCONSENSUSAT S000407 (-)CAATBOX1 S000028 (-)GATABOX S000039 (+)CATATGGMSAUR S000370 (+)EBOXBNNAPA S000144 (+)MYCCONSENSUSAT S000407 (-)CATATGGMSAUR S000370 (-)EBOXBNNAPA S000144 (-)MYCCONSENSUSAT S000407 (+)GATABOX S000039


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(+)ROOTMOTIFTAPOX1 S000098 (-)CACTFTPPCA1 S000449 (+)POLLEN1LELAT52 S000245

401 AATTAGTCTTCCTTTCCATAAAGGGAATGCACCTTGCATTGGCCTCAGAC (-)DOFCOREZM S000265 (+)AGATCONSENSUS S000316 (+)TAAAGSTKST1 S000387 (+)DOFCOREZM S000265 (-)CAATBOX1 S000028 (-)CCAATBOX1 S000030

451 CTCTCTTCTCCTTGACCAAAGTGTGAAGTGATTCTGCTGCCTCAGAACTT (+)NODCON2GM S000462 (+)OSE2ROOTNODULE S000468 (+)ELRECOREPCRP1 S000142 (+)WBOXATNPR1 S000390 (+)WBOXNTERF3 S000457 (+)WRKY71OS S000447 (-)TBOXATGAPB S000383 (+)DOFCOREZM S000265 (-)CACTFTPPCA1 S000449 (+)GTGANTG10 S000378 (-)CACTFTPPCA1 S000449 (+)GTGANTG10 S000378 (+)ARR1AT S000454 (-)DOFCOREZM S000265 (+)PYRIMIDINEBOXHVEPB1 S000298 (-)GT1CONSENSUS S000198 (-)GT1GMSCAM4 S000453

501 TTTTCCTTTGTAACAGCAATCAATTGCAGCACAAATACCTTCACAAACAC (-)GT1CONSENSUS S000198 (-)DOFCOREZM S000265 (-)MYBCORE S000176 (+)CAATBOX1 S000028 (-)ARR1AT S000454 (+)CAATBOX1 S000028 (+)EBOXBNNAPA S000144 (+)MYCCONSENSUSAT S000407 (-)EBOXBNNAPA S000144 (-)MYCCONSENSUSAT S000407 (-)CAATBOX1 S000028 (-)GTGANTG10 S000378 (+)2SSEEDPROTBANAPA S000143 (+)CANBNNAPA S000148 (+)PROXBBNNAPA S000263

551 CGCCACAGTGGCCTGCTTGGCCCTTTTCTCTCACTGCCATATCTGTACAG (+)SORLIP1AT S000482 (-)CACTFTPPCA1 S000449 (-)SORLIP1AT S000482 (-)SORLIP2AT S000483 (+)PYRIMIDINEBOXOSRAMY1A S000259 (-)DOFCOREZM S000265 (-)POLLEN1LELAT52 S000245 (-)GTGANTG10 S000378 (+)CACTFTPPCA1 S000449 (-)GATABOX S000039 (-)CACTFTPPCA1 S000449 (+)GTGANTG10 S000378

601 TGAAGTGGACGCATGTGGGCCCTTTTGCTTGTCTTCCTTCCACAGACAAA (-)CACTFTPPCA1 S000449 (+)EBOXBNNAPA S000144 (+)MYCATERD1 S000413


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(+)MYCCONSENSUSAT S000407 (-)EBOXBNNAPA S000144 (-)MYCATRD22 S000174 (-)MYCCONSENSUSAT S000407 (+)SITEIIATCYTC S000474 (+)SORLIP2AT S000483 (-)SORLIP2AT S000483 (+)PYRIMIDINEBOXOSRAMY1A S000259 (-)DOFCOREZM S000265 (+)EBOXBNNAPA S000144 (+)MYCCONSENSUSAT S000407 (-)EBOXBNNAPA S000144 651 TGATCCTCACAATTACCCTTGCGTTGTGTGCCCCGAAATATCATCTCAAT (-)GTGANTG10 S000378 (+)CAATBOX1 S000028 (-)GT1CONSENSUS S000198 (+)LTRE1HVBLT49 S000250 (-)ROOTMOTIFTAPOX1 S000098 (-)GATABOX S000039 (+)CAATBOX1 S000028 (-)RAV1AAT S000314

701 GTTGTCACATGACAAATCCACAAACATAGCAGACCGCACAACACCATTAA (+)SEBFCONSSTPR10A S000391 (+)BIHD1OS S000498 (+)TGTCACACMCUCUMISIN S000422 (-)WRKY71OS S000447 (-)GTGANTG10 S000378 (+)EBOXBNNAPA S000144 (+)MYCATRD22 S000174 (+)MYCCONSENSUSAT S000407 (-)EBOXBNNAPA S000144 (-)MYCATERD1 S000413 (-)MYCCONSENSUSAT S000407 (+)WRKY71OS S000447 (-)BIHD1OS S000498 (-)ARR1AT S000454 (+)RAV1AAT S000314 (-)WUSATAg S000433

751 TGCAACACTTCATTGCCCAGTGGGGTGTCTGCGCGTGTGCATGTGTGTGT (+)RAV1AAT S000314 (+)CACTFTPPCA1 S000449 (-)CAATBOX1 S000028 (-)CACTFTPPCA1 S000449 (+)CGCGBOXAT S000501 (-)ABRERATCAL S000507 (-)CGCGBOXAT S000501 (-)DPBFCOREDCDC3 S000292 (-)RYREPEATLEGUMINBOX S000100 (-)RYREPEATBNNAPA S000264 (+)EBOXBNNAPA S000144 (+)MYCATERD1 S000413 (+)MYCCONSENSUSAT S000407 (-)DPBFCOREDCDC3 S000292 (-)EBOXBNNAPA S000144 (-)MYCATRD22 S000174 (-)MYCCONSENSUSAT S000407 (-)2SSEEDPROTBANAPA S000143 (-)CANBNNAPA S000148

801 GTTTGCGCGCACATGCGTGTGTGTGCGTGCGTGCATGCGTGCATGCGTGC (+)CGCGBOXAT S000501 (-)CGCGBOXAT S000501 (+)EBOXBNNAPA S000144 (+)MYCATRD22 S000174 (+)MYCCONSENSUSAT S000407


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(-)EBOXBNNAPA S000144 (-)MYCATERD1 S000413 (-)MYCCONSENSUSAT S000407 (-)DPBFCOREDCDC3 S000292 (-)RYREPEATLEGUMINBOX S000100 (-)RYREPEATBNNAPA S000264 (-)RYREPEATLEGUMINBOX S000100 (-)RYREPEATBNNAPA S000264

851 GTGGGTGTGTTGTTTACTTTGCATGATGCAGCAAGCCCCAAGCTTTTGAT (-)RAV1AAT S000314 (+)CACTFTPPCA1 S000449 (+)TBOXATGAPB S000383 (-)-300ELEMENT S000122 (-)DOFCOREZM S000265 (-)PROLAMINBOXOSGLUB1 S000354 (-)RYREPEATBNNAPA S000264 (-)DOFCOREZM S000265

901 GGGTGAGGCTGTTGTCATTGCCCCAACCAAATCTGCCTGCTTTATCCGCC (+)GTGANTG10 S000378 (+)MYBCORE S000176 (-)RAV1AAT S000314 (+)BIHD1OS S000498 (-)WRKY71OS S000447 (+)E2FCONSENSUS S000476 (-)CAATBOX1 S000028 (+)MYBPZM S000179 (+)CIACADIANLELHC S000252 (+)REALPHALGLHCB21 S000362 (-)ARR1AT S000454 (-)DOFCOREZM S000265 (-)TAAAGSTKST1 S000387 (-)GT1CONSENSUS S000198 (+)SREATMSD S000470 (-)IBOXCORE S000199 (-)GATABOX S000039 (-)MYBST1 S000180 (-)REBETALGLHCB21 S000363

951 AGCATCTCACAGGGCCTGATTGATTAGAGAACAAGAAGTTCACTAGCCTG (-)GTGANTG10 S000378 (+)SORLIP2AT S000483 (+)ARR1AT S000454 (-)CAATBOX1 S000028 (+)ARR1AT S000454 (-)GTGANTG10 S000378 (+)CACTFTPPCA1 S000449

1001 CTCTTAGGGGGGGGTGCAGAAGGTTTTGGAGAAATGGTGGATGCACTCCT (+)NODCON2GM S000462 (+)OSE2ROOTNODULE S000468 (+)POLLEN1LELAT52 S000245 (+)CACTFTPPCA1 S000449

1051 TGCTCACCCTTGGCTGGACTTTCACGCGTCTCTGCACCTAAACATCTTCA (-)GTGANTG10 S000378 (+)EECCRCAH1 S000494 (-)DOFCOREZM S000265 (-)GTGANTG10 S000378 (+)ABRERATCAL S000507 (+)CGCGBOXAT S000501 (-)CGCGBOXAT S000501 (-)SURECOREATSULTR11 S000499 (-)GTGANTG10 S000378 (+)CACTFTPPCA1 S000449 (+)EBOXBNNAPA S000144 (+)MYCCONSENSUSAT S000407


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(-)EBOXBNNAPA S000144 (-)MYCCONSENSUSAT S000407

1101 CTTGAATGAACTGGATGAGGAATTTCCTAAAGGAGCAACAACATTGTCAC (+)EECCRCAH1 S000494 (-)GT1CONSENSUS S000198 (+)TAAAGSTKST1 S000387 (+)DOFCOREZM S000265 (+)RAV1AAT S000314 (+)RAV1AAT S000314 (-)CAATBOX1 S000028 (+)SEBFCONSSTPR10A S000391 (+)BIHD1OS S000498 (-)WRKY71OS S000447 (-)GTGANTG10 S000378

1151 CCGAAGAGCATGGGTTCACACTTATCACTCAAAATCACGCCTTTTAAGTT (-)NODCON2GM S000462 (-)OSE2ROOTNODULE S000468 (-)SEF3MOTIFGM S000115 (-)GTGANTG10 S000378 (+)CACTFTPPCA1 S000449 (-)IBOX S000124 (-)IBOXCORE S000199 (-)GATABOX S000039 (-)GTGANTG10 S000378 (+)CACTFTPPCA1 S000449 (-)ARR1AT S000454 (-)GTGANTG10 S000378 (+)PYRIMIDINEBOXOSRAMY1A S000259 (-)DOFCOREZM S000265

1201 TGTGAGATTGCTCGCATTACCACTTTACTTTATACGTCCATTCCTGCACA (+)GTGANTG10 S000378 (+)ARR1AT S000454 (-)CAATBOX1 S000028 (-)GT1CONSENSUS S000198 (+)CACTFTPPCA1 S000449 (+)NTBBF1ARROLB S000273 (-)DOFCOREZM S000265 (-)TAAAGSTKST1 S000387 (+)CACTFTPPCA1 S000449 (+)NTBBF1ARROLB S000273 (-)DOFCOREZM S000265 (-)TAAAGSTKST1 S000387 (+)ACGTATERD1 S000415 (-)ACGTATERD1 S000415 (-)INTRONLOWER S000086

1251 CACCCACTCACACACCACCCCAGTCAGACACAATGGCCTCTCTCATTGCA (+)CACTFTPPCA1 S000449 (-)GTGANTG10 S000378 (-)WBOXHVISO1 S000442 (-)WBOXNTCHN48 S000508 (-)WBOXNTERF3 S000457 (-)WRKY71OS S000447 (+)CAATBOX1 S000028 (-)CAATBOX1 S000028 (+)-300ELEMENT S000122 (+)PROLAMINBOXOSGLUB1 S000354 (-)TBOXATGAPB S000383 (+)DOFCOREZM S000265

Figure 4.10: Promoter analysis for the presence of transcription factors using the software “SIGNAL SCAN” for region upstream of ATG (bold and underlined) (Database used: PLACE).


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4.3.2 Construction of binary vector p1304-RBB-BKT and p1304-RBS-BKT

The various stages of construction of binary vector p1304-RBB-BKT and p1304-

RBS-BKT are presented in gel images in Figure 4.11 to 4.13. The actual promoter

length of 1,282 bp in pTZ-RBB, 419 bp in pTZ-RBS, 136 bp of transit peptide region

as per the sequence result is mentioned as 1.25 kb, 0.45 kb and 0.15 kb in the text.

The final constructs were checked with PCR using the primers TF1& TR1 (1.4 kb)

and TF2 & TR1 (0.6 kb) respectively (Figure 4.13 D). Diagrammatic representation

of the RBB-BKT and RBS-BKT region is given in figure 4.14.

Figure 4.11 Construction of pTZ-RBB and pTZ-RBS (A) Lane 1-2: Amplification of 1.4 kb RBB using TF1 and TR1 ; Lane 3-4: Amplification of 0.6 kb RBS using TF2 and TR1; Lane M: 10 kb marker (B) Ligated constructs: Lane 1:pTZ-RBB; Lane 2-3:pTZ-RBS; Lane M:control pTZ (C) Restriction digestion pTZ-RBS using EcoRI and BamHI: Lane 1-2:pTZ-RBS with insert release of 0.6 kb; Lane M: 3 kb marker (D) Restriction digestion pTZ-RBB using EcoRI and BamHI: Lane 1-2:pTZ-RBB with insert release of 1.4 kb; Lane M: 3 kb marker


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Figure 4.12 Construction of vectors pTZ-RBB-BKT and pTZ-RBS-BKT (A) Lane 1:bkt insert release from pRT-BKT by XhoI-XbaI M: 3 kb marker (B)Lane 1: linearised pTZ-RBB;Lane 2: Linearised pTZ-RBS;M: 3 kb marker (C) ligated plasmids: Lane 1: pTZ-RBB; Lane 2:pTZ control; Lane 3:pTZ-RBS-BKT; Lane 4:pTZ-RBB-BKT (D) Insert release through XbaI-KpnI digestion from Lane1: 2.4 kb from pTZ-RBS-BKT; Lane 2 :3.2 kb from pTZ-RBB-BKT

Figure 4.13: Construction of vectors p1304-RBB-BKT and p1304-RBS-BKT (A) Lane 1:p1304-BKT digested with XbaI-KpnI Lane M:10 kb marker (B)Ligated plasmids: Lane 1: p1304-RBS-BKT; Lane 2: p1304-RBB-BKT;Lane 3: p1304-BKT (C) Digestion of p1304-RBB-BKT(Lane1) and p1304-RBS-BKT (Lane 2) with XbaI-KpnI; Lane M:10 kb marker (D) Amplification of RBCS promoter regions from p1304-RBB-BKT and p1304-RBS-BKT: Lane M: 3 kb marker; Lane 1&3: Amplification from p1304-RBB-BKT using TF1& TR1; Lane 2&4: Amplification from p1304-RBS-BKT using TF2 & TR1


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Figure 4.14: Diagrammatic representation of RBB-BKT and RBS-BKT region in the constructs. Filled box represent 136 bp long transit peptide region and unfilled region represent bkt gene (Scale 500 bp=1” in promoter region)

4.4 Discussion

β-carotene ketolase (bkt) is the major gene involved in the production of keto-

carotenoids including astaxanthin. BKT is the crucial enzyme which is involved in the

conversion of β-carotene to echinenone and then to canthaxanthin. Further the

pathway proceeds with the conversion of canthaxanthin to astaxanthin by the use of

CHY (Hirschberg, 2001). So far cloning of the bkt from H. pluvialis has been made

only from the cDNA of H. pluvialis (Kajiwara et al, 1995; Lotan and Hirschberg

1995), and also from the partial fragment of the cDNA of bkt for the expression

studies (Huang et al, 2006; Vidhyavathi et al, 2008). The present study used the

genomic clone of bkt2 from H. pluvialis. Since the ketolases known to date prefer an

unsubstituted β-ionone ring, astaxanthin synthesis is possible only when ketolase

concentration in transgenic plants is significantly higher than the endogenous

hydroxylase level (Zhu et al, 2007). The cloned bkt (GQ214765) which is used in the

current study has 99% sequence homology to already reported bkt cDNA clone

(NCBI Accession No.D45881) which was categorized as bkt2. Most ketolases are

rather poor in converting the hydroxy derivative zeaxanthin to astaxanthin via

adonixanthin. bkt2 is reported to utilize both zeaxanthin and β-carotene as the

substrate(Kajiwara et al, 1995). A recent study (Huang et al, 2006) demonstrates the

presence of at least three of the bkt transcripts in the same H. pluvialis strain. They

propose the existence of multiple bkt genes in H. pluvialis that are up-regulated by

different stress conditions which may be responsible for high astaxanthin production

in this alga. Different bkt genes can also have different degrees of affinity for different

substrates, such as β-carotene and zeaxanthin. β-carotene ketolase belonging to bkt2

category due to its bi-functional nature can act either on β-carotene to produce


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canthaxanthin or on zeaxanthin which is already present in Dunaliella to produce

astaxanthin.

Nucleotide and amino acid sequence analysis also revealed the reading frame

of the bkt is exactly similar (99%) to the nucleotide and amino acid sequence of the

known bkt accession number D45881(Kajiwara et al, 1995). Since, the expression of

the whole BKT is complete even if gene start from the 3rd ATG (264th nucleotide) of

bkt, the amplification of the bkt was carried out from this third starting codon (Fraser

et al, 1997; Kajiwara et al, 1995). The sequence analysis of the cloned bkt revealed

that there was some nucleotide polymorphism when aligned and compared with the

known bkt (D45881). But there was no change in the reading frame. The protein

sequence synthesized from this sequence is also matching with the result of Kajiwara

et al (1995). The presence of six exons and five introns in the cloned bkt gene was

observed.

The CaMV 35S promoter was used to express selectable marker gene like hpt

in C. reinhardtii (Kumar et al, 2004), reporter genes like lacZ gene in H. pluvialis

(Teng et al, 2002) and for the over-expression of bkt (Kathiresan, 2009) in H.

pluvialis. Though Meng et al (2005) identified the partial region of the promoter

region for bkt in H. pluvialis, it has not been studied experimentally for its expression

levels. p1304-CaMV-BKT which is the binary vector construct used for over-

expression of bkt in H. pluvialis (Kathiresan, 2009) was also maintained as a control

in the present study in transforming Dunaliella. However a signal peptide was absent

in this construct.

In Dunaliella β-carotene accumulation is observed in inter-thylakoidal

membranes of the chloroplast. So the localization of BKT in chloroplast is imperative

for BKT expression in Dunaliella. Nuclear-encoded chloroplast-targeted proteins,

such as the light-harvesting Chl a/b protein or the small subunit of Rubisco (RBCS2),

following cytoplasmic translation are normally imported into the chloroplast (Dean et

al, 1989) with an amino-terminal transit sequence which are later cleaved off.

Attaching such a sequence to the amino terminus of the bkt and placing the resulting

coding region under the control of an endogenous promoter should result in the

production of chloroplast localized bkt.


145

Promoters of the Rubisco enzyme have been suggested as strong promoters

useful for the preparation of DNA constructs for transforming plants. There is often

substantial increase in RBCS2 expression after induction with light, and this is

mediated by both phytochrome and blue light photoreceptors (Fluhr and Chua, 1986).

Light-responsive regulatory elements are positioned to the 5'side of the transcriptional

initiation site and possess at least two autonomous control sequences. Light is a major

environmental factor favouring β- carotene accumulation in Dunaliella. β- carotene is

the precursor for keto-carotenoid formation and substrate for bkt. So the presence of

an endogenous rubisco promoter with chloroplast targeting increases the chances of

BKT expression.

In this regard, cloning of Rubisco smaller subunit promoter region along with

transit peptide region from D. bardawil which was carried out in the present study

proved as an appropriate step for expression BKT in Dunaliella. The presence of

several cis-regulatory elements and transcription factors was compared to that of

previously reported RBCS2 promoters from both plants and algae. The cloned

sequence was having 98% similarity to D. teritiolecta sequence (NCBI accession no.

AY530156). Although Signal P analysis revealed the presence of a cleavage site

between 62 & 63 bp, amplification of the peptide region was extended till 135 & 136

bp as per the results from further analysis using the same software and also the reports

from other studies (Walker et al, 2005a).

Promoter analysis through SIGNAL SCAN revealed the presence of several

cis elements like TATA box, CCAAT motif, I box and G box across the promoter.

For many genes, the critical binding site for the transcription complex is TATA box,

usually located 25-30 bp 5' of the transcription start site. The cloned RBCS2 promoter

was having TATA box 52 bp upstream of transcription start site (TSS). The cloned

promoter region had CAGAC motif at position -553 and a CACCACA motif at

position -740, although both showed one base substitution similar to D. teritiolecta

(Walker et al, 2005a). Two similar motifs were found in approximately the same

positions in the C. reinhardtii RBCS2 promoter, suggesting that these conserved

motifs may play a role in light-responsive gene expression in some of the microalgal

RBCS2 genes. Although not completely conserved between the two species, the

presence of motifs like DOF are known to be involved in photo-responsiveness of

other photosynthesis genes upstream of the defined transcription start site. This


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indicates that the full and active promoter sequence has been identified in the cloned

promoter of the present study.

Attempts to express genes fused to heterologous promoters were unsuccessful

in the diatom P. tricornutum (Apt et al, 1996). In Chlamydomonas, the same resulted

in low transformation frequency (Hall et al, 1993) or unstable expression (Tang et al,

1995). The only report of stable transformation of microalgae with heterologous genes

under the control of heterologous promoters is the expression of GUS-GFP and hpt

driven by CaMV 35S promoter as in C. reinhardtii (Kumar et al, 2004) and H.

pluvialis (Kathiresan et al, 2009). However, it is generally accepted that, in

chlorophytes and diatoms, stable expression of heterologous genes can only be

optimally achieved when adequate homologous promoters and other regulatory

regions are included.The promoter sequence of the chloroplast Rubisco gene was

reported as an efficient promoter for foreign gene expression in Porphyra yezoenesis.

Relatively high expression rate of introduced GUS was observed for a Rubisco driven

uidA. The ability of the Dunaliella RBCS2 promoter regulatory regions for driving

expression of the ble selectable marker gene was assessed in

C. reinhardtii, and promoter deletion analysis was undertaken to determine the

optimal promoter length (Walker et al, 2005a). The optimal promoter length for

maximum expression from the D. tertiolecta RBCS1 was 300 bp, which gave over

four fold higher expression than the full length promoter construct, suggesting that

there may be an essential element within the region from 180 to 300 of this promoter.

Based on the above report, a deletion construct (p1304-RBS-BKT) was prepared with

400 bp of the RBCS2 promoter region, upstream of ATG for a better expression of

BKT in Dunaliella.

H. pluvialis, the alga which produces high amount of astaxanthin, for which

pathway for the astaxanthin production is well known, was selected as the source for

the isolation and cloning of bkt in the present study. The target organism for BKT

expression, D. bardawil and the source organism H. pluvilais belong to the same

family chlorophyceae, which further ensures similarity in codon bias for successful

foreign gene expression. The construction of the binary vector described in the

present chapter with bkt gene under the control of endogenous RBCS2 promoter with

chloroplast transit peptide thus make it a suitable vector for expression of BKT in

Dunaliella .

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CHAPTER IV Binary vector construction with bkt H. pluvialisshodhganga.inflibnet.ac.in/bitstream/10603/39819/9... · 2018. 7. 2. · H. pluvialis was shown to possess three different