isolation and characterisation of legumin promoter sequence...

Indian Journal of Experimental Biology Vol. 38, April 2000, pp. 363-372

Isolation and characterisation of legumin promoter sequence from chickpea (Cicer arietinum L.)

Ajit Kumar Shasany & Kirpa Ram Koundal*

*National Research Centre, Plant Biotechnology, Indian Agricultural Research Institute, Pusa, New Delhi II 0 0 12. India

Received 6 July / 999; revised 3 November 1999

Seed spec ific promoters are useful for express ion of fore ign genes in the seeds. We have iso lated a Cicer

a rietinum legumin promoter from A.EMBL genomic library and subcloned in pBluescript II KS (-) in Eco RV and Pst I site. The 2.762 kb Hind II Pst I fragment was sequenced completely by dideoxy chain termination method by creating a set of unidirectional deletions of the inserts in pAKKIII . The insert contains mainl y upstream sequence (2240 bps) and only a part o f structural gene (522 bps) sequence. The 522 bps of the structural gene shows - 80% homology with pea legumin A and this is almost the same as chickpea legumin in its sequence. The amino acid sequence deri ved from the part of the structural gene was similar to the chickpea 5' part o f the legumin structural gene with a few vari ations. A 2 1 amino acid signal peptide was also deduced like many other legumes. Transcription start site (CAT) was located at 55 bp upstream of the initiation codon ATG. One codon downstream to ATG codon Hind III site was present. TAT A box was observed at- 30 pos ition, with a consensus of CCTAT AAAT AACC. The consensus CATGCAAG, a part of legumin box was noticed at - II 0 bp pos ition. At - 295 to -265 bp upstream AGGA box like sequences were observed and a 56 bp perfect repeat was located between - 91 3 bp and - 972 bp. Strong homology with pea promoter sequence near the CAT sequence was noti ced which gradually decreased towards the upstream region. Thus the cloned fragment contains a full length promoter which can be utili sed for express ion of foreign genes in seeds of chickpea.

The seed storage prote ins especiall y the major one like legumins ( II S) from various legumes have been studied in detai Is, inc luding the ir biosynthesis and structure. The genes for these proteins are expressed to high level s in the cotyledons of legumes and the corresponding mRNAs are accumulated temporarily at the middle to late phases of seed development 1

• As these prote ins are the products of group of developmentall y regulated genes, which have ti ssue specific expression, the level of express ion changes with the response to the environment. The spatial and temporal expression of the legumin genes2 make these excellent model systems for the study of the molecular mechanism re lated to differential gene regulati on in pl ants 1•

Most of the knowl edge on legume storage proteins and the ir genes comes from Pea, Soyabean, French bean and Broad bean1

• The 32 1 nucleot ides towards the upstream from the coding region of leg A, B, C of Pisum were analysed and found to be identica l' . This part of leg A contains TATA, AGGA/CAAT boxes

*Corrcspondant author Fax: 091-11-5766420, E-mail: krk_pbio @iari.ernet.in

and sequence homologous to adena virus enhancer. Al so the legumin box containing 30 bp having consensus sequence of CATGCATG, has been identified, which is responsible for effic ient expression of legumin genes4

·5

. Thi s box is conserved in all the legumin genes. Leg J in Pi sum has only TATA and legumin boxes'. Similarly TATA box at -25 to -30 bp and CAAT box at - I 00 bp have been identified in the 5'region of the glycinin genes. A 9 bp 5' CAACACAA T 3' sequence, which is responsible for seed spec ific expression is present beside the legu min box 1• Sequence compari son in Glycine, Pisum and Vicia revealed the presence of similar consensus regulatory sequences4

•

Identification, cloning and characterisation of the legu min promoter in chickpea is the first step to study the gene express ion in a model system. Seed specificity may also be used to produce the transgenic plant resistant to Heliothis armigera by directing the expression of Bt 6 endotoxin gene in seed, which may reduce the insect insurgency . However, the molecular biology studies in this aspect is very limited in chickpea, which is the major pulse crop of India. So

364 · INDIAN J EXP BIOL, APRIL2000

far there is no report on the chickpea promoter sequence in literature. [n thi s paper, the isolation and characteri sation of a sequence which contain the full length promoter of legumin ( I IS) seed storage protein gene of chickpea has been di scussed .

Materials and Methods Enzymes and chemicals-Restri cti on enzy mes,

modifying enzymes, ligation kit and plasmid pBluescript II KS (-) were procured from Stratagene (USA). Nick translation kit was obtained from Du Pont (UK). Erase-a-base sys tem kit was purchased from Promega Crop Inc. (USA) and sequenase vers ion 2.0 was from USB (US A). 32P dCTP and 35S dATP used were the product of BARC (India).

Genomic library and probes - Chickpea (Cicer anettnum L.) cv Pusa 256, genomic library constructed in EMBL-3 a lambda replacement vector6

wasused in the present investi gati on. Initi ally, inserts from pDUB6 and pDUB8 containing pea legumin eDNA in serts of I I 00 bp (from C-tenninal) and 952 bp (from N terminal) respective ly, at Bam HI s ite were used to screen the genomic librar/.

In the latter stage a fragment of 0.8 kb from 5' region of chickpea legumin gene and a 0.5 kb DNA fragment ex tending into the structural gene was used to screen the genomic library (Fig. I) . These probes were developed in our laborator/ and the two fragments were eluted from the low melting agarose gel after Eco RI , Hind III and Eco RV restriction of the recombinant pBluescript II SK(-).

Phage DNA isolation, restriction and Southern hybridisation-Phage DNA was isolated as described by Sambrook et al.9 The DNA samples were restricted with Sal I first and a set of restricti on enzymes later and run on 0.8% agarose ge l. Southern hybridi sation using insert from pDUB6, pDUB 8 (0.8 kb 5' probe and 0.5 kb structural gene probes) was performed9

.

Subcloning in pBluescript II KS(-)--Th e 2.76 kb fragment generated by digesting the gelliomic clone F with Hind II and Pst I was ex tracted and purified from

0 0 .8Kb 1.30Kb

IE 0.8 Kb ~~ O.SK~

Probe Probe

low melting agarose ge l. P lasmid was double digested with Pst I and Eco RV, phenol: chloroform ( 1: 1) ~tracted, prec ipitated, dried and dissolved in sterile

di stilled water. The li gat ion mixture consisted of I ~I

vector (I OOng), 2 ~I of fragment DNA (200 ng), I ~I ligat ion buffer, I ~I of 100 mM ATP, I ~I of T4 DNA

ligase (4 weiss unit) and 4 ~I terile wate r. The

mixture was briefly spun and incubated at l5°C

overnight. Next day competent E. coli DH Sa cells

were transformed with 5 ~I li gation mixture and

grown in Luri a aga r plates conta ining I 00 ~g/ml

amp icilli n and coated with 2% X-ga l (50 ~I ) and 0 . 1 M IPTG ( 10 ~1 ). The plates were incubated at 37°C for 16 hr and the white bacteri al colonies deve loped were checked for the presence of recombinant plasmid.

Un idirectional deletion with Exonuclease ///The subclone containing the in sert was rest ricted with Kpn I and Hind II. Then unidirectional deletion was carried out according to manufacturers guidelines (Erase-a-base system kit, Promega) . After ligation and transformati on into E. coli, DH Sa, the deleted subc lones were checked for the size of inserts by restricting with Bss HI. In the same way unidirectional de le ti on was carried out from the other end of the insert.

Double stranded DNA sequencing- Plasmid DNA minipreparation was carried out according to Stephen et al. 10

. DNA sequenc ing using 35S dATP and Sequenase version 2.0 DNA sequenc ing kit was carried out according to the dideoxy chain terminat ion method 11

• The sequence data were submitted to EMBL Nucleotide Sequence Database (accession# Y 131 66).

Results and Discussion Out of nine EMBL-3 recombinant c lones screened,

using eDNA in serts of pDUB6 and pDUB 8, e ight hybridised st rongly while one clone hybridised fa intly . The strong hybridisati on with these pe:1 legumin eDNA c lones indicated strong homology

.... 0 .c. ><

________ ___L_J L 2.75Kb

Fig. 1-0.8 kb and 0.5 kb DNA inserts, obtained from pARK3 used as probe for promoter ana lysis

SHASANY & KOUNDAL: LEGUMIN PROMOTER SEQUENCE CFROM CHICKPEA 365

between chickpea and pea legumin genes. DNA samples of the recombinant phages were isolated and restricted with Sal I. Two separate blots were Southern hybridised with inserts of pDUB6 and pDUB8 separately to reconfirm the presence of structural genes.

The variation in intensity of hybridisation signal between the clones may be due to the absence of full length fragment homologous to the probe. In addition, variation exists between the gene families and also within the family which can explain the difference in intensity of signal 12 described interclass homologies within a species as about 50-60% at the nucleotide level. Whereas intergeneric comparisons of a particular group on class show 70-80% homology 13

.

These homology studies led to put the Pisum leg A,

Vicia legumin A and Glycine glycinin genes in one group and Pisum leg J, Vicia legumin B and Glycine glycinin genes in another group 13

•

To confirm the presence of structural gene in the Sail fragmented clones (Fig. 2a) were again Southern hybridised with 0.5 kb and 0.8 kb probes described earlier (Fig 1). The clones B,D,F,I hybridised with 0.5 kb probe containing structural gene from N terminal (Fig. 2b), where as, clones D and F hybridised with 0.8 kb legumin promoter probe (Fig. 2c). This result substantiated the presence of promoter and structural gene in the insert of phage clone D and F and the phage clones B and I contain either a part or full structural gene. From the restriction and hybridisation pattern, assuming D and F are same, clone D was selected for further analysis. The phage clone was digested with a set of restriction enzymes singly and in combination and the hybridisation patterns were studied (Fig. 3a). A fragment of about 2.76 kb showed hybridisation (Fig. 3b, Lane 9) with 0.8 kb probe. This is the only fragment showing the signal from the restriction of cloneD with Pst I and Hind II when the other blot was hybridised with 0.5 kb probe the same 2.76 kb fragment showed the signal along with a 0.8 kb fragment (Fig. 3c). The hybridisation signal for 2.76 kb fragment was stronger in comparison to the other minor fragment. Therefore it was concluded that the 2.76 kb fragment contains the 5' upstream promoter region along with the part of structural gene. The structural gene part seemed to be less than 0.5 kb as indicated by an additional faint band in the autoradiogram.

Subcloning 2. 76 kb fragment containing promoter sequence-The genomic clone D was restricted with Pst I and Hind II to excise out the fragment. The desired fragment was eluted from low melting agarose gel and

Fig. 2b

IVf A ·a C 0 E F G H 1

Fig. 2c

Fig. 2-Restriction pattern of A.EMBL genomic clones with Sail. DNA samples from positive clones identified in tertary screening were restricted with Sal I, run in agarose gel and two separate blots were prepared. One of the blot was hybridized with 0.8 kb and the other with 0.5 kb labelled DNA probes. (A) Lane M

Marker A DNA Hind Ill digests . Lane A to I Genomic clones restricted with Sal I. (B) Autoradiogram of the blot A hybridized with 0.8 kb probe. (C) Autoradiogram of the blot A hybridized with 0.5 kb probe

366 INDIAN J EXP BIOL, APRIL2000

Fig. 3b

N I2345S- 7a -9

Fig. Jc

Fig. 3-Restriction pattern of 'A EMBL-3 genomic clone D restricted with different enzymes run in 0.8 agarose gel and southern blot hybridization. (A) Electrophoretic pattern of restricted genomic clones Lane N, Marker 'A DNA res tricted with Hind III and EcoR I Lane 1-9 clone DNA restricted with EcoRY Hind II , Hind Ill + EcoRI , Ora I, Xba I, BSP 106 1 PS+ I and Pst l + Hind II respect ively. (B) Autoradiogram of A hybridized with 0.8 kb probe. (C) Autorad iogram of A hybridized wit h 0 .5 kb probe

ligated to pBluescript II KS(-) restricted with Pst I and Eco RV. The E. coli DH Sa cell s were

transformed, plated on 2% Xgal (50 IJ.I ) and 0 .1 M IPTG ( IOIJ.I ) plate containing ampicillin and white recombinant colonies were isolated . Plasmids from these c lones were isolated, restricted with Pst I and Sal I, analysed in 0.8% agarose gel (Fig.4a) and Southern hybridi sed with 0 .8 kb promoter probe (Fig.4b). As seen in Fig. 4 all four clones hybridi sed with the probe indicating the presence of promoter sequence.

Unidirectional deletion by Exonuclease Ill-As described in the materials and methods the subclone

N ,J 23451\1-

Fig. 4b

Fig. 4-Agarose gel electrophoresis of subclone pA KK Ill DNA digested wi th Pst I and Sal I. (A) Lane N, Marker 'A D A, Hi nd IH and Eco RI diges t Lane I , pBiuescripl II KS(-) Lane 2-5 , pA KK Ill clone DNA . (8 ) Autoradiogram of A hybridized with 0 .8 kb probe

MN I 2 3 ~ 5 G 7 8 9

Fig. 5-Agarose gel electrophoresi s of undirec tional deletion of pA KKIII DNA with exo-nuclease Ill and S I nuclease. Lane M, Marker /..DN A Hind Ill di gest. Lane N, Marker /..DNA Hind III and EcoRI. Lane 1-9 Deleted subelones of pA KKK Ill , digest restri cted with Bss III


was digested with Kpn I and Hind II. Kpn I protects the vector from exonuclease III digestion where as Hind II produces blunt terminus at the insert end which is susceptible to exonuclease III. So a set of overlapping deletions were created for dideoxy sequencing (Fig. 5).

Sequencing of 2. 76 kb fragment (promoter)-A total of 2762 bases of the insert cloned in pBluescript II KS(-) were sequenced by sequencing and overlapping the deleted sequences. The complimentary strand was also sequenced to confirm the sequence data. The complete nucleotide sequence data (Fig. 6) and the restriction map derived from the sequence data (Fig. 7, pAKK III) depicts the consensus of the promoter and the amino acid sequences of a part of the structural gene .

Two Hind III sites were found at 34 and 2283 nucleotide position from the 5' end of the sequence. On further analysis of the nucleotides near the Hind III site, (at 2283 position) , an initiation codon (ATG) was detected (at 2277 position). The sequence was compared with the available sequences of the legumin genes and 5' upstream sequences of different !egA gene family of various crop plants like pea 14

·15

, LeA and Le B4 family of Viciafaba 16

.4 and glycinin group I genes of soyabean 17

.

Lycett et at. 3 reported a Hind III site near the start codon in pea legumin A gene. Accordingly sequence near Hind III site was compared with the leg A sequence of pea legumin . Based on the result ATG sequence and Hind III site near to it were numbered at + 36 and + 42 positions, while the position of Hind III site farther to it was numbered as -2203 nucleotide upstream.

Structural gene-The sequence of the legumin A gene of chickpea has been worked out previousll and the intron/exon splice junctions have been determined comparing with the boundary consensus sequences 18 and the nucleotide sequences of leg A gene of pea 14

• By comparing the initial nucleotides from ATG codon of the 2.762 kb insert in the sub clone pAKK III, with that of the pea leg A, the presence of 21 amino acid signal sequence was observed (Fig. 6) which shows typical features , starting with a positively charged amino acid, followed by a hydrophobic core, 13 tum creating

'd d . I 'd .. . 8 19 ?O rest ue an a stgna peptt e recogmt10n stte · ·- . Legumin storage proteins in pea are produced as precursor polypeptides which are cleaved at the cleavage site of signal peptide as well as at the a-13

polypeptide processing site21'22

• Similarly, in chickpea legumin protein was predicted to be 496 amino acid long including 21 amino acid transit peptide and the

MW of the protein was expected to be 53 KD with a and 13 peptides having molecular weight of 33 KD and 20 KD, respectivell.

In pea comparing the gene sequences with the sequences of several legumin cDNAs 14 the presence of three intervening sequences of 88 bp, 88bp, and 99 bp long were reported. The first two intervening sequences are present within the sequence encoding the a-subunit and the last one is present in the sequence encoding the 13-subunit. In chickpea8 we observed the presence of three short introns of 89, 74 and 229 bp long. These were located by comparing pea leg A and soyabean G I gene; and by comparing the intron-exon boundary sequences with consensus splice site sequences 18

• These introns were observed at the homologous position as in pea which indicates the phylogenetic relationship of these two species. All the splice site sequences obey GT/AG rule and show additional homology to the donor and acceptor conserved sequences derived from plant genes rather than animal genes2

·1. In the 2.762 kb fragment about

90 bp intervening sequence was observed at homologous position as IVS I of pea. When compared to the intervening sequences, the 90 bp intervening sequence was obtained at the same position. An extra T, at the stretch of 8 T increased the number to 90 bases from 89 as described earlier (Fig. 6). About 522 bases from the transcription start site was present in the clone. This shows about - 80% homology with the pea legumin sequences . Structural sequences of same position with chickpea legumin has been described earlier8

. It was similar except at some places like at +89 and + 115 position G bases are present instead of C, at + 154 position G is present in place of A; at +274 position T is deleted; at +303 position a G is inserted and finally a ' T' insertion inside the intron at +354 position (Fig. 8). In Fig. 8 the part of structural gene present in the subclone pAKK III has been compared with the part of structural legumin gene of chickpea8

. As predicted earlier 2.76 kb fragment includes less than 0.5 kb of the structural gene. About 479 bases are present from the Hind III site of the structural gene which is also near the ATG initiation codon .

Promoter sequences-Similarity in legumin gene and upstream regulatory sequences of various plants like pea, soybean and broad bean led to the belief that

10 20 )0 40 so 60 70 .so 90 100 -2241 CTGCAGCAGT TGGTTTCT.4.A GTATGMGAG TGG~ TCAAAGTTCA GGGGCACAAC TAACACAAAA TGCTGACACT GCCAAGCATG ATGCCGAAGC -2141 TGTTCAAAAC CCCAATGAM GATGTGAAGG TGATGAATCT GMGAGTTGG AAGCTGCTCA GAGTCTTGAT CGAAAACTGC CGAAAAAGTC AMCA(.,'CTT -2041 GAAGTGGATG GCAATGCAAC CAAGGGGGAT GGTCATGTTG AAACCAAMT AGTGGATAGC ACTGAAGAGA AGAACGGGGG AATCTAGCTT GAAGAGTAAT -1941 ACTACCGAAT AAGAMATTG AAGGTTGCAT GTTTCATTGT TTCCTTCCTA GCATTGTMT TTGTTATTGT CCATATCGTG TAGGCATGTA TTATTTATAA -1141 ATTAT!IACCT ATAGTTCCTT CTTCAAGGGT GATTATATCC CGAGGATTGT GACATTTAAT CCTTGTGAGA TCATGGTAGT TCATCATAAT GTTMTGTGA -1741 TTTTT'tCTTG TAAAAGCTCT CACTTTGTTC GTCTTCAGAT TAATAAGGCC TGTTTAGTGA TGGAGTACTT TATTATTTTA · rATTCCCTGA TTGGTTAMC -1641 CTACCATGTT CTGTGCGCAT CMTTTGCTA ATGTTTGATA CGCCTCGTCA CAAGCATTAG CCTAAATAAC ATGATCTATG CTTCATMTG GACGCMAGG -IS41 GACTGGGATT CTTCCCTTTT CTTTGGGAAC TTGTTTATGG- CAGATCGTGT GTAGAAGCTG GTTTGTCTTG TATTGTCTTT CGTTATAAAA AAATTTACAG -1441 GGCTATGTTT ATTAAMAAT TGACGAGTTC CGCAAATTGC CCTTCTGTCA CTCATCTTTT GTTTTTGTCA TGTTCATCTA CTATTMCCA AGTGGACGTA -1341 ATTTGTGCCA ATGGTTAATG TATTTGTGTT AAGATTGATG AATTGTTTGG GTACTATTTG ATTTGATGCC IIAGCTCTATG TATCGAATGA ACTTTAGATT -1241 ATCATGGTAA AAAGTAMTA GTTAAGATAA AAAAGTATTC ATCTMTTAG AMMMTTC AMTCCATCG ATAAAMCCG CTMCGTACA GTGTTTACAG

-1141 TTTTTCCCGC CAACTCTTCC CTCTTTGGTT TTTACMTGA TTGMCGG-GG TCCGAAATM TTTCTGTAGA ATCATI"TOAA GGM.AGGCAG ~-TTACACCCT -1041 TACATTTTAT GCAIIIIIIfo UCTCAATAC ATTGCTICTA CCTIIGAITA TTATTTATTT AAGTAGCAGT CGIIIIIIt.I ICTCAATACA ITGCUCIAC .-941 CITIGATTAT TATTTATTTA ~GAG GMAGAATTT CAGGTCCATG AATATCTATA AATAGGCCTA TTATCTGAGC TCAGGAIAAT TTAAAAATGC -141 MCGTTGATT GTGTTTGAGC GTGACATAGI TGGCGCTTGG CATTTTTAAA AGCTAGTTAA ATGGCTGGGA ATTCTAIATA IGTGCCAMT GMIAITGAT -741 GCAGAGTCCT TTATTCATTG ATTTCTACCC TTGTAATCCA TTGCTCACTA GTTCACTGAT TTAAGTTTAT CATTGATGAG GAAGMTTGG GGTGIAGCAG ~I TTGAAITGGG TAGATCTTTT ATACGTGGGA GAAGAGMM CATGGCATCA CCCAAATCAT AITCGAAGAA CTAAGITAAT ATGG-GTCACG GACCMITCI -541 ITGIAGTGAG TCAAGCACAT GTAITAAAAA CTTACGACCC OAMMATCT GAAAAGTTTA TCAATATATO GMCAAATTT TAMMAITG AAAITGATAT ~I ITCMAMGT GGTTAITAIT ACAITAGAIT ITGACTTAIT TAGAGTMAC TCAITITAAA TGTAAMAGT AAAATGTTTA TCAAGCACTA TGTAAITACA -341 CATATATTTT AAGGAITMT GITCTTCGTA AATCTTTGCA GATATGMA!i AGAGAAQCAG OOAAGAMAC AAA ... IGGAGC CATGGTCCCC CCICCCCCAC -241 CGATTTAMC TAATAATAAT GCCTAGGCAA GAAITGCMT ACAAACMGC ATGCITTITG ITGGAITATG GATAIGITli& M(ZCCACCTT GCTCTATCAG -141 ACATAGGTGT MTGCACAAG GCITCCATAC CCATGCMGC TGMGMTGT QMTGCTCA GCTCCACACC ITCTTTGACG IGICCATCCT TCGCCAACCT

~ · ·~ :!i A'T'T'rrCCO'.A It.t'd;I;",."'rr.•. CTTcrC.4.TTA AGfiTICTCCG OOCACAAAC CAACAT"rCTT TTCAATATCT GTCATCAIQ GCTMfJ.Cl!CITC'....C.ACTCTCT+60 MAKLLALS

CTT TCA ITT TGT TIT CIA CTT TIT GGT AGC TOT TTT GCT IT G AGA GAT CAA CCT GAA CM AAT GAG TGI CAG CTT GAA CAC CIC AA T GCC ClT GAA CCr GAT AAC L SF C F L L F G S C FA L R 0 Q P E Q N E C Q L E H L N ALE P 0 N

CGT ATA AAG TCA GAA GGT GGA CTT A IT GAG ACT IGG AAC CCA AGC AAC AAG CM TIC CGA TOT GCC GGT GIG GCC CIC ICT CGT GCT ACC CTT CM CCA AAT R I K S E G G · L I E T W N P S N K Q F R C A G V A L . S R A T L Q P N

TCCCICCGC AGA CCTITCIAC ACC AACGCT CCC CAG GM AITITC ATC CM CAA G al!Q;;TTATAI IQA n:ITAITAl:Aniiiiii6.Q.TAl:.ut.ITAlill:it.ni SLRJ.PFY TNAPQE IFIQQ ITATAI~~~~TAIIII~.ut.6.Q.An~GI~GGA~TTTGGC~GTAITCCCTGGTIGTGTT~~TTT~~CCACGT~

GNGYF GMVFPGMVETFE EPRE TCI GAA CM GGA GAG GGA AGC AAG TTT AGA GAC AGT CAC CM AAG OTT l' +S2S S E QG EGS KFRDSHQKV

Fig. 6-Promoter DNA sequence and downstream structural gene sequence of the 2.76 kb insert. The deduced amino acid sequence and the intron sequence (Dark boxes) are presented. Consensus sequences like TATA box, legumin box, AGGA boxes Adeno & SV 40 consensus sequences, 56 bp unidirect repeat, CAT, ATG sequences are underlined.

w 0'1 00

z 2 ;J> z '-tTl X '"0 c:l

0 r ;J> '"0 ;:o:l p N

§


the chickpea regulatory sequences will definitely have the consensus sequences as those found in the other species. The consensus sequence for the cap site in this gene was found to be comparable and in agreement with the LeB4, G I and Leg A genes. The Cap site was found to be 35 bp upstream to the start codon ATG (Fig. 6). Similarly a search of the sequence for the canonical subsequences observed at the S' end of most of the genes from animal source and legumin genes revealed a T A TA box at position -30. The consensus sequence is homologous to the TATA box of higher plants 17

• The TATA consensus was found to be 5' CCCTATAAATAACC 3' compared to 5' CTCTATAAATTACC 3' in pea leg A gene. Another upstream consensus found in animal is CAAT box, the homology in some plants are either poor or not found at all. So Messing et al ?3 proposed a new consensus called AGGA box. In the sequence under study at -161 position GAAAG sequence was found and a stretch of AG rich sequence was observed between -265 to -295 nucleotide position (Fig. 6).

The sequence motif responsible for expression of legumin gene in seed of different crop species are conserved. This conserved sequence of 28 bp are found between -89 to -I 16 for leg A gene of pea and at -90 to -117 position for G 1 gene of soybean. The conserved sequence for pea legumin is given as "TCCATAGCCA TGCAAGCTGCAGAATGTC"24

.

In the present fragment the sequence found at -89 to -117 nucleotide posttJon "TCCA TACCCA TGCAAGCTGAAGAATGTC" (Fig. 6). Except two nucleotides variation all the bases were same to that of pea legumin. The most conserved sequence "CA TGCAAG" is found in this region. These sequences in lectin and other seed protein genes are listed and called as RY repeats5

.

These consensus are responsible for expression in the seed25 and termed as legumin box .

When about 700 nucleotide sequences from Hind III site (at +42 bp position) were aligned (DNASIS programme) with the leg A upstream sequence of pea in the computer about 65% homology was observed. The comparison of 700 bases upstream from Hind III

~~~===~Y.ba I 0.68 pe I 0.68 am HI 0.69

Sma I 0.70

st I 0.70 pAKK Ill

5.72 Kb

Bgl II 3. 18 Hind Ill 2.QQ

ind Ill 0.74

mn I 0.61

ca I 1.27

Fig. ?-Restriction map of pAKKIII. Dashed area indicates the fragment sequence while dark part indicates the vector sequence indicating col E I ori gin and ampicillin resistance marker.


site of pea and chickpea promoter has been shown in Fig . 9. The homology was perfect near the Hind ill site, where the translation starts, which then gradually decreased towards upstream. Other strong homologies were observed in the legumin box (as described earlier), from -117 to -149 bp having similarity with the SV 40 consensus and Adeno virus enhancer sequence3

. All the consensus sequences including AGGA box like sequences at about -281 bp position (Fig. 6). As shown in Fig. 7 and Fig.9 the promoter portion can be separated out ( -2.25 kb) from pAKK ill and cloned inTi plasmid derived vector for tissue specific gene expression. Based on homology with plant promoters and the presence of consensus sequences like TATA, AGGA and legumin boxes the 2762 bp sequence was confirmed having a promoter sequence including the enhancer like elements in the

upstream. Study of foot pnntmg and gene actiVIty due to

mutations in these control regions will be useful 26.

Model plant system could be created by producing transgenic Tobacco and Petunia with different deletion constructs of the promoter. A number of storage protein genes have been transferred to tobacco and Petunia and shown to be tissue specific and developmentally regulated24

'26

. The regulatory motifs within the upstream sequence as in glycinin A2B 1 gene were found out by foot printing experiment involving the seed embryos fac tors27

'28

. Lessard et al. 29a1so identified the regulatory sequences of B-conglycilin gene of soyabean. The gene containing 159 nucleotides of the 5' flankin g sequence was expressed at a low level in immature embryo of Petunia. When the gene was flanked by 257

TCTCTTTCA TITTGTTITCTA CTITITGGT ACC TGTTITGCTTIG AGA GAT TCT CTI TCA TIT TGT TIT CT A CTT TIT GGT AQ£:; TGT TIT GCT TIG AGA GAT

S L S FC F L LF G S CF A L R D

CAA CCT CAA CAA AAT GAG TGT CAG CTT GAA CAC CTC AAT GCC CTT AAA CCT OAA CCT QM CAA AA T GAG TGT CAG CTI GAA CAC CTC AAT GCC CTT .GA.A CCT

Q P E Q N E C Q LE H L N ALE P

OAT AAC COT A TA AAG TCA GAA GGT GGA CTT A TT GAG ACT TGG AAC CCA AGC OAT AAC CGT ATA AAG TCA GAA GGT GGA CTT A TT GAG ACT TGG AAC CCA AGC

D N R IKS E GG LIE TWNP S

AAC AAG CAA TIC CGA TGT GCC GGT GTG GCC CTC TCT CGT GCT ACC CIT CAA AAC AAG CAA TIC CGA TGT GCC GGT GTG GCC ci·c TCT CGT GCT ACC CIT CAA

N KQFR C AGV ALSRA T L Q

OCA AA t TCC CTC CTG CAG ACC A TI CT A ACA CCA CGC TCC CCA GAA ATI TIC CCA AAT TCC CTC mG MiA Q:A :riC IAA .cAC .CAC ili:I .C.CC .cMi GAA A TI TIC

P N S L R R P F Y TN AP Q E IF

ATC CAA CAA G GT TCC TTA TAT TGA TCT TAT TAC ATT TTT TT- ACA TAC ATA Afc CAA CAA G GT TCC TTA TAT TGA TCT TAT TAC ATT TTT TIT ACA TAC ATA

I Q Q

ATG TTA TAT AGT AGTGGCTAA TATTTTGCTATAACAATT ACAGGT AATGGA ATGTTATATAGTAGTGGCTAA TATTTTGCT ATAACAATT ACAGGT AATGGA

G N G

TAT TIT GGC ATG GTA TIC CCT GGT TGT GTI GAG ACT TIT GAG GAG CCA CGT TAT TIT GGC ATG GTA TIC CCT GGT TGT GTI GAG ACT TIT GAG GAG CCA CGT

Y FG M VF P G CV E T FEE P R

GAA TCT GAACAA GGA GGA AGC AAG TIT AGA GAC AGT CAC CAA AAG GTI 3' +525 GAA TCT GAA CAA GGA GGA AGC AAG TIT AGA GAC AGT CAC CAA AAG GTI 3' +525

E S E Q G E S K F R D S H Q ·K V

Fig. &-Comparison of structural gene part in the isolated sequence with the corresponding part of structural gene of chickpea reported earlier.

SHASANY & KOUNDAL: LEGUMIN PROMOTER SEQUENCE CFROM CHICKPEA

GGGGT?GTAGCAGT?TGAAT?TGGGTA?GA?T??CT?J?ITATACGTGGGAGAA?GAGAAAA CP ITCGT?CCAITCTTTCTCTGGAAATTACTCCCTATGIT?TA??T???AGAAITTGAAAA P

.c ?AJGGCA?TCACCCAA? ?ATCA ?TAITCGAA ?G?AACTAAGITAATATGG ?GTC? ?A ?CGG CP CITITG?AGT?A???AAITAGCACT?IT?AAATGTAA??AAG?T?AT ?TGGCATCTTATC?A P

ACCAAITC? TTTGTAGTGAGTCAA? ?GCACATGTAIT?A?? ?A ?AAACITACGACCCGAA ?? CP AACAA ?CCGGITG ? A ?TGA ? ? AAA TTTCACA T ?T ?ITCAGGTAGTAA ? ?TA TGA ? ? ? ? AA IT P

? AAAATCTGAAAAGTTTATCAATA?TATGGAACAAATTTTAAA?A?AAITGAAATTGATAIT CP GATAA? ?TGGAAAGATGAT? ?ATAGTAT ?TAA? TAA?? ?TAAATATATTTGAAA ?AGAT~?? P

TCAA? AAA? GTGGITAT ?TAT ?TACA ?IT?? ?AGAIT?ITGACITATTTAGAGT? ?AAAC?? CP ACAA TAAA TGT? A IT A TA TCTA TAAA TTT ACAAGGITCIT? A ??TA TTT ACA ?T ACAAACAA P

? T? CA TTTT AAA TG? ?T? AAA ? AAGT A ? ? ? A? ? AA TGTTT A TCAAGCAC? ?T ? A ? ?TGT A ? ? CP ATGCA??GT?AATGTTTCAAACAA?TATGCAGTAA?G?TAAITAA?CACTTTAAITTGAAGG P

A ITACA ?CATATAIT? ? T?? ? T ?AAG?? G? ?? AIT? AATG?ITCITC? ?GTAAATC? ?T ?T? CP AITA?ATCA?AT?ITGGTAACTGAAGTAGCTAAITGAAAGTTTAITCITATAAATCITTGTA P

?TGCAG?ATATGAAAGAGAGAAGCAGGGAAG?AAAACAA??AATGGAGCCATGGTCCCCCCT CP ATGCAGAATATGIAAGAAAGAAACATGGL&QTATAAGAAGTAA???AGCCATGGT? ?CCCCT P

CCCCCACCGATITAAACTAATAATAATGCCTAGGCAAGAAITGCAATACAAACAAGCATGCT CP ?? GCCACCGATTT???C????? ???A?G?CTA? ?TAAGAAITG??????? ??CAAGTATGCT P

TTTTGT ?TGGA IT A TGGA T A TGITQ 2 A'J.1 1 A'J. .'ZAQC.CACCITGCTCT A TCAGACA T A.QQ.IQI CP CTTTGTCTGG? T AA TGGAGA TGA TGAAGCCA ITAGCCACC?TCCTCTA TCAGACA TAQQ.IQI P

AATGCACA ??AGGCITCCATACCCATGCAAGCTGAAGAATGTCTAATGCTCAGC?TCCACAC CP AAAGCA ?AITATGCITCCATAGCCATGCAAGCIGCAGAATGICCAATTCICAACAICC?CAC P

CITCTTTGACGTGTCCATCCITCGCCAACCTATTTTCCCIAIAAAIAACCGCITCTCATTAA CP TTTCAATGACGTGTCCAACCITCACCACCCTCTCITCICTATAAAITACCACIICTCAIIAA P

+I +36 GGITCICCGCAJCACAAACCAACAITCTTTTCAAIATCTGRCATCAIQGCTAAGCIT3' CP GGITCTCCGCAICAC ? AACCAACA ITCTCTI ? AGT A TCTCTCITCAIQGCT AAGCTI 3' P

Fig 9-Compari son pea legu min promoter and isolated chickpea promoter CP: Chickpea. P: Pea

371

nucleotides of the upstream region the level of expression increased at least 20 fold. Analysis of the sequence spacing the nucleotides 159 to 257 revealed four repeats of a 7 base pair GC rich sequence (AGCCACA), thi s play an important part in determining the level of express ion of the gene in transgenic plants. The functional significance of such sequences needs to be establi shed by mutat iona l analysis in gene transfer rnethods30

. Such approaches will bring about the most rapid progress towards understanding plant development in molecular terms .

introduced genes, in a given ti ssue at a specific time, ti ssue specific promoter such as the above described one will be of immense help for chickpea improvement which is a major crop of India.

As for majority of the transformation experiments and those cases where the transgenic pl ants have been produced, the promoter used has been a derivative of CaMV 35S. In monocots, the promoters used has been either a deri vative of actin or ubiquitin . Most of the promoter to a very large extent are constitutive in nature. In order to ensure the express ion of the

Acknowledgement The authors are grateful to the Project Director,

NRCPB for provid ing the fac ilities and Prof Donald Boulter, Univ. of Durham, UK for providing the legumjn eDNA c lones.Thanks are also due to Mandaokar, Lekha, Sangha, Rekha, and Gupta for providing help. The financial assistance from ICAR, CSIR, New Delhi in the form of fe llowship to AKS is also acknowledged .

References l Goldberg, R.B . Regulation of plant gene expression . Phi/s.

Trans. Royal Sor. Lon d. B. Bioi. Sci. 314 ( 1986) 343 .


2 Casey, R., Domoney C & Ell is N. Legumin storage proteins and their genes. In : Oxford Survey of Plant Molecular and Cell Biology Eds. Vol. 3: (1986) 1-95.

3 Lycett, G.W., Croy, R.R.D, Shirsat, A.H, Richards, D.M & Boulter D. The 5' regions of three pea legu min genes. Comparison of DNA sequences. Nucl. Acids Res, 13 (1985) 6733 ..

4 Baumlein, H., Wobus, U, Pustell, J & Kafatos, F.C. The legumin gene family : Structure of a 13-type gene of Vicia faba and possible legumin gene specific regulatory element ,. Nucl. Acids. Res, 14 (1986) 2707.

5 Dick inson, C.D., Evan, R.P. & Nielsen, N.C. RY repeats are conserved in the 5'-tlankeing regions of legume seed protein genes. Nuc/. Acids Res ,. 16 ( 1988) 37 1.

6 Koundal, K.R ., Mehta, S.L & Sharma, R.P. Construction of chickpea genomic library. In dian 1 Exp. Rio/, 27 ( 1989) 858.

7 Lycett, G.W., Delauney, A.J , Zhao, W, Gatehouse, J.A, Croy, R.R.D & Boulter, D Two eDNA clones coding for the legu min protein of Pi sum sativum L. contain sequence repeats. Plant Mol. Bioi, 3 ( 1984) 9 1.

8 Mandaokar, A.D. Cloning and characterisation of storage protein genes in chickpea (Cicer arietinum L. ). Ph.D. Thesis, Division of Molecular Biology and Biotechno logy. IARI, New Delhi -110012 (1994).

9 Sambrook, J., E.F. Fritsch and T. Maniatis: Molecu lar Cloning: A laboratory manual. Cold Spring Harbour Laboratory Press, New York ( 1989) .

I 0 Stephen, D., Jones, C & Schofield , J.P. A rapid method for isolating hi gh quality plasmid DNA suitab le fo r sequencing. Nucl. Acids Res, 18 ( 1990) 7463 .

II Sanger, F., Ni cklen , S & Coulsen , A.R. DNA sequencing with chain terminating inhibitors. Proc. Nat/. Acad. Sci.(USA), 74 (1977) 5463.

12 Domoney, C. , Ell is, T.H .N & Davies, D.R. Organi sation and mapping of legumin genes in Pi sum. Mol. Gen. Genet, 202 (1986 ) 280.

13 Casey, R. & Domoney, C. The st ructure of storage protein genes. Plant Moi.Biol. Rep, 5 ( 1987) 261.

14 Lycett, G.W., Croy, R.R.D., Shirsat, A.H. & Boulter, D. The complete nucleotide sequence of legumin gene from pea ( Pisum sativum). Nuc/. Acids Res, 12 ( 1984) 4493.

15 Bown, D., Elli s, T.H.N & Gatehouse, J.A. The sequence of a gene encoding convicilin from Pea ( Piswn sativum L.) shows that convicilin difters from vicilin by an insertion near the N terminus. Biochem 1, 251 ( 1988) 717.

16 Schlesier, B., Bassuner, R., Hai, N.Y. & Muntz, K. The eDNA derived primary structure of two distinct legumin as

sub unit precursors from tield bean (Y icia fab a L.). Nuc/. Acids Res, 18 ( 1990) 7146.

17 Nielsen, N.C. , Dickinson, C.D, Sho, T.J, Thonh , Y.H, Scallon , B.J , Fischer, R.L, Sims, T.L., Drews, G.N & Goldberg, R.B. Characterisation of glycinin gene family in soyabean. Plant Cell, I ( 1989) 3 13.

18 Brown J.W.S. : A catalogue of splice junctions and putative branch point sequences fro m plant introns . Nucl. Acids Res, 14 (1986) 9549.

19 IPerlman, D. & Halvorson, H.O. A putative signal peptide recognition site and sequence in eukaryotic and prokaryotic signal peptides. 1. Mol. Bioi, 167 ( 1983) 39 1.

20 Von Heijne. Signal sequences the li mit of vari ation. 1. Mol. Bioi, 184 (1985) 99.

21 Croy, R.R.D., Gatehouse, J.A., Evans, l.M . & Boulter,D. Characteri sat ion of the storage protein subunits synthesised in vi tro by polyribosomes and mRNA from developing pea (Pisum sativum L.) liS Vi cillin . Plan/a, 148 ( 1980) 57 .

22 Higgins, T.J .Y. Synthesis and regu lation of major proteins in seeds. Ann. Rev. Plant Physio /, 35 (1984) 19 1.

23 Messi ng, J., Geraghty, D., Heidecker, G. , Hu , N.T, Kridl, J & Rubenstein , I. Plant gene structure, In : Genetic Engineering of Plants (Plenum Press, New York), 1983 , 22 1. ·

24 Shirsat, A., Wilfold , N, Croy, R &. Boulter, D. Sequences responsible for the ti ssue specific promoter activity of a pea legumin gene in tobacco. Mol. Gen. Genet, 2 15 ( 1989) 326.

25 Lelievre, J.M ., Oliveira, L.O & Nielsen , N.C. 5'CATGCAT-3' elements modulate expression of glycinin genes. Plant Physiol, 98 (1992) 387.

26 Baumlein, H. , Boerjan, W, Nagy, I, Panitz, R, Inzc, D & Wobus, U. Upstream sequences regu lat ing legumin gene expression in heterologous transgeni c plants. Mol. Gen. Genet, 225 (1991) 121.

27 ltoh , Y., Kitamura, Y, & IFukazawa, C. The glycinin box: a soyabean embryo fac tor binding motif within the quantitative •egulatory region of the I I S storage globulin promoter. Mol. Gen. Genet, 243 (1994) 353.

28 ltoh, Y., Kitamura, Y, Arahira, M & Fukazawa, C. Cis-acting regulat ing regions of soyabean seed storage II S globulin gene and their interaction with seed embryo fac tors. Plant Mol. Bioi, 2 1 (1993) 973.

29 Lessard, P.A., Allen , R.D, Fujiwara, T & Beachy, R.N . Upstream regulatory sequences from two beta-conglycinin genes. Plant Mol. Bioi, 22 ( 1993) 873.

30 Meakin , P.J . & Gatehouse, J.A. Interacti on of seed nuclear protein with transcriptionally enhancing regions of pea ( Piswn sativum L) Leg A gene promoter. Plan a, 183 ( 199 1) 471.

isolation and characterisation of legumin promoter sequence...

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