Indian Journal of Experimental Biology Vol. 37, November 1999, pp. 1117- 1122

Isolation and analysis of genomic clones coding for legumin storage proteins and their copy number determination in chickpea, Cicer Cl;rietinum L.

Le kha D Kumar, A D M andao ka r & K R Ko unda l

National Research Centre, Plant Biotechnology, Indian Agricultural Research In stitute, New Delhi 110012, India

Received 8 March 1999; revised 28 June 1999

The genes encoding legumin storage proteins of chickpea were identified initiall y by Southern blot hybrid izatio n of restricted total genomic DN A of chickpea with pea legumin cDNA probes (pDUB6 and pDUB8) . Since these probes showed strong homology to chi ckpea DNA, they were used for screening chickpea genomic library to isolate and ascertain the positi ve clones. Confirmation and detailed analysis of these clones were then done by restricti on analys is and Sout hern hybridization. The gene copy number of legumin was es timated and found to be 10 to 12 in chickpea genome.

Chickpea is the major pul se crop in India and provides a substantial portion of the dietary protein . But unfortunately, like most legumes, chickpea seed storage proteins being deficient in sulphur containing amino acids, methionine and cystein suffer from an essential amino acid imbalance. Thus, improvement of nutritional quality of these proteins is very essential. Based on their sedimentation coefficient storage proteins of legumes are classified into two major groups i.e. lIS and 7S, otherwise known as legumin and vicilin respectively. These proteins are salt soluble, and deficient in sulphur containing amino acids' and synthesized by multi gene family . The advent of recombinant DNA technology has made it possible to isolate storage protein genes and transfer it across species barriers. The isolation of genes are now possible by several ways . The most commonly used approach is by constructing and screening of genomic libraries using suitable probes2.

Materials and Methods Plant material-Seeds of chickpea (Cicer

arietinum L.) var. 256 were surface sterilized using 0.01 % mercuric chloride and germinated on paper towels in dark. A week old etiolated seedlings were harvested, frozen in liquid N2 and used for the study .

Isolation and southern blot hybridization of genomic DNA-Chickpea genomic DNA was isolated from 7 days old etiolated seedlings according to the modified protocol3

. Genomic DNA was further purified by CsCh gradient (0.90g/mJ of DNA)

ultracentrifugation in Vti 65 rotor at 60,000 rpm for 16 hr at 20°e. The DNA band was collec ted and dialysed against TE buffer ( I OmM Tri s, I mM EDT A,

pH S.O) at 4°C overnight and precipitated using 0.1 volume of 3M sodium acetate and 2 volumes of ethanol. The yield of pure DNA was es timated by taking absorbance at 260 nm. Isolated chickpea genomic DNA (10 flg) was restricted with the independent sets of enzymes, each contallllllg BamHI, EcoRI, EcoRV, HindIII, Pspl, Pstl and Sau3A. The restricted samples were e lectrophoresed on 0 .7% agarose gel blotted on to gene screen plus filters. The membranes were hybridi zed using pea legumin cDNA probes, pDUB6 (leg6) and pDUB S (legS) separately (obtained from Prof. Donald Boulter, Department of Biological Sc iences, University of Durham, UK). The hybridi zati on was carried out at 65°C overnight. The membranes were washed with 2xSSC, I xSSC and 0.5xSSC all with 0.1 % SDS at 65°e. The washed filt ers were ex posed to X-ray film in dark for 2 days and film was developed using Kodak developer and fixer.

Isolation of genomic clones from chickpea genomic library - The chickpea genomic library constructed earlier4 was amplified to get a titre va lue of 1.1 x 106

pfu/fll. A total number of 5x 105 plaques were screened at a time for each of the probes . The plaques were lifted on to nitro-cellulose membrane filters and screened using radiolabe lled pea legumin cDNA probes (pDUB6 and pDUBS) separate ly. The hybridization and washings were carried out as

1118 INDIAN J EXP BIOL, NOVEMBER 1999

described for the Southern blot hybridization of genomic DNA. Sixteen plaques lighted up by screening filters with leg8 and 12 plaques were identified using leg6. To separate out the positive clones further from the mixture of primary clones, secondary and tertiary screenings were done. These well separated clones from the secondary screening were ultimately spotted in duplicates on to the gene screen plus membrane di scs and probed separately with pDUB6 and pDUB8 probes.

Analysis of the legumin genomic clones by Southern hybridization - In order to confirm the presence of legumin gene, the recombinant phage DNA was isolated from all the clones which showed hybridization with both the probes and were subjected to further ana lysis by restriction digestion and Southern analysis. All the clones showed a complex restriction pattern when digested with Sail. The restricted samples separated on 0.7% agarose gel in duplicates gave five to six bands in different clones suggesting internal sites for SaIl. These blots were separately hybridized to pD UB6 and pDBU8 cDNA probes .

Estimation of gene copy nU1l1ber of legumin ill chickpea-Total genomic DNA ( 10 flg) of ch ickpea was digested separately with EcoRV, HindIII and EcoRI enzymes. The gene copy equivalent to 10 fl g of chickpea DNA (C= 1.75 pg) was est imated and electrophoresed on 0.7% agarose gel. DNA was then blotted on nylon membrane and hybridized with 1.5 kb EcoRV/XhoI DNA fragment of chickpea legumin gene, leg35

. The intensity of hybridization was compared with known amollnt of legumin DNA using gel scanner.

Results and Discussion Identificalion of legumin sequences in chickpea

using pea legumin cDNA probes - Genomic DNA of chickpea restricted with di fferent set of enzymes and hybridized separately to pea legumin cDNA probes, pDUB6 and pDUB 8 (Fig. I ) showed differential hybridization with leg 8 (Fig. 2) and leg 6 (Fig.3). Though the probes were heterologous, they showed very strong hybridi zation with 1-2 kb and 4-5 kb fragments of genomic DNA suggesting strong homology with chickpea genomic DNA . Such kind of intergenic homologies have been shown in other legume genera like Vicia faba and Glycine max6

.

These prooes were therefore, used for screening the genomic library of chickpea.

Isolation of legumin genomic clones from chickpea genomic library-Chickpea genomi c library constructed in EMBL3 vector was used for screen ing for legumin gene4

. Considering the insert size of 15-20 kb, approximately 5x I 05 independent recombinants from the genomic li brary have been screened in order to have a resonab le chance of including the desired sequences taki ng into accou nt that legumjn storage protein genes belong to multigene family . Hybridi zati on with leg6 and leg8 separately showed a total of 10 positive clones . These 10 clones were then spotted in dup licate and hybridized separate ly to both the probes . All the 10

2

Fig I- Agarose gel electrophorcsi s of purified pca legumin pDUB6 and pDUB8 Lane 1- pDUB6 cut with BamHI. lanc 2- pD UB ell! with B am HI. Lower bands indicate the pea legumin cDN A insert s.

8 765432

A 8

Fig 2A-Agarosc gel e/t:ctrophorcsis of chickpc<1 ~cn()m ic DNA digested with vari ous rcstri etion cnLymcs. Lanc~ 1-7 indi catc the digcs ti on of DNA wi th BamHI. BgllI. EeoRI. Hind li l. Msp l. p,t l. Sau3A I respecti vcly. lanc 8 shows uncut DNA. B. Southern Blot hybridisa!ion of ge l shuwn ill A probed with pDUB8 probe.

KUMAR el af.: GENOMIC CLONES FOR LEGUMIN STORAGE PROTEINS; CHARACTERIZATION II 19

clones hybridized with leg8 (Fig. 4A) and designated as L01 to LOIO. However, when hybridized with leg6 cDNA probe only 6 out of 10 clones showed positive signal (Fig. 48) and these were designated as LO 1 to LD6. This indicated that these clones carry sequences homologus to the sequences encoding the ~-subunit of the legumin polypeptide of pea and since leg8 (which codes for a subunit of legumin) also hybridized to these clones suggesting that LD I to LD6 contain at least one full length gene sequence. The clones L07 to LO to showed strong hybridization with leg8 probe but did not show any hybridization with leg6 probe. This indicated that these clones carry sequences homologous to only 5' end of the legumin gene (encoding a subunit) and not to 3' end of the gene (encoding B subunits). The presence of such

987654321M 9876543Z1M

A 8

Fig 3-Agarose gel electrophoresis of chickpea genomic DNA digested with various restriction enzymes (A) and Southern blot hybridisation (B) with pDUB6 probe. Lanes 1-8 indicate DNA restrict ion with BamHI, EcoRI, EcoRV, Hindlll , Hinfl, Mspl , Pstl, Sau3AI respectively. Lane 9 -undigested DNA, M - Molecular weight marker "-DNA digested with Hindll!.

Fig 4-Autoradiogram showing terti ary screening of chickpea genomic library probed with pDUB6 (A) and pDUB8 (B).

intergeneric homology has been demonstrated between other legume genera like Pisum and GlycineX

and also between Vicia and Glycine. It would be interesting to know the extent of homology between legumin genes of chickpea and the rest of other legume species for which the sequences are known . This can give an insight into their phytogenetic relationship and the position of eicer in the evolutionary tree with reference to other legumes.

Analysis of legumin genomic clones by Southern hybridization - In order to confirm the presence of legumin gene, the recombinant phage DNA was isolated from all the six clones (LD I to LD6) which showed hybridization with both the probes and were subjected to further analysis . The recombinant phage DNA of all the six clones showed a complex restnctlOn pattern when di gested with Sail, suggesting that there are some internal sites of Sail in the genomic insert and Southern hybridizati on with pea legumin probe indicates that some of these clones contain more than one legumin gene.

The clone LD 1, LD2 and LD3 upon restriction with Sail gave different restriction pattern (Fig. 5A), indicating that these three clones are different from each other. Southern hybridization with legumin cDNA (Leg6) probe also showed the different hybridization pattern of these clones (Fig. 58). The LD I clone showed the presence of single hybridi zing band (Fig. 58 lane 1) indicating the presence of legumin gene in large fragment. Clone LD3 also showed hybridization at single fragment of about 9 kb (Fig. 58, lane 3) . Interestingly, clone LD2 showed hybridization at 3 bands of 9, 5 and I kb (Fig. 68 lane 2). As these fragments can not be the part of single gene and therefore at least two legu min genes must be presented in LD2 clone. The less intensity of 5 and I kb fragments could be because of the presence of internal site within a gene. The intensity differences between 9 kb and these two fragments also suggest the presence of legumin genes from different family in this clone.

Domoney et al.9 described interc lass homologies within a species at about 50-60% at the nec leotide level. In Phaseolus vulgaris the genomi c members of phaseolin gene family are very c losely re lated with 71 % to 95% homolog/ D. Genetic evidence which showed that phaseolin genes are closely linked led them to suggest that duplications and other events must have led to the formation of phaseolin multigene

1120 INDIAN J EXP BIOL, NOVEMBER 1999

family. In Pisum sativum also, the genes coding for legA and legJ which belong to different classes of legumin share sequence homolog/ ' . In Vida faba legumin A and B subfamilies were found to be recognizably 50% homologou s'2, though they showed considerable sequence divergence at the amino acid level.

In case of LO I and LD3 the number of genes cannot be assessed as there is only single hybridizing band . Since intensity in those bands is high it could be possible that more than one gene is present in these two clones as legumin genes are present in clusters's. This possibility can be tested by digesting these clones with different restriction enzymes and then hybridizing with legumin probe.

The other three clones, LD4, LD5 and LD6 were

lA 2A

18 28

Fig 5-Agarose gel electrophoresis of DNA iso lated from three genomic clones and diges ted wi th Sail (A) and Southern hybridisati on wi th pea legumin pDUB6 probe (8 ).

restricted with Sa il and electrophoresed on agarose gel (Fig. 6, 1 A and I B). The clone LOS was found to be different from the L04 and LD6 whereas these two clones showed identical restrict ion pattern . To have further insight these restricted c lones were blotted on hybond membrane and hybridized separately with leg8 and leg6. As shown in Fi g 68 , these three clones have differential hybr idi zation pattern. Though the restriction pattern of LD4 and LD6 was similar (Fig. 6A) hybridi zat ion with e ither of the probes gave different pattern indi cat ing that all the three clones are different. Hybridi zati on of these three clones with both the probes gave s imil ar pattern . Since leg8 and leg6 codes for 5' and 3' end of legumin polypeptide respect ive ly and hybr id ization of both the probes to same restriction fragment suggests the presence of complete legumin gene(s) in a ll the three clones. The detailed characteri zation of these fragments with sequencing will reveal the exact nature .of the legumin genes.

Estimation of gene copies of legumin ill chickpea -Total genomic DNA of c hickpea di gested with restriction endonucleases and gene copy equivalent to 10 I1g of DNA is shown in Fig. 7a. This DNA was then blotted and hybridized with 1.5 kb fragment of leg3 gene (Fig. 7b) . The probe hybrid ized to number of restriction fragments of c hickpea genomic D A and intensity of bands indicates the presence of family of legumin genes. The intensity of

3 2

A B

Fig 6-Agarose gel e lectrophoresis o f DNA iso lated from genomic clones and d iges ted wit h Sail ( I A & 21\ ) and outhcrn hybrid isati on with pea legumin eDNA prohe pD UI3X (18 ) and pDU86 (28) .

KUMAR el al.: GENOMIC CLONES FOR LEGUMI STORAGE PROTEINS: CHARACTERIZATION 11 2 1

A

Fig 7-Estimation of gene copy number of chi ckpea legumin

B

1234567 8 •• _, ____ ~_., ......... _._. ____ ... ___ ~ • ________ .. ft.

A. Agarose gel electrophoresis of restricted chickpea genomic DNA and gene copy number equ ivalents. Lanes 1-5 are loaded with 3.7, 7.5, 15 .0,30.0 and 45.0 pg of leg3 insert DNA (corresponding tu 0.5, 1,2, 4 and 6 gene copies in I 0 ~g of genomic DNA). Lane 6, 7 & 8 shows the digesti on of chickpea genomic DNA with EcoRV , HindIII , and EcoR I respeCli ve ly. B. Autoradiogram of a gel shown in A, hybridi zed with chickpea legumin (Ieg3) probe.

hybridization was compared with known amount of legumin DNA using gel scanner. The number of genes thus corresponds to 10-1 2 copies in chickpea genome.

The copy number obtained in thi s study differs with those available for Glycine max where five copies of glycinin ( II S) protein gene have been estimated '6 . However, the resu lts presented here is in good agreement with those publi shed for peal7 in which eight legumin genes per haploid genome have been reported . Legumin genes are classi fied into class I and class II family and high degree of sequence homology wi thin the class and cons iderable degree of homology between the classes have been reported9

.

This suggests the presence of conserved sequences among legumin genes and thus 1.5 kb fragment of leg3 which belongs to class I gene fami Iy could also have hybridized to class II gene family of chickpea. Therefore, 10-12 copies of chickpea legumin genes obtained in th is study might be the members of both class I and class II famil ies of chickpea. Further iso lation and characterization of genes from class II family will give complete picture of legumin genes of chickpea.

The organization of seed storage protein genes is si milar to that of other plant genes l4

. These genes occur singly and in clusters on the :nme as well as on

different chromosomes as has been reported in Vicio Jaba 's . Hence the genomic clones analysed in this study may contain complete gene or similar lin ked legumin genes in a single clone. Additional stud y may provide further insight to establi sh their structural and functional relationship.

Acknowledgement The authors are grateful to Dr R.P. Sharma. Project

Director, NRCPB for providing the encou ragement and fac ilities to carry out thi s research.

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