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Open Access
Development of resistance against banana bract mosaic virus in musa spp. var. grand naine using small interfering RNA (siRNA)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2019) Jadhav Pritam, Ramesh; KAU; Soni, K B
The study entitled “Development of resistance against Banana bract mosaic virus in Musa spp. var. Grand Naine using small interfering RNA (siRNA)” was carried out during 2015-2019 in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective of the study was to develop resistance against Banana bract mosaic virus in banana var. ‘Grand Naine’ using siRNA mediated technology. Embryogenic calli of banana var. Grand Naine were used for agrobacterium mediated transformation of ihpRNA cassette as the embryogenic cells have single-cell origin and do not produce chimeric plants. The rapid and efficient protocol standardised for somatic embryogenesis in Nendran developed in the Department of Plant Biotechnology, College of Agriculture, Vellayani was followed for inducing somatic embryogenesis in banana var. Grand Naine. Immature male flowers from position 0 to 11 were used as explants. Pale white embryogenic callus was initiated within 45 days on Murashige and Skoog (MS) medium supplemented with 6-Benzyladenine (BA) (8 mgL-1) and Thidiazuron (TDZ) (0.6 mgL-1) under dark conditions. The percentage of embryogenic calli obtained was 13.88. For regeneration of somatic embryo, the embryogenic calli were transferred to MS medium supplemented with BA (2 mgL-1) and incubated under light (16 hr), which resulted in 100 per cent germination and plantlet regeneration. The plantlets were transferred to coir pith compost and were hardened for one month in mist chamber. Plants were then transferred to polybags with soil and cow dung (1:1) mixture and kept in shade net house for secondary hardening. An intron hairpin RNA (ihpRNA) vector was constructed to produce small interfering RNA (siRNA) against the coat protein gene of BBrMV. The construct was designed using the partial coat protein gene sequence of BBrMV isolated from the infected banana var. Grand Naine. The coat protein gene was amplified from the cDNA prepared from the total RNA isolated from the infected plants by RT-PCR. For this coat protein gene specific primers were designed using the whole genome sequence of BBrMV retrieved from NCBI GenBank. The partially amplified coat protein gene fragment of 745bp was sequenced and analysed using BLASTn tool for similarity with the sequences of BBrMV deposited in NCBI genome database. Sequence was closely related to BBrMV infecting cardamom with 97.83 percent similarity. The sequence was subjected to miRNA target prediction for selection of the target site to be used in preparation of ihpRNA construct. The sequence that frequently occurred in probable dicer substrate sites was selected. It was then checked for the presence of restriction sites of AscI, PacI, KpnI and SpeI as these sites were the cloning sites of the primary vector, pSTARLING. Based on this information the primers with anchored restriction sites were designed to amplify a fragment of 326bp towards the 5’ end. The sense fragment of coat protein gene (326bp) was amplified with the primers anchored with AscI and PacI sites and the antisense fragment (326bp) was amplified with the primers having KpnI and SpeI sites. The respective restriction sites were anchored at 5’ end of forward primer and 3’ end of reverse primer. The use of two restriction sites helped in proper orientation of sense and antisense strand in ihpRNA construct. The amplified sense and antisense fragments were eluted from agarose gel and cloned in pJET1.2 cloning vector. The cloned fragments were released with sticky ends from pJET1.2 using the corresponding restriction enzymes and integrated in pSTARLING vector flanking the cre intron to favor the formation of the hairpin structure. Presence of the inserts was confirmed by restriction digestion and PCR. The ihpRNA casette in pSTARLING consisted of ubiquitin promoter, ubiquitin intron, sense coat protein strand, cre intron, antisense coat protein strand and termination sequence in the order within the NotI restriction sites. For agrobacterium mediated transformation, the complete cassette was released using NotI and ligated at NotI site within the lacZ gene of the binary vector pART27 containing antibiotic resistance markers nptII and Spec. Integration of cassette within lacZ gene facilitated the selection of the binary vector with the ihpRNA cassette by blue-white screening. The white positive colonies were confirmed with PCR. The binary vector with the insert was transferred to Agrobacterium tumefaciens strain GV 3103 by freeze-thaw method. Transformed colonies were picked and the presence of the vector and the ihpRNA insert was confirmed by PCR and restriction digestion. Embryogenic calli were transformed with A. tumefaciens strain GV 3103 carrying the ihpRNA construct and the transformed embryos were selected under antibiotic pressure (kanamycin 200 mg L-1). Transformed calli were transferred on MS medium containing 2 mg L-1 BA which gave a maximum regeneration of 12 percent. The regenerants were confirmed for the presence of ihpRNA construct using PCR with the primers for insert, npt II and cre intron. The formation of siRNA in the transformed plant was analysed using Northern hybridisation. The small RNAs were isolated from the leaves of transformed plants and separated on 15 percent acrylamide gel containing 7M urea and electroblotted on the positively charged nylon membrane. It was then subjected to hybridization with biotin labelled probes designed against the target site. The small RNAs were detected after DAB staining. The study was successful in developing ihpRNA construct for resistance against BBrMV in Musa spp. var. Grand Naine. This is the first report on development of ihpRNA construct for siRNA mediated resistance against BBrMV. The transgenics developed in this study need to be evaluated for virus resistance by challenging with viruliferous aphids. The technology developed in this study can be applied in other banana varieties also for imparting virus resistance. Compared to other recombinant DNA techniques, RNAi offers specificity and efficacy in gene silencing. Since the small gene fragment used for construct preparation do not code for any protein, this technology does not arise much biosafety concerns. In banana, where traditional breeding for virus resistance is very difficult, this technology is a promising alternative.
Open Access
Metabolite Profiling and gene expression analysis for gingerol production in selected somaclones of ginger (zingiber officinale rosc.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Sreeja, S; KAU; Shylaja, M R
Ginger (Zingiber officinale Rosc.) is one of the oldest known spices and is much valued for its medicinal properties. Ginger rhizome is a source of active biological compounds which are responsible for its medicinal properties. Gingerol is the major pungent polyphenol in ginger which has got a wide array of pharmacological properties. Research on somaclonal variation done at Kerala Agricultural University could locate ginger somaclones with high gingerol content and in the course of investigations high somaclonal variation was observed for quality components. Ginger has a very big genome of 23,618 Mbp which is little exploited and reports on the genomic base of gingerol biosynthesis are scanty. The present investigations hence aim at profiling the metabolites in selected ginger somaclones using high throughput analytical platforms and to analyze the gene expression with respect to gingerol production. One released ginger variety from KAU (Athira), two selected ginger somaclones (B3 and 132M) and parent cultivar (Maran) formed the experimental materials for the study. Studies were carried out at Centre for Plant Biotechnology and Molecular Biology, Distributed Information Centre of College of Horticulture and Arjuna Natural Extracts Pvt. Ltd., Aluva during August 2013 to July 2017. The profiling of aroma principles using Gas Chromatography-Mass Spectrometry (GC-MS) and pungency principles using High Performance Liquid Chromatography (HPLC) at various growth stages viz. five months after planting (5MAP), six months after planting (6MAP) and seven months after planting (7MAP), revealed that aroma and pungency principles accumulated in ginger rhizomes at the rhizome formation stage (5MAP). Clone to clone variation was observed in the number and quantity of aroma and pungency principles accumulated in the rhizome. Total gingerol content in somaclone B3 (19.07%) was high when compared to the control cultivar Maran (17.49%) irrespective of the growth stages. Gene expression for Chalcone synthase in selected somaclones done using real time PCR assay showed highest gene expression in somaclone B3 when the control cultivar Maran was set as calibrator. Somaclone B3 recorded 54 per cent increase in Chalcone synthase gene expression over the control cultivar Maran. Suppression subtractive hybridization done to identify differentially expressed genes in somaclone B3 and control cultivar Maran could prepare Expressed Sequence Tag (EST) libraries both for rhizome and leaf. Analysis of EST sequences (25 rhizome ESTs and 19 leaf ESTs) using various bioinformatic tools revealed that there were no differentially expressed genes for gingerol production in rhizome ESTs. But eleven differentially expressed proteins involved in signaling response, protein trafficking, photosynthesis, ATP formation and transposon mediated mutation were observed in rhizome ESTs. The analysis of leaf ESTs showed differential gene expression in somaclone B3 for 3-ketoacyl CoA thiolase (ACAA1) gene which is involved in gingerol biosynthetic pathway. Hence the higher expression of 3-ketoacyl CoA thiolase gene is responsible for the high gingerol content in somaclone B3 as compared to control cultivar Maran. Eighteen other differentially expressed proteins involved in biological processes like transportation of plant secondary metabolites and their intermediates, mobilization of sucrose into pathways involved in metabolism, lipid biosynthesis, transportation of cellular material to microtubules, biogenesis of metabolic pathways in Calvin cycle were observed in leaf ESTs. The differentially expressed gene (ACAA1) can be further validated using northern blotting and quantitative real time PCR by designing specific primers from the ESTs. Expressed sequence tags and corresponding differentially expressed proteins can be used as molecular markers. Post translational modification in differentially expressed proteins can be used to study the mechanism of gingerol production. Forty four sequences deposited at NCBI form the base sequences for further research.
Open Access
Development of small interfering RNA (siRNA) mediated resistance in banana against banana bract mosaic virus
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2016) Lekshmi, R S; KAU; Soni, K B
The present study entitled “Development of small interfering RNA (siRNA) mediated resistance in banana against Banana bract mosaic virus (BBrMV)” was carried out during 2012-2016 in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The study was carried out with an objective to develop siRNA mediated technology for the development of banana plants resistant to Banana Bract Mosaic Virus (BBrMV). The study was conducted in banana cv. Nendran. A protocol for somatic embryogenesis in banana cv. Nendran was standardized by using immature male flowers as explants. Pale white friable callus with rich cytoplasm was initiated in Murashige and Skooge (MS) medium supplemented with BA (0.1 – 0.5 mgL-1) and picloram (0.5 – 2 mgL-1) incubated in dark with a maximum explant response of 30 per cent. For embryogenesis, the developed embryogenic calli were transferred to semisolid MS medium supplemented with BA 2 mgL-1 and IAA 0.5 mgL-1 which resulted in a maximum of 10 per cent embryogenesis. The glassy elongated monocot embryos were germinated in half strength semisolid MS medium (0.3 per cent Gelrite) supplemented with BA 2 mg L-1 and IAA 0.5 mg L-1 and incubated in dark. A maximum germination rate of 80 per cent was obtained in this medium. The germinated embryos were transferred to MS medium with BA 2 mg L-1 and NAA 1 mg L-1 resulted in 100 per cent Plant regeneration. The plantlets were transferred to coirpith compost in pot trays in mist chamber for one month for hardening and then transferred to polybags with soil and cowdung (1:1) mixture. To develop siRNA technology to silence the replicase gene of BBrMV, an intron hairpin RNA (ihpRNA) construct was developed. For this a partial mRNA sequence of replicase gene was isolated from BBrMV banana plants. Gene specific primers designed based on the whole genome sequence information retrieved from the GenBank, NCBI. Total RNA from infected banana leaves was isolated and cDNA was prepared using RT-PCR. The partial gene fragment isolated was sequenced and analysed using the bioinformatics tool BLAST. The sequence was subjected to miRNA target prediction and restriction mapping to select suitable region for the construct and further processing. Based on this information a fragment of 400 bp towards the 5’ end was amplified by designing a set of primers with anchored restriction sites. The primers anchored with BamHI and PacI sites were used for the amplification of sense strand and primers anchored with KpnI and SpeI sites were used for antisense strand amplification. The sense and antisense fragments amplified were cloned to pTZ57R/T cloning vector. In the next step the inserts were released from pTZ57R/T using the corresponding restriction enzymes and were integrated in pSTARLING (primary vector), on either side of the cre intron which facilitated the formation of the hairpin (ihpRNA) construct. Presence of the inserts was confirmed by restriction digestion and electrophoresis. The ihpRNA construct in pSTARLING now contained ubiquitin promoter, ubiquitin intron, sense strand of replicase gene, cre intron, antisense strand of replicase and termination sequence in the order with the NotI restriction sites. This construct was released from pSTARLING and ligated to the digested NotI site in the lacZ gene of the binary vector pART27 containing antibiotic resistance marker nptII and spec. The binary vector was confirmed for the insert by transferring to DH5α and colony selection by blue-white screening. The binary vector with the insert isolated from the white colony, was transferred to Agrobacterium tumefaciens strain LBA 4404 via freeze-thaw method. Transformed colonies were picked up and confirmed the presence of the vector and the ihpRNA insert by PCR. Somatic embryos were transformed with LBA 4404 carrying the ihpRNA construct the ihpRNA construct and the transformed embryos were selected with antibiotic pressure (Kanamycin 100 mg L-1). Transformed embryos were subjected to regeneration. A maximum regeneration of 25 per cent was obtained after transformation. The regenerants were confirmed for the presence of ihpRNA construct using PCR with specific primers for sense-intron-antisense fragment, npt II and cre intron. The study was successful in developing a siRNA construct for resistance against BBrMV and obtaining transformed Nendran banana plantlets.
Open Access
Validation of apomixis and transcriptome analysis for detection of the genes related to apomixis in black pepper (piper nigrum L.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Rohini Rajkumar, Bansode; KAU; Valsala, P A
Black pepper (Piper nigrum L.) universally honoured as “Black Gold” and also known as “King of Spices” is one of the most important spices in the world. It is a perennial climber belonging to the family Piperaceae and is valued throughout the world for its spice value and medicinal properties. Black pepper is grown in Southern India mainly in Kerala. Here productivity is declining due to various reasons and continuous vegetative propagation leads to accumulation of diseases and finally results in unproductive vines. Therefore, use of quality planting material of improved varieties is necessitated for the enhancement of productivity. Apomixis is a mode of asexual reproduction where the sexual organs are utilized, but the seeds develop without fertilization. It combines the advantage of seed propagation and vegetative propagation, and can be utilized for developing disease free planting material without losing the clonal integrity. The present study on “Validation of apomixis and transcriptome analysis for detection of the genes related to apomixis in black pepper (Piper nigrum L.)” was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2011-2014 with the objective to validate apomixis in black pepper varieties, Panniyur-1 and Panniyur-2 through controlled pollination studies and to identify differentially expressed genes associated with apomixis through transcriptome analysis. Studies were conducted on bush pepper plants of the selected varieties maintained in the green house. Floral biology of Panniyur-1 and Panniyur-2 were studied. The female phase in Panniyur-1 started 8th day after spike initiation and completed on 27th day. Stigma became receptive on 9th day and receptivity was indicated by creamy white colour. Beginning of male phase indicated by anther emergence on either side of the ovary occurred on 19th day of spike emergence and active dehiscence of anthers occurred on 20th day. In Panniyur-2, beginning of female phase started on 14th day after spike initiation and stigma became receptive on 15th day. Beginning of male phase occurred on 22nd day of spike emergence and active dehiscence of anthers occurred on 23rd day. So in Panniyur-1 and Panniyur-2 the active female and male phase is separated by 10 and 8 days, respectively. Attempts were made to confirm apomixis in Panniyur-1 and Panniyur-2 varieties by allowing berry development in controlled condition by bagging of spike initials. Berry development occurred under bagged condition. In this study, upper six berries were considered as apomictic and lower six berries were considered as pollinated. Efforts were made to regenerate apomictic and non- apomictic progenies under in vitro and ex vitro. In vitro embryo culture resulted in embryo germination and multiple shoot induction in SH medium with hormones. But plants were lost due to microbial contamination and phenolic interference. So ex vitro germinated seedlings were used for further studies. Histological examination of pollinated and unpollinated ovaries was done through microtomy. The results revealed that in case of pollinated berries, the sexual fertilization occurs and embryo develops inside the embryo sac in the micropylar end. Whereas, in apomictic embryo sac more than one aposporous initial cells were observed which were arising from a somatic cell located in the nucellus. So, it can be concluded that facultative apomixis exists in black pepper varieties P1 and P2 and the embryo develops parthenogenetically. The molecular characterization of apomictic and non-apomictic seedlings was done through Inter Simple Sequence Repeats (ISSR) and Simple Sequence Repeats (SSR) assay. Isolation of good quality genomic DNA from apomictic, pollinated progenies and mother plant was carried out from the young leaves using modified Rogers and Bendich (1994) as reported by Mogalayi (2011). In ISSR and SSR assay certain primers showed polymorphism among mother plant, apomictic and pollinated progenies. Similarity matrix was calculated and corresponding dendrogram was also constructed for both markers using UPGMA cluster analysis for P1 and P2 varieties. In ISSR assay the similarity coefficient between P1 mother plant and apomictic progenies ranged from 69 to 89 per cent while it was 64 to 66 per cent between mother plant and pollinated progenies. In P2 variety similarity coefficient between mother plant and apomictic progenies ranged from 76 to 90 per cent while it was 62 to 69 per cent between mother plant and pollinated progenies. In SSR assay similarity coefficient between P1 mother plant and apomictic progenies ranged from 50 to 100 per cent while it was 71 to 100 per cent between pollinated progenies and mother plant. Similar results were obtained for P2 variety also. So apomictic progenies may differ from mother plant in certain characters. RNA mediated transcriptome analysis of the apomictic and pollinated berries was done to detect differentially expressed genes. Good quality RNA was isolated by modified LiCl precipitation method. Total RNA from the apomictic and pollinated berries were taken for DDRT-PCR analysis. The first strand cDNA was synthesized from the above RNA samples using HT11C (AAGCTTTTTTTTTTTC). Each first strand cDNA was used for the second strand amplification with 8 different arbitrary primers. The PCR product was resolved in 6 per cent denaturing urea polyacrylamide gel and visualized after silver staining. The differentially expressed cDNA fragments were retrieved from the gel and reamplified and electrophoresed. The agarose gel electrophoresis showed 7 transcript derived fragments (TDFs) ranging from 200-600 bp. TDFs were cloned using pGEMT vector and were sequenced by outsourcing. The sequence data were analysed by BLASTn and BLASTx. The sequences showed homology to NADH dehydrogenase subunit J, acetyl-CoA-benzylalcohol acetyltransferase and purine permease 4 but no significant sequence similarities for apomictic genes deposited in NCBI database were found.
Open Access
Isolation, characterisation and evaluation of PINI and BP genes in ralation to inflorescence architecture in black pepper (Piper nigrum L.)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2019) Smitha Bhasi; KAU; Swapna Alex
The study entitled “Isolation, characterisation and evaluation of PIN1 and BP genes in relation to inflorescence architecture in black pepper (Piper nigrum L.)” was conducted during 2015-2018 at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram. The objective of the study was to isolate and characterize PIN1 (PINFORMED1) and BP (BREVIPEDICELLUS) genes in black pepper and to evaluate the role of these genes in branching of spikes by studying their differential expression pattern in branching and non-branching varieties of black pepper. Spike samples at their three developmental stages viz. stage-1 (12-15 days after emergence of bud; 1-2 cm length), stage-2 (22-25 days after emergence; 6-8 cm length) and stage-3 (32-35 days after emergence; 9-12 cm length) were collected from bush pepper plants of branching type (Thekken) and non- branching varieties (Panniyur-1, Karimunda and Aimpiriyan) and used for the study. Morphological, histological, molecular (genome, transcriptome and proteome levels) and hormonal analyses were carried out. On morphological analysis of spike characteristics based on IPGRI descriptors, Thekken exhibited six fold increase in number of berries per spike (480) compared to Panniyur-1 (79). Maximum spike length (14 cm) was exhibited by Panniyur-1, and minimum by Karimunda (7.5 cm). Thousand berry weight was maximum for Aimpiriyan (135.6 g) and minimum for Thekken (100 g). Microscopic analysis showed the presence of thick fleshy bracts in Thekken compared to Panniyur-1. Spikelet emergence was noticed at stage-3 in the spikes of Thekken. Histological analysis revealed the presence of a distinct mass of meristematic tissue in stage-1 of Thekken that gradually differentiated during stage-2 and emerged into a spikelet during stage-3. For genome level analysis, DNA isolated by CTAB method was amplified by Polymerase Chain Reaction (PCR) with gene specific primers for PIN1 and BP, designed using Primer-3 software. Two amplicons of size 124 bp and 650 bp were obtained using PIN1 specific primer. Sequence of the 124 bp amplicon on BLASTn analysis revealed 94% identity to PIN1 gene of Canna sp. while the 650 bp amplicon revealed 83% identity to auxin efflux carrier gene of Sorghum bicolor. Amplification of the genomic DNA with primer designed for BP gene produced an amplicon of 145 bp size and its sequence revealed 88% identity to homeobox protein knotted-1 like gene family to which BP belongs. Twenty five per cent variation was noticed between the PIN1 sequences of Thekken and Panniyur-1, whereas only five percent variation could be noticed between BP sequences. For transcriptome level analysis, cDNA reverse transcribed from total RNA isolated using Trizol method was used. Differential expression analysis was carried out using Real Time PCR. The expression pattern of PIN1 was similar in both Thekken and Panniyur-1. However, the expression levels were significantly higher in all stages of Thekken with maximum (14 fold) at stage-1. The expression pattern of BP varied in Thekken compared to Panniyur-1. It showed twenty seven fold over expression in stage-1 and three fold down regulation in stage-2 of Thekken compared to Panniyur-1. Rapid Amplification of cDNA Ends (RACE) of the 124 bp amplicon of PIN1 generated two fragments of size 280 bp and 580 bp. Protein domain analysis of the fragments revealed the presence of zinc finger domains. Proteome analysis using SDS Polyacrylamide gel electrophoresis (PAGE) showed unique protein fragments in all the stages of inflorescence in Thekken (100 KDa fragment in stage-1 and stage-2; 48 KDa and 55 KDa fragments in stage-3). Hormonal analysis of auxin using High Performance Liquid Chromatography (HPLC) revealed that IAA content in Thekken (25 ppb) was one-fourth compared to Panniyur-1 (90 ppb) and one-third compared to Karimunda (60 ppb). Enzyme Linked Immuno Sorbant Assay (ELISA) showed an increase in total cytokinin content in Thekken from stage-1 (2.8 ng/g) to stage-2 (3.5 ng/g), in contrast to non-branching varieties (3.6 ng/g to 3.0 ng/g). The present study is the first report on the isolation and characterisation of coding sequences of PIN1 and BP genes in black pepper. The fragments of PIN1 and BP genes isolated were deposited in the NCBI database. The 580 bp PIN1 sequence revealed numerous zinc finger domains similar to PIN1 gene of Arabidopsis indicating similar function. PIN1, being an auxin efflux carrier, its overexpression noticed in Thekken at the transcriptome level correlated with the lower auxin content in the hormonal analysis. The twenty seven fold expression of BP noticed in Thekken correlated with the increase in cytokinin content and the emergence of a differentiated meristamatic tissue that was prominent in the histological sections of stage-2 of Thekken. The unique proteins of sizes 100 KDa, 48 KDa and 55 KDa in Thekken in proteome analysis suggest the presence of novel proteins in Thekken. The differential expression of PIN1 and BP genes revealed in the present study indicates the significant role of these genes in inducing the spike branching trait in Thekken.
Open Access
Gene expression analysis in relation to Fusarium wilt resistance in banana (Musa spp.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Jusna Mariya, P L; KAU; Keshavachandran, R
Banana is one of the important fruit crops of India. Banana is susceptible to several fungal pathogens, nematodes, viruses and insect pests. The greatest threats to global banana production is Fusarium wilt or Panama wilt caused by Fusarium oxysporum f. sp. cubense. Control of the pathogen is difficult and mainly involves the use of disease free suckers. Although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult due to its triploid nature and sterility factors of banana. The study entitled "Gene expression analysis in relation to Fusarium wilt resistance in banana (Musa spp.)" was carried out at the Centre for Plant Biotechnology and Molecular Biology, Vellanikkara during the period 2009-2013 with an objective to identify differentially expressed genes in disease resistant genotype of banana, Palayankodan using the molecular technique called suppression subtractive hybridization (SSH). Total RNA and mRNA were isolated from healthy and inoculated plants (with Fusarium oxysporum f.sp. cubense) and were used respectively as 'driver' and 'tester' in SSH reaction. The reactions were performed utilizing the PCR select" cDNA subtraction kit provided by CLONTECH, USA. Control subtraction was carried out first using PCR select" cDNA subtraction kit, which gave satisfactory and expected results. For experimental subtraction, the double stranded cDNAs synthesized from Zug mRNA from normal 'driver' and treated 'tester' were digested with RsaI enzyme. Two tester populations were created and each ligated to two different adaptors. This was followed by two hybridization reactions and finally a selective PCR amplification. Only differentially expressed cDNAs were amplified exponentially. This was confirmed by analyzing the PCR products on agarose gel, which showed a smear ranging from 0.9 to 1.3 kb in the subtracted sample and was different from smear pattern of unsubtracted ones. The cDNA fragments from subtracted sample were cloned in pJET and pGEMT vectors and sequenced. Fifty clones were sequenced and analysed after vector and adaptor editing. In silica analysis using bioinformatics tools revealed that some of the cloned sequences showed similarity with known sequences which play important roles during disease resistance conditions directly or indirectly. These included resistance gene candidate NBS type protein, mitogen activated protein kinase, phytoene desaturase, glycerol 3-phosphate dehydrogenase, neutral invertase, 1- aminocyclopropane-l-carboxylase synthase, superoxide dismutase, MADS-box protein, ubiquitin 2, actin, NADPH oxidase, phytoene synthase, ACC synthase, sucrose phosphate synthase, phosphatidic acid phosphatase-like protein, ORF III like polyprotein, bHLH transcription factor like protein, cytochrome oxidase, isochorismatase hydrolase, basic helix-loop-helix family protein, constitutive triple response I-like protein, granule bound starch synthase, alpha amylase precursor, rop protein, GTPase family protein, S-adenosyl-L-methionine synthase protein, ADP-glucose pyrophosphorylase glucose-l-phosphate adenylyl trans, ethylene signal transduction factor and ribosomal protein. Clones were classified into 6 major groups based on function of protein. Sequences had conserved domains for the above mentioned proteins. Genes involved in defense, signal transduction, metabolism, hypothetical protein, transcription factor and translation. For further exploitation of these sequences it is necessary to clone full length cDNA. ESTs thus generated in the present study will be of great use in future for further downstream applications.

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