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| Isolation of protease inhibitor and lectin genes |
In order to broaden the pool of plant defense genes and to identify new and more potent inhibitor proteins for pest control, a program has been initiated for screening indigenous legumes for presence of insect specific protease inhibitor proteins and isolation of their genes from the mature seeds.
Genes for protease inhibitor and lectin have been isolated from cowpea, sequenced and characterized prior to their utilization in engineering resistance against insects (Fig. 1). A partial list of genes and promoters cloned at NRCPB is given in Table 1.
Table: 1 Genes and promoters isolated and sequenced
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| Fig. 1. Effect of protease inhibitors on the development of larvae (A)and emergence of adults of Helicoverpa armigera from PI untereated (B) and treated (C) larvae. |
Complete promoter of the legumin gene was cloned and characterized. This promoter was found to have consensus sequences like legumin box and AGGA box in addition to other usual structural features. This promoter has potential application in driving seed specific expression of any target gene in transgenic programmes. |
| Contact : Prof. K.R. Koundal ( kirparam@rediffmail.com ) |
| Tomato fruit specific promoter cloned and characterized |
| To delay fruit ripening and for extending shelf life of tomato through genetic engineering. It is imperative to clone and deploy fruit- specific promoters (Fig.2). These promoters will be useful to precisely limit expression of a desired gene in fruits at a particular stage of development, which is essentially required to develop transgenic tomatoes with an ability to ripen slowly and without the exogenous application of ethylene. Consequently, monetory losses to the tune of about Rs 15,000 crores per annum only on account of post- harvest losses from fruits viz. tomato, banana, papya and mango could be saved by developning transgenics with delayed fruit ripening in above fruit crops. To achieve this target the center has isolated and characterized tomato fruit specific promoters which can be used for the production of fruits with delayed ripening. |
| Contact : Dr. K.C. Bansal ( kailashbansal@hotmail.com ) |
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| Fig.2. Transient gene expression assay in tomato |
| Promoter trapping using T-DNA insertional mutagenesis |
| Populations of Arabidopsis tagged with a promoterless GUS ( b -glucuronidase) have been generated. Some new mutant lines, exhibiting altered phenotypes have been identified. Several mutant lines exhibiting tissue specific expression of GUS have been identified. A transgenic line of Arabidopsis exhibiting anther specific GUS expression (Anth-85) has been identified (Fig.3). The T-DNA insertion is in the locus At2G24800, a putative peroxidase gene. |
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| Fig. 3 Anther specific expression of GUS in Anth 85 mutants. |
We have identified a T-DNA tagged line of Arabidopsis with lateral organ junctions and shoot apical meristem specific expression of reporter gene (Fig. 4). The insertion is in the upstream region of locus At2g39230, encoding for a putative PPR protein. Our studies have led to the identification of a unique TATA-less promoter different from that are known so far in monocots or dicots. Also, our work for the first time implicates a PPR protein gene in lateral organ development and boundary formation. |
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Fig. 4 Lateral organ junction specific expression in loj mutant |
A mutant line, M-57 showing GUS expression in the vascular regions of young roots, was identified. The insertion was identified to be in the intergenic area between loci At4G13940 and At4G13930, coding for SAHH (S-Adenosyl-l- Homocysteine Hydrolase) and SHMT (Serine Hydroxy Methyl Transferase) genes, respectively. The 452-bp fragment immediately upstream of the T-DNA insertion when cloned and mobilized as a GUS fusion was capable of driving a similar root-specific expression of reporter gene in transgenic Arabidopsis plants and their progenies (Fig.5 A & B). This promoter region does not appear to be associated with any gene and does not appear to contain any known root-specific promoter element. Thus the promoter element identified by us appears to be cryptic and novel in nature. |
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| Fig. 5 A & B Root specific expression of GUS driven by the 452 bp cryptic promoter |
| Contact : Dr. Srinivasan ( sri@iari.res.in) |
| Isolation and characterization of novel genes and strains of Bacillus thuringiensis |
Bacillus thuringiensis isolate were collected from the high altitude and dry region of Ladakh and looked for the presence of novel cry 1 type genes therein. An isolate TA-22 responded positively to both cry 1Ab and cry 1Ad specific primers (Fig 6). Using another set of primers designed to detect all cry 1A-type genes, such that both the genes would be amplified, a 1.7 Kb product was obtained and cxloned in pGEM-T Easy vector. Twelve positive clones were randomly selected based on blue-white selection. In order to differentiate between the clones carrying cry 1Ab gene and those carrying cry 1Ad gene, all the clones were tested with both cry 1Ab and cry 1Ad specific primers. |
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Fig 6. Detection of a novel cry1a-type gene Lane1: HD-1 (cry1Ab), Lane2: HD-1 (cry1Ad), Lane3: TA-22 (cry1Ab), Lane4: TA-22 (cry1Ad) |
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| Contact : Dr. Sarvjeet Kaur ( dr_sarvjeetkaur@yahoo.com ) |
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