Development of transgenic Indian mustard (Brassica juncea) expressing siRNA against mustard aphid serine protease
KrishiKosh
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Title |
Development of transgenic Indian mustard (Brassica juncea) expressing siRNA against mustard aphid serine protease
M Sc |
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Creator |
VIDUSHI RASTOGI
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Contributor |
Ramcharan Bhattacharya
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Subject |
planting, genes, genetic processes, enzymes, bacteria, wood, mustard, amino acids, solutes, rna
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Description |
T-8619
Indian mustard [Brassica juncea (L.)] is an important oilseed crop of India. Among the various biotic constraints, the mustard aphid (Lipaphis erysimi Kaltenbach) causes severe yield losses throughout the cropping season. Nymphs as well as the adult insects are responsible for the damage. Besides, mustard aphids are known to act as vectors for diseasecausing luteoviruses. Chemical insecticides available against aphids are not only environmentally hazardous but also incur a sumptuous expense on the part of farmer. No source of resistance against mustard aphids is yet known within the crossable germplasm. Hence, conventional breeding programs are inapplicable in breeding for aphid resistance. A few insecticidal transgenes viz. plant lectins and agglutinins have been reported to be effective against aphids. But these experiments have not been encouraged to further field applications. Thus, advantage of transgenic technology is yet to be realized in breeding aphid resistant cultivars. Therefore, a new biological mechanism to impart genetic resistance against sap sucking pests like aphids is desirable. RNA interference (RNAi) is a powerful technique to inhibit homologous gene transcript. The unique advantage of technique lies with its specificity, which depends on the sequence of one strand of the dsRNA 40 corresponding to a part or the entire target gene transcript. The degradation of dsRNA is mediated by dsRNA-specific endonucleases known as Dicers through the production of small interfering RNAs (siRNAs).RNAi is already an established functional genomics tool in insects. It was envisaged that the host mediated delivery of siRNA molecules targeted to indispensable aphid genes can lead to development of aphid resistant crop cultivars. Serine proteases are vital enzymes of aphids being involved in digestion of proteins drained out of plant phloem. A plant transformation vector which would generate dsRNA molecule corresponding to mustard aphid serine protease gene for RNAi mediated silencing was available in the laboratory. To aim the expression of siRNA to phloem, the phloem specific suc-2 promoter of Arabidopsis thaliana was cloned in the construct upstream of the partial coding sequence of the serine protease gene. Here we describe Agrobacterium tumefaciens mediated transformation of Brassica juncea var. Varuna using that construct. The transformants with pBI121 were used as control in various experiments. The regenerating transformants were selected through positive selection by kanamycin resistance. The transformation with the dsRNA construct was confirmed in the regenerating shoots through PCR employing primers specific to both gene and the promoter. To affirm the transcription of the inserted T-DNA kanamycin resistance gene was amplified by Reverse Transcription PCR. To detect the transcription of serine protease gene in both orientations, reverse transcription PCR was performed .However ; the transcript could not be detected probably because of its processing by plant DCL. |
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Date |
2016-09-21T16:16:25Z
2016-09-21T16:16:25Z 2012 |
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Type |
Thesis
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Identifier |
http://krishikosh.egranth.ac.in/handle/1/78166
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Format |
application/pdf
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Publisher |
IARI, NATIONAL RESEARCH CENTRE ON PLANT BIOTECHNOLOGY
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