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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
 
Creator VIDUSHI RASTOGI
 
Contributor Ramcharan Bhattacharya
 
Subject planting, genes, genetic processes, enzymes, bacteria, wood, mustard, amino acids, solutes, rna
 
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
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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.
 
Date 2016-09-21T16:16:25Z
2016-09-21T16:16:25Z
2012
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/78166
 
Format application/pdf
 
Publisher IARI, NATIONAL RESEARCH CENTRE ON PLANT BIOTECHNOLOGY