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Isolation, genetic mapping and expression studies of a peptide deformylase gene, DEF2, from bold-seeded and small-seeded varieties of the oilseed crop, Brassica juncea (Indian mustard) and analysis of its over-expression and down-regulation on seed size of transgenic plants
BHARAT JOSHI, MALOTI HEMBRAM, ARUNDHATI MUKHOPADHYAY, MANU AGARWAL, SHAILENDRA GOEL and ARUN JAGANNATH
Abstract:
B. juncea (Indian mustard) is an important oilseed crop with seed size or seed weight being a major contributor to crop yield. The nuclear-encoded DEF2 gene produces a peptide deformylase that is targeted to the chloroplast. It is an essential enzyme with a role in embryo development, chloroplast function and photosynthesis. In this study, we analyzed the role of DEF2 gene in modulation of seed size in B. juncea utilizing four germplasm lines, including two bold-seeded Indian varieties, Varuna and Pusa Bold, and two small-seeded east-European varieties, EH2 and Heera. Isolation of DEF2 sequences from Varuna and Heera revealed the presence of two homologs of which, one homolog showed significantly higher expression levels during seed development stages in the bold-seeded varieties as compared to the small-seeded varieties. Genetic mapping studies based on polymorphisms between corresponding homologs identified one homolog co-localizing with a QTL for seed size in B. juncea. These observations indicated a possible correlation between the DEF2 gene and seed size phenotype. To validate the functional effects of DEF2 on seed size, transgenic plants were developed for antisense-mediated down-regulation of the DEF2 gene in Varuna and over-expression of the gene in EH2. While 35 out of 37 single-locus antisense transgenic lines in Varuna showed decreased seed size, 11 out of 17 single-locus EH2 transgenic plants over-expressing the DEF2 gene showed increased 100-seed weight of T1 seeds compared to the corresponding untransformed (control) plants. Homozygous lines were developed for a promising single locus EH2 transgenic plant and analyzed for traits viz., seed size, oil content, fatty acid composition, protein content and seed yield per plant. Significant increase in seed size was obtained for the DEF2 over-expression line in comparison to the untransformed control plants. The EH2 transgenic line with increased seed size generated in this study would be useful as a combiner for use in transgenic-based heterosis breeding programs in Indian mustard.
Keywords: seed size, DEF2, Brassica juncea, transgenics
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