Fusion gene construct preparation with lectin and protease inhibitor genes against aphids and efficient genetic transformation of Brassica juncea using cotyledons explants
2017
Globally, Brassica juncea is one of the significant oilseed crops, which is commercially important and cultivated under diverse agro-climatic region preferably for its high oil content as well as superior oil quality. Biotic stress is one of the dominant factors which hinders plant growth and yield globally. Though substantial progress has been made in breeding and genetic manipulation of plants for imposing resistance, the task remains as a challenge even today. Here, in this study, two major defense genes of plant origin, lectin from lentil and protease inhibitors from chickpea, which have insecticidal activity against phytophagous insect pests have been isolated and stacked into one ORF by overlapping extension PCR and a fusion gene construct is prepared. For tissue specific expression of this fusion gene, phloem specific promoter rolC was utilized and the construct was mobilized into Agrobacterium tumefaciens strain GV3101. Further, genetic transformation of B. juncea cv. Varuna was performed with fusion gene using Agrobacterium-mediated genetic transformation. The regeneration of the transformed putative plants was screened with hygromycin selection. Fusion gene integration in randomly selected transformed plants gives 17% overall transformation efficiency by PCR and Southern hybridization. A quantitative real-time PCR analysis showed that the transcript level of the fusion gene was within 1.8–2.8-fold. The expression of the fusion gene was significantly higher in the third line (2.8-fold). The aphid resistance test demonstrated that inhibition of larval survivability is about 40%, and the extent of leaf damage measured was also diminished in the transgenic plants compared to non transformed control plants. This study provides insight the multi-toxin approach developed through overlap extension PCR in B. juncea and may facilitate genetic engineering for the potential improvement of resistance against aphids.
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