Assessment of transgenic Brassica juncea cv. Varuna harbouring MAPK3 gene against Alternaria brassicae after preconditioning with phytohormones and MAPK inhibitor
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Date
2019-09
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
Alternaria blight is one of the most devastating fungal disease which hampers the productivity of Brassica juncea in India. The disease is caused by a semi-biotrophic pathogen, Alternaria brassicae. The absence of resistance source against Alternaria blight has made the plant researchers shift their interest towards de-novo sources of resistance. The MAPK 3 gene is an important gene expressed in plants during biotic stress for strengthening the defense system of plant. The MAPK 3 gene was overexpressed in Brassica. juncea cv.Varuna for the evaluation of resistance provided by transgenic lines against Alternaria blight. The two important plant hormones salicylic acid and jasmonic acid plays a major role in transducing the activation of plant defense systems against pathogen attack. These phytohormones regulate the expression of MAPK cascade genes that are the key role player of defense signaling in plants. The codon usage bias is an evolutionarily conserved phenomenon that affects the expression level of genes. The knowledge regarding the codon usage biasness pattern of genes helps us to know whether the proteins will be expressed in the heterologous system or not that guides in the development of transgenic plants. In the present investigation the copy number of MAPK 3 gene in transgenic B.juncea cv. Varuna plants were calculated using real-time PCR and Southern blotting. The effect of salicylic acid, jasmonic acid, and MAP kinase inhibitor was studied on the transgenic and non-transgenic B.juncea cv.Varuna plants at different stages of disease progression. The antioxidant activities of APX, GPX, CAT, proline was higher in phytohormones treated transgenic plants. The MAPK cascade genes viz. MAPKKK 1, MAPKK 4, MAPKK 9, WRKY 33, WRKY 22, OASTL-B, ACD-2, CSD-2 that are involved in defense pathway were observed to be expressed higher in transgenic plants as compared to the non-transgenic plants. The codon usage bias analysis of MAPKs and WRKY genes of A. thaliana and B. rapa was carried out by using the bioinformatics approaches. The studies revealed that the pattern of CUB in these genes are affected by natural selection and mutational pressure. The clustered heat map grouped the MAPKs and WRKY genes having similar RSCU values and similar functions under the same cluster. The codon AGA coding for arginine was found to be preferred in most of the MAPKs and WRKY genes. The codon usage bias in A.thaliana and B.rapa MAPKs and WRKY genes was observed to be low based on ENc values. The expression level of MAPKs and WRKY genes was observed to be low based on the RSCU values. These studies will give direction for the engineering of defense pathway so that the Alternaria blight resistant B. juncea plants could be developed.
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