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20211
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Subject: Agricultural Biotechnology × Author: Lokshman, Milan Kumar × End date: 2024 × Start date: 2020 × Type: Thesis × Thesis Type: M.Sc ×
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    ThesisItemOpen Access
    Molecular cloning and Agrobacterium mediated transformation of Brassica spp. with full length calreticulin gene along with its n, p and c domains
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-09) Lokshman, Milan Kumar; Pandey, Dinesh
    Biotic and abiotic stresses in crop plants can significantly affect yield under resource poor conditions. The ability of plants to grow under stress conditions is an adaptive trait that can help to survive the crop with sufficient yield and quality. Recently, Calreticulin protein is identified as one of the differentially expressed genes in plants to minimize stress induced damages. Anti-oxidative activity of Calreticulin could be responsible for decreasing disease severity. The anti-oxidative enzymes scavenge these ROS produced during infection and environmental stress in plants. Calreticulin help in faster scavenging of ROS by activating antioxidant genes and enzymes inside the cell. Therefore it could be an attractive candidate for improving defense responses in plants. Calreticulin is mainly an endoplasmic reticulum resident chaperone protein. This protein has three distinct domains namely, N, P and C domains with structural and functional specificity. However, the actual domain responsible for providing stress resistance has not been analysed and there is no systematic study on actual mechanistic pathway of functioning of these genes. Hence, the present studies were conducted to understand role of calreticulin-3 by isolating and cloning of N, P and C domains and full length Calreticulin-3 gene followed by their transformation in to two varieties of Brassica spp. (Brassica juncea cv. Varuna and Brassica rapa cv. Bhawani ) by floral-dip method with main aim of investigating antioxidative role of this gene and its domains and subsequent antioxidative and stress tolerant activities of transformed Brassica plants. In order to achieve this target, total RNA was isolated from B. juncea var. PAB9511 followed by synthesis of cDNA. PCR was performed using the synthesized cDNA as template for obtaining full length calreticulin-3, N, P and C domains. Then these constructs were cloned into pGEMT-Easy vector and digested using restriction endonuclease enzymes SpeI and BstEII. The restricted fragments of each insert were then successfully cloned into plant expression vector, pCAMBIA1302. Recombinant pCAMBIA1302 with the N, P, C domains and entire Calreticulin gene was transformed to Agrobacterium GV3101. The transformation were carried out by giving infection of Agrobacterium harbouring these four constructs, in floral tissues of Brassica genotypes Varuna and Bhawani at early inflorescence stage. Seeds were collected from each plants and they were screened to identify transformed ones by growing on selection media containing antibiotic Hygromycin B. Transformation was confirmed by performing PCR for selection marker Hygromycin B after genomic DNA extraction from leaves of seedlings growing on selection media. Transformation efficiency was calculated as approximately 1% for B. juncea and approximately 0.55% for B. rapa. These transformed Brassica plants could further be tested for its role in conferring resistance against Alternaria blight and other different pathogenic infections or environmental stresses and downstream signalling pathway involved in executing this role could further be identified and studied.