Response of brassica plants transformed with calreticulin gene and it’s N, P, C domains on the expression of antioxidant enzymes and tolerance to alternaria blight disease

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dc.contributor.advisorPandey, Dinesh
dc.contributor.authorDarkal, Nishika
dc.date.accessioned2022-12-21T17:52:05Z
dc.date.available2022-12-21T17:52:05Z
dc.date.issued2022-09
dc.description.abstractCalreticulin protein has recently been discovered as one of the plant genes which has role in biotic and abiotic stress tolerance due to it’s antioxidative action. infected plants produce ros as a result of environmental stress, which are scavenged by anti-oxidative enzymes. through the activation of anti-oxidant genes and enzymes inside the cell, calreticulin aids in the quicker scavenging of ros. calreticulin which is primarily a chaperone protein present in the endoplasmic reticulum has three domains named as n, p, and c, each having a distinctive structural and functional role in this protein. however, the precise domain causing stress resistance has not yet been examined, and the real molecular mechanism by which these genes work has also not been well investigated. therefore, the current research was carried out to investigate the response of fully grown transformed brassica juncea var. varuna plants overexpressing calreticulin and it’s n, p and c domains on the expression of antioxidant enzymes and tolerance against alternaria blight disease. in order to achieve this target, the seeds from brassica juncea var. varuna plants previously transformed with n, p, c and crt3 genes were grown and transformed plants were selected after screening through pcr using primers specific for selectable marker (hygromycin b). after screening, 2 plant samples for both crt3 and c, and 1 for each n and p were obtained as hygromycin b positive transformants. for conducting the expression analysis studies in these plants, total rna was isolated followed by synthesis of cdna, and using the cdna as template mean fold expressions of respective genes was calculated through rt-pcr ct values. the mean fold expression of crt3 and it’s n, p and c domain’s genes in transformed plants were observed to be significantly higher (2-3 folds) as compared to control/untransformed plants (1 fold), which confirmed the domain/gene specific in-planta transformation of brassica plants. the transformed plants were then subjected to alternaria blight disease stress by spraying a. brassicae spore suspension over their leaves. the disease indices of brassica plants transformed with crt3 and c, p, n domains were found to be 38%, 44%, 55%, and 62% respectively at 21 dpi stage as comparison to untransformed/control plants (94%), which revealed their inhibitory effect on the development of the alternaria blight disease. for further molecular analysis the rna and subsequently cdna was prepared from 12 hpi, 24 hpi, 48 hpi and 7 dpi transformed plant samples. the rtpcr analysis of these cdnas showed a significant rise in mean fold expression levels of n, p, c and crt3 genes (2.02-3.14 folds) from 12-48 hpi periods of infection as compared to control plant that exhibited only slight or nonsignificant rise in mean fold expression levels at the same time intervals (1.01-1.41 folds). also domain/gene specific rtpcr results conferred that the n, p, c and whole-length calreticulin-3 gene have potential in imparting transformed plants the ability to combat alternaria blight disease. the rtpcr analysis study of sod and cat antioxidative enzymes further revealed that plants transformed with the n, p, c, and crt3 genes displayed significant antioxidant levels as compared to untransformed/control plants, which are also accompanied by the reduction in disease indexes of respective domains/gene specific transformed plants. as a result, it was interpreted that there is positive correlation between expression of calreticulin and it’s domains, and expression of antioxidant enzymes which may subsequently help to mitigate the alternaria blight disease of brassica. further the proteomic studies and the downstream signaling pathway involved in executing it’s defensive role could be identified and studied in such plants.en_US
dc.identifier.urihttps://krishikosh.egranth.ac.in/handle/1/5810190736
dc.keywordsbrassica, antioxidanten_US
dc.language.isoEnglishen_US
dc.pages66en_US
dc.publisherG.B. Pant University of Agriculture and Technology, Pantnagar, District Udham Singh Nagar, Uttarakhand. PIN - 263145en_US
dc.research.problemAntioxidanten_US
dc.subBiotechnologyen_US
dc.themeBrassicaen_US
dc.these.typeM.Scen_US
dc.titleResponse of brassica plants transformed with calreticulin gene and it’s N, P, C domains on the expression of antioxidant enzymes and tolerance to alternaria blight diseaseen_US
dc.typeThesisen_US
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