Computational Characterization, Functional Analysis and Molecular Cloning of Chitinase Gene From Tomato

Abstract

Tomato (Solanum lycopersicum L.) belongs to the nightshade family Solanaceae, native to South America and is world's largest vegetable crop after potato and sweet potato. It is the third most economically important after grasses and legumes, India ranked second in the world in terms of area and production of tomato during 2019. Vascular wilt caused by Fusarium oxysporum f. sp. lycopersici (Sacc.). C. (FOL) is one of the serious disease of tomato and has known to brought extensive losses in tomato production globally. The losses caused by this disease are variable as per climate, region, season, management practices etc. The disease is known to cause 60 to 70 % of fruit yield loss. In Uttar Pradesh yield loss due to this disease is 25.14 - 47.94 %. Chitinases are sub grouped under pathogenesis related (PR) proteins produced in response to pathogenesis and known to degrade chitin the principle component of fungal cell wall and inhibit fungus growth. Different tomato genotypes show variation for synthesis of chitinases depending on the presence of chitinase genes and accordingly shows susceptibility and resistance against FOL attack. Hence, by this we focused on chitinase and other biochemical activities due to fusarium induced biotic stress, expression pattern of chitinase genes among 14 tomato genotypes, cloning of selected chitinase gene and comparative in-silico characterization tomato, potato and Arabidopsis chitinase genes. The percent disease intensity of fusarium on 14 tomato genotypes was analysed and Among both the years was reported by genotype FEB-4 shown highest 82.22% followed by Phule Keasri (75.97%). The wild species S. Pimpenelifolium reported lowest 13.06% PDI., followed for both the years. Due to fusarium infection the morphological, growth and yield related characters viz. plant height, stem internode length, average fruit weight, number of fruits plant-1, fruit length, fruit width, days to first flower, number of inflorescence (truss) plant-1, number of flowers truss-1 etc. were affected drastically than the normal. Higher level of biochemical activities was reported for β 1,3 glucanase, chitinase, peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, proline and various proteins in fusarium inoculated plants roots than the uninoculated. Comparative in-silico study of 116 chitinase genes from tomato, potato and Arabidopsis was undertaken and investigated interrelations among chitinases in these species. The chitinase genes viz. Solyc03g116190.1, Solyc02g082930.2, Solyc10g055820.1, Solyc04g072000.2, Solyc02g082920.2, LeChi9 were expressed in these tomato genotypes in varied proportions and amount in order to produce defense related proteins as a result of fusarium infection. The tomato chitinase gene Solyc03g116190.1 of product size 141bp shown differentiating expression profile among all genes in this study was successfully cloned in to E. Coli.