TRANSFORMATION, HETEROLOGOUS EXPRESSSION AND FUNCTIONAL EVALUATION OF GERMIN-LIKE PROTEIN GENE IN MODEL LEGUME MEDICAGO TRUNCATULA

Abstract

Germin-like proteins are widely distributed in various plant tissues like leaves, roots, seeds, fruits and nectar glands, etc. They are expressed during different developmental stages of plants as well as during different biotic and abiotic stress interactions. The current study was planned to explore the function of an Oryza sativa rice root expressed Germin-like protein, OsRGLP1 gene by heterologous expression in Medicago truncatula. For genetic transformation, recombinant vectors pCOsRGLP1 and pGOsRGLP1 both harboring OsRGLP1 gene under transcriptional control of CaMVS35 promoter and Agrobacterium tumefaciens strain AGL-1 were used. Different transgenic lines were produced through Agrobacterium mediated transformation, and confirmed through PCR amplification of OsRGLP1 gene. Reverse transcriptase PCR analysis revealed differential expression of transgene in different transformed lines. Inheritance pattern analysis of seed collected from T0 progeny revealed that transgenic lines followed a Mendelian inheritance pattern. Morphologically under normal growth regime, difference was observed between T1 transgenic and wild type plants for various characters that included the number of leaves, length of stem and number of flowers developed, while non-significant difference observed for seed germination percentage, number of days taken for seed germination and pod formation. Enzymatic characterization of T1 transgenic progeny exhibited significantly increased superoxide dismutase activity (SOD) whereas no oxalate oxidase activity was observed. It was also noted that additional SOD activity attributed to OsRGLP1 transgenic lines was heat resistant. Further, this additional added SOD activity in transgenic lines was insensitive to KCN and sensitive to H2O2 indicating its resemblance to FeSOD rather than Mn or CuSOD. The functionally active status of transgene was also monitored by high accumulation of H2O2 in xviii transformed progeny. Some selected transgenic lines were also tested for their reaction against a highly pathogenic strain of Fusarium oxysporum, a serious soil borne root affecting pathogen active against a wide range of agricultural crops including legumes. Wild type plants were highly affected by pathogen infection showing a percent disease index value of 50 and bore only 2-3 pods per plant, whereas transgenic lines demonstrated high level of tolerance and provided protection against damaging effect of infection with PDA value ranging from 10 to 18 with 14 to 16 pods formed per plant. Significant loss of fresh weight was also observed in infected wild type as compared to transgenic plants. Microscopic observation of roots of infected transgenic plants demonstrated reduction in the attachment and penetrance of fungal hyphae to plant root cells. Transcriptional analysis of subset of defense-related proteins revealed that the expression of OsRGLP1 gene was responsible for activation in the expression of PR-1, PR-1580, PR-1A670 and Def-1 and down regulation for PR-10 in transgenic plants. The findings of the study may suggest that OsRGLP1 is a heat resistant FeSOD-like protein responsible for providing protection against fungal pathogen Fusarium oxysporum, as one of its functions.