| Tomato is an important cash crop in China’s agricultural production,and it often suffers from various adverse environmental threats during the growth process,which cause significant economic loss to the agricultural production.With the development of biotechnology,improving the resistance of plants through genetic engineering has become a very feasible method,and the key of this technology is the mining and research of resistance genes.In this study,the SlR1-A-like gene was cloned from tomato for the first time.We constructed the overexpression vector successfully and obtained the transgenic N.benthamiana plants.The function of the transgenic plants on cold stress and infection by P.infestans was researched.The specific research results are as follows:(1)Bioinformatics analysis showed that the SlR1-A-like gene was 1701 bp in length and encodes 566 amino acids.The SlR1-A-like protein was a hydrophilic,non-transmembrane protein with a NB-ARC functional domain,which has a molecular weight of 65.7kDa and the isoelectric point was 5.69.(2)The results of tissue expression analysis showed that SlR1-A-like gene was the highest expressed in tomato leaves.Analysis of the expression pattern of SlR1-A-like gene under abiotic stress and hormone induction showed that the gene expression responsed to different treatments.The effect of up-regulation of SlR1-A-like gene expression was most significant after cold stress and SA treatment.Subcellular localization results showed that SlR1-A-like protein was localized in the nucleus and cytoplasm.(3)The transgenic N.benthamiana showed the wilting degree was more severe than wildtype N.benthamiana under cold stress.Determination of related indicators showed that transgenic N.benthamiana contained less osmotic regulators such as proline and soluble sugar than wild-type N.benthamiana.The electrolyte leakage and MDA content of transgenic N.benthamiana were significantly higher than those in wild-type N.benthamiana,which proved that the damage of membrane system in transgenic N.benthamiana was more serious.The results of photosynthesis-related indicators showed that the photosynthesis capacity of transgenic N.benthamiana was significantly reduced under the cold stress.At the gene level,the expression level of the antioxidant enzyme genes in the transgenic N.benthamiana was low,the antioxidant enzyme activitys decreased,which led to the accumulation of reactive oxygen species.At the same time,the expression of cold-resistant genes in transgenic N.benthamiana was significantly lower than that of wild-type N.benthamiana.(4)The results showed that transgenic N.benthamiana was less susceptible than wild-type N.benthamiana after inoculated by P.infestans.According to the results of statistical analysis,the disease index and lesion diameter of the transgenic N.benthamiana were significantly lower than those of the wild-type N.benthamiana,which proved that the transgenic N.benthamiana was more resistant to P.infestans.The results of reactive oxygen staining showed that the content of reactive oxygen species in transgenic N.benthamiana was lower than that of wildtype N.benthamiana.At the gene level,the expression level of the antioxidant enzyme genes in transgenic N.benthamiana was relatively high,so the antioxidant enzyme activitys increased,which could timely remove excess reactive oxygen species in the plant and protect plant cells from damage.At the same time,the expression level of pathogenesis-related genes in transgenic N.benthamiana was significantly higher than that of wild-type N.benthamiana.(5)The SlR1-A-like gene was successfully silenced in tomato Heinz 1706 plants through the virus silencing vector.Cold stress and P.infestans inoculate experiments were performed on the silenced tomato plants.In contrast to the overexpression experiments,the silenced plants showed resistance to cold stress and susceptibility to P.infestans. |
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