Research On Molecular Mechanism Of Sly-miR482a Negatively Regulating Plant Fruit Development And Resistance To Botrytis Cinerea

During the whole growth and development process,plants are extremely susceptible to the influence of the surrounding environment.Gray mold of tomato(Solanum lycopersicum)caused by the pathogen Botrytis cinerea is one of the main factors that harm the production of tomato.In the previous study,we found that the expression of mi R482a(sly-mi R482a)was changed in tomato leaves infected by Botrytis cinerea.In order to further understand the function of sly-mi R482 a,we have used molecular biology techniques,cell biology techniques and genetic engineering techniques to conduct in-depth research on sly-mi R482 a influencing tomato growth and tolerance to Botrytis cinerea,and obtained some research results.The results are as follows :1.Using qPCR,we found that in tomato roots,stems,leaves,flowers and fruits,the expression abundance of sly-mi R482 a in flowers and fruits is much higher than that of the other three tissues.In addition,we also found in tomato leaves,the infection of Botrytis cinerea can up-regulate the expression of sly-mi R482 a.The above results indicate that mi R482 a is a bifunctional gene involved in the developmental regulation of plant flowers and fruits,as well as in the regulation of plant immune responses against Botrytis cinerea.2.To further understand the mechanism by which mi R482 a responds to Botrytis cinerea infection,we inoculated Botrytis cinerea on transgenic Arabidopsis thaliana and transgenic tomato overexpressing sly-mi R482 a.The results showed that compared with the wild type,the surface area of transgenic Arabidopsis and tomato showed larger lesion area,and the results of trypan blue staining and real-time PCR showed that the Botrytis cinerea hyphae proliferated faster in the transgenic plants.It is indicated that overexpression of sly-mi R482 a increases the sensitivity of plants to Botrytis cinerea infection.3.The target gene of sly-miR482 a was mainly the gene encoding the resistance protein,and the expression in the transgenic Arabidopsis thaliana and transgenic tomato leaves overexpressing sly-mi R482 a was significantly decreased,which was negatively correlated with the expression of mi R482 a,indicating that sly-mi R482 a Immunization of plants against Botrytis cinerea infection is reduced by targeting resistant proteins.4.In the transgenic Arabidopsis thaliana overexpressing sly-mi R482 a,the phenotype of delayed twitching and seed setting rate decreased,and the seedling rate decreased in transgenic tomato,indicating that mi R482 a negatively regulates flower development or fruit development of plants;The pectate lyase gene,a target gene of sly-mi R482 a,can positively regulate the development of flower hair or fruit,indicating that sly-mi R482 a is likely to delay the development of flower and fruit by targeting pectate lyase gene.In this study,transgenic Arabidopsis thaliana and transgenic tomato material overexpressing sly-mi R482 a were used to investigate the effect of sly-mi R482 a on Arabidopsis thaliana and tomato resistance to Botrytis cinerea infection,revealing that sly-mi R482 a targets(solyc04g0073201.1、solyc01g102920.2.1、solyc08g080850.2.1、At5g17680.1 、 At1g17600.1 、 At4g14610.1)in response to Botrytis cinerea and encoding proteins negatively regulate the resistance of Arabidopsis and tomato to in response to Botrytis cinerea infection,and negatively regulate the pectate lyase gene(solyc11g008140.1.1、At3g53190.1)delays the development of flowers and fruits in Arabidopsis thaliana and tomato.These studies will lay the foundation for further understanding of the regulatory mechanisms of sly-mi R482 a and molecular control of Botrytis cinerea infection.