Identification Of LRR-RLK Gene In Vernicia Fordii And Optimization Of TRV-mediated Transient Silencing System

Tung tree(Vernicia fordii)is an important woody oil species in China.The oil extracted from its seeds,known as tung oil,has significant industrial value.Although the V.fordii is widely planted due to its fast growth,early fruiting,high yield,and good oil quality,the wilt disease of tung tree is one of the important factors that hinder its development,and it is difficult to be effectively controlled at the root cause.In contrast,the V montana has a lower incidence of the disease,but it grows and fruits slowly,and its oil quality is inferior to that of the V.fordii.Currently,the prevention and control of tung tree wilt disease rely on grafting V fordii onto V.montana rootstocks that are resistant to the disease,which can alleviate the spread of the disease to a certain extent.Therefore,it is of great significance to study the genes related to Tung Fusarium wilt at the molecular level for the breeding of tung oil disease resistance and the improvement of its yield.In order to initially explore the molecular mechanism of V.fordii disease resistance,the research group screened out the key gene VfLRR-RLK(Vf06G1605)that responds to the infection of Tung Fusarium wilt based on the genome and transcriptome data of tung tree.In this study,using V.fordii as the research material,used bioinformatics,gene cloning,and transgenic technologies to clone and investigate the biological functions of members of the Leucine-rich repeat receptor-like protein kinase(LRR-RLKs)gene family.This study explore the molecular mechanism of VfLRR-RLK(Vf06G1605)in regulating the resistance of V.fordii to wilt disease.The main research findings are as follows:1.Identification of the LRR-RLK gene family in V.fordii was conducted.Using bioinformatics methods,167 members of the LRR-RLK family were identified from the V.fordii genome database.These genes encode proteins ranging from 255-1937 amino acids in length,with molecular weights ranging from 28.08-216.77 kDa and isoelectric points ranging from 5.08-9.69.2.A VfLRR-RLK gene member was cloned from V.fordii.By analyzing transcriptome data and using RT-PCR cloning technology,one VfLRR-RLK gene member,Vf06G1605,was isolated.The full-length CDS sequence of this member is 1134 bp,encoding 377 amino acids,and belongs to the LRR VI-2 subfamily of the LRR-RLKs gene family.3.A plant overexpression vector was successfully constructed and transformed into Agrobacterium,which was then used for floral infiltration to infect wild-type Arabidopsis.After resistance screening and PCR identification,positive transgenic plants were obtained.Phenotypic observation showed that the root system of transgenic plants was more developed than that of wild-type plants.Leaf and plant inoculation experiments showed that the transgenic plants displayed a certain degree of resistance to Fusarium oxysporum.VJLRR-RLK(Vf06G1605)may reduce plant ROS levels and promote plant root development by activating the plant ROS antioxidant system,thereby improving plant tolerance to pathogenic bacteria.4.The optimization of the transient silencing system mediated by V fordii TRV was conducted,and a VIGS vector containing VfLRR-RLK(Vf06G1605),pTRV2VfLRR-RLK,was established.Tobacco rattle virus(TRV)was used as the viral vector to silence the VfLRR-RLK(Vf06G1605)gene fragment.The gene was successfully silenced in V fordii leaves via the back infiltration method.It was found that the phenotypic observation of plants was seriously affected when the concentration of infection solution was OD600=1.2-1.5,while it had no effect on plant phenotypes when the concentration of infection solution was OD600=0.6-0.8.The infiltration solution concentration range that could achieve silencing effect without affecting the plant phenotype was estimated to be between 0.6 and 1.2.This study provides a basis for further study on the disease resistance function of VfLRR-RLK(Vf06G1605)gene in Fusarium wilt.