Screening The Arabidopsis Thaliana CDNA Library For Proteins Interacting With TYLCCNV Pathogenicity Factor βC1 Using Yeast Two-hybrid Method

The Chinese tomato yellow leaf curl virus(TYLCCNV)is a Geminiviridae family virus that belongs to the Begomovirus that causes severe crop damage in a number of crops.βC1encoded by β satellite DNA accompanying TYLCCNV is a key pathogenic factor and engages in numerous plant regulatory pathways.Most studies on βC1 are currently being conducted in tobacco,the natural host of TYLCCNV,but the completeness of genome sequencing and genome annotation,knockout mutants,and other genetic resources of the allotetraploid tobacco are relatively insufficient,using it as a model plant to study βC1 is still inconvenient.Therefore,we will take a further study on the complex functions of βC1 with A.thaliana,a popular model plant with them most abundant genetic information and genetic resources.It does not belong to the TYLCCNV host range and cannot be infected by TYLCCNV,but the phenotypes of transgenic A.thaliana overexpressing βC1,such as curled leaves,show that βC1 interacts with important plant components and generates viral symptoms,and that the process that influences plant growth and development is comparable to that of natural hosts.Yeast two-hybrid method was employed in this work to screen the A.thaliana cDNA library for proteins interacting with βC1 and study the complex functions of βC1.The research content mainly includes four aspects:(1)Constructing the expression vector of βC1.The two vectors constructed,pGADT7-βC1 and pGBKT7-βC1,are not cytotoxic to yeast.However,the vector,pGADT7-βC1,shows autoactivation of reporter genes,His3 and Ade2,pGBKT7-βC1 doesn’t.So pGBKT7-βC1 was selected for subsequent experiments.(2)Constructing the expression vector of cDNA library.The four synthesized primers,Rec1/Rec4 and Rec2/Rec3,were paired by phosphorylation and annealing respectively to synthesize homologous arm Rec.Then the Rec was cloned into the vector pGADT7,namely pGADT7 Rec,which was used to express dsDNA.(3)Preparing the cDNA library of yeast.m RNA was isolated from total RNA extracted from A.thaliana,and dsDNA synthesized by reverse transcription and PCR amplification using m RNA as a template was transformed into yeast together with pGADT7 Rec.Finally,all yeast were collected and stored at-80 ℃,namely the cDNA library.(4)Screening the Arabidopsis thaliana cDNA library.Mating the bait strain containing pGBKT7-βC1 and library strain,the positive clones were screened by SD medium,PCR and other methods.The library plasmids were sequenced,and the candidate proteins were analyzed by sequence alignment.Through sequencing,comparison and analysis of the screened candidate proteins,a total of 16 candidate proteins were screened from the yeast cDNA library of Arabidopsis thaliana,which mainly include vascular transport-related pathways,r RNA maturation pathway,transcription factors,plant hormone pathways,and plant stress response pathways factors.Our work provides basis for further study of the mechanism of βC1,and have certain application value in plant disease resistance.