Establishment Of Screening System For Volatiles That Induces Tea Plant Defense Against Tea Green Leafhopper And Preliminary Study On(E)-Nerolidol Induction Mechanism

The tea plant(Camellia sinensis)is an economically important woody crop in Asia,and the tea green leafhopper,Empoasca(Matsumurasca)onukii Matsuda(Hemiptera: Cicadellidae)is a serious pest of tea plants,which is lack of efficient and harmless prevention and control technology.Plants release large amounts of volatile organic compounds(VOCs)in response to attackers.Several VOCs can serve as volatile signals to elicit defense responses in undamaged tissues and neighboring plants.Therefore,the efficient volatile elicitors can provide a new idea for the prevention and control of tea green leafhoppers.In this study,12-oxophytodienoate reductase gene CsOPR3 was confirmed as a marker gene related to the resistance of tea plants against the leafhopper.Based on CsOPR3,a rapid screening system was set up in Arabidopsis.Using this screening system and bioassay,the inducement activity of a candidate substance,(E)-nerilodol,was identified.Then a preliminary study was conducted on(E)-nerilodol induction mechanism.The main results are as follows.1.The protein level of CsOPR3 was increased by wounding,jasmonic acid and tea green leafhopper treatment,and the transcript level of CsOPR3 gene was increased by tea green leafhopper treatment.CsOPR3 was cloned,which containing an open reading frame of 1197 bp.The 35S::CsOPR3 overexpression vector construct was inserted into the opr3 mutant,and two T2 homozygous lines were obtained.After wounding treatment,the content of JA in the opr3 mutant was supplemented by the overexpression of the CsOPR3 gene.At the same time,the number of tea green leafhopper on tea branches was significantly reduced after exogenous JA treatment.Therefore,the CsOPR3 gene can be used as a molecular marker in the rapid screening method to determine the volatile elicitors.2.A 1510 bp promoter sequence upstream of tea plant transcription was cloned.This promoter sequence contained a typical core promoter element(TATA-box),an enhancer element(CAAT-box),a transcriptional start site,and other defense-responsive cis-acting elements.The promoter was fused to a GUS gene,and two independent lines in Arabidopsis(OPR3p::GUS lines,pL1-1 and pL2-5)were finally generated.JA treatments remarkably increased GUS activity in transgenic Arabidopsis leaves by more than four times.After JA treatment,the foliar tissues of pL1-1 and pL2-5 leaves were stained blue by GUS staining.Thus,a visual rapid screening system for volatile elicitors was established.Using this screening system and bioassay,the candidate,(E)-nerolidol,was identified.3.After(E)-nerolidol exogenously treated,the transcription and protein levels of CsMAPK and CsWRKY3 in tea plant was increased,hydrogen peroxide burst,the transcription level of CsCHIT1 and CsOPR3 was increased,and the content of jasmonic acid,jasmonoyl-isoleucine(JA-Ile)and abscisic acid(ABA)were increased.In this study,CsOPR3 was selected as a marker gene for tea plants defense against tea green leafhopper.Based on CsOPR3,a visual and rapid screening system was set up in Arabidopsis.This screening system was verified by the active substance JA and(E)-nerolidol.Exogenous(E)-nerolidol treatment activated the early defense signal,increase the transcription level of defense-related genes,and promote the synthesis of defense signal molecules in tea plants.The above results provide a new idea and theoretical basis for the prevention and control of tea green leafhoppers.