Cloning And Expression Of A Novel Gene Trehalase From Empoasca Vitis(G(?)the)

Empoasca vitis?G???the?is widely distributed in various tea regions in China.It is one of the most important tea pests in tea gardens.This pest is difficult to control,mainly relying on chemical pesticides,resulting in a serious"3R"problem.Therefore,there is an urgent event to find the new ideas for the prevention and treatment of Empoasca vitis.Studies have shown that the Trehalase of insect is a key enzyme in diapause hormone regulation and chitin synthesis,which is of great significance to the growth and development of insects.In this study,RACE technology and fluorescence quantitative PCR technology were used to clone and obtain the trehalase gene from the Empoasca vitis.It clearly clarified its expression and provided new ideas for the prevention and control of leafhoppers,laying the foundation for new technologies for controlling pests.Part 1,wehave selectedthe reference genes of Empoasca vitis and selected 5 kinds of internal gene?18S,ACT,EF1?,RPL10,?-TuB?from Empoasca vitis transcriptome data,and design the specific primers.We test the stability of internal gene by Fluorescent Quantitative PCR technique to detect different processing?different developmental stage,low temperature stress,starvation stress treatment,pesticides stress?.And using geNorm,NormFinder,Bestkeeper,?Ct method and RefFinder software to analyze the reliability of the reference genes.The results showed that the stability of RPL10 and EF1?was the best reference gene at different developmental stages.Under pesticide stress,18S and ACT were the best reference genes.Under the starvation stress,EF1?and RPL10 could be the most stable genes.Under low temperature stress,RPL10 and EF1?were the most stable genes.Part 2.Cloning and analysis of the trehalase gene from the Empoasca vitis.The total length of the trehalase gene was 2469 bp,including ORF 1797 bp in the open reading frame,which encoded a total of 642 amino acids.The start codon and stop codon were ATG and TAG,respectively.The sequence contains the classic tagged sequences“PGGRFREFYYWDSY”and“QWDYPNAWPP”of the trehalase gene and a glycine-enriched region"GGGGEY".The predicted molecular weight of the encoded protein is 74185.44,the theoretical isoelectric point is 5.80,the instability coefficient is38.91,the molecular formula is C₃₃₆₁H₅₀₉₁N₈₆₉O₉₇₂S₃₀,the total fat is 77.59,and the total average hydrophilicity is-0.381.This amino acid sequence has high similarity to other insect trehalase genes that have been published.The analysis revealed that the gene predicted two high-span transmembrane regions,and therefore the trehalase gene was membrane-bound trehalase and was named EvTre2.Part 3.Expression analysis of EvTre2 gene under different treatments,The EvTre2is expressed under different treatments.Under the low temperature stress treatment,the EvTre2 expression in each low temperature treatment was higher than that in untreated adults,and the expression level was the highest in samples treated at 4°C for one hour.In the pesticides stress,the EvTre2 expression was increased in the samples treated with1400 mL/hm,3000 mL/hmand 5000 mL/hm of the three pesticides,and reached the highest at a concentration of 0.3%.Under different developmental stages,the nymphs'expression was lower than that of adults.Under starvation stress,starvation was increased at each time point,especially in the 60 h starvation samples.The results show that EvTre2 is closely related to the resistance mechanism of Empoasca vitis.In summary,the present study successfully cloned the membrane-bound trehalase gene from Empoasca vitis.for the first time,and conducted a bioinformatics analysis and expression analysis to further study the EvTre2 gene in the Empoasca vitis.The role of the Empoasca vitis laid the foundation for the prevention and treatment of Empoasca vitis provide a new idea.