Cloning And Response To Stress Of Core Genes In RNAi Pathway Of Empoasca Onukii

Empoasca onukii is a Hemiptera genus,one of the most common tea plant pests in major tea areas in China.The use of sucking mouthparts to suck tea tree juice and spawn in young shoots seriously harms the health of tea trees and affects the quality and yield of tea.However,due to its small size,strong athletic ability,good concealment and rapid reproduction,Empoasca onukii is difficult to control.RNA interference(RNAi)has great potential in insect function gene identification and population management,and is known as the future of pest control.However,RNAi still faces the problem of low interference efficiency in the application of Hemiptera insects.Insects often encounter various stresses in natural growth environments,such as high temperature,low temperature,photoperiod,food shortage,mechanical damage,etc.These unfavorable factors may affect the efficiency of RNAi.In order to apply RNAi to mine key functional genes of Empoasca onukii and develop new measures for pest control,it is important to explore the factors affecting the efficiency of RNAi for the successful application of insect RNAi.In this study,(ORF)of three core genes EoSID-1,EoDCR-2,and EoAGO-2 of Empoasca onukii RNAi pathway was cloned by RACE-PCR;RNAi was analyzed by real-time PCR(RT-qPCR).The relative expression levels of core genes in different tissues,different ages,and different stresses such as temperature,photoperiod,and starvation length of Empoasca onukii were presumed to affect the efficiency of RNAi.Finally,the core gene of RNAi pathway was studied for exogenous dsRNA.In response to the situation,the feasibility of using microinjection for RNA interference was discussed.The main findings are as follows:1.The full-length sequences of three Empoasca onukii RNAi core genes were cloned and named EoSID-1,EoDCR-2 and EoAGO-2.Through NCB1 BLAST homologous sequence alignment and functional domain validation:ORF of the gene EoSID-1 is 2346 bp,encoding 781 amino acids,with a signal peptide at the N-terminus,with 11 transmembrane structures;EoDCR-2 ORF is 4881 bp,encoding 1626 amino acids,and has the same functional domain as the nematode;the EoAGO-2 ORF is 3306 bp,encoding 1101 amino acids with PAZ and Piwi domains.The RNAi core gene of herbivorous insects is relatively conservative,and the above three genes have high homology among different species.Phylogenetic analysis showed that EoSID-1,EoDCR-2 and EoAGO-2 were associated with Recilia dorsalis,Graminella nigrifrons and Nephotettix cincticeps.The kinship is relatively similar.2.Empoasca onukii quantitative PCR results at different ages and different sites showed that EoSID-1,EoDCR-2 and EoAGO-2 were expressed in different tissues of the 4th instar Empoasca onukii,and expressed in the spider mites to adult and adult heads,chest and abdomen.EoSID-1,EoDCR-2 and EoAGO-2 were expressed in 4th instar to adult.The three genes were the lowest in the 4th day of Empoasca onukii,and the lowest in the 4th instar.EoAGO-2 had the lowest expression in the female chest,and all of them had the highest relative expression in the male head,abdominal expression is lowest.3.Empoasca onukii After different temperature treatments.EoSID-1 had no significant difference at 4℃,25℃,36℃;EoDCR-2 The expression level was the highest at 25℃,and the expression was inhibited at 4 0 C at low temperature;EoAGO-2 was significantly inhibited at high temperature4.EoSID-1,EoDCR-2 and EoAGO-2 were most expressed under normal photoperiod(14 hours light-10 hours dark)under different photoperiod treatments;EoDCR-2 was in normal photoperiod and complete Dark and full light were significantly up-regulated compared to expression;EoSID-1 had no significant difference in expression under three photoperiods;EoAGO-2 normal light was significantly up-regulated compared to complete darkness5.EoSID-1,EoDCR-2 and EoAGO-2 were the lowest expression in the treatment of different starvation periods at 3 hours of starvation,EoAGO-2 starvation for 3 hours was significantly inhibited;EoSID-1 was the highest in 6 hours of starvation,but in four different hungers.There was no significant difference in duration treatment;the relative expression of EoDCR-2 was the highest when it was starved for 12 h,and was significantly up-regulated compared with the lower 0 and 3 hours6.In order to explore and establish the use of microinjection to deliver dsRNA to Empoasca onukii,the expression of Empoasca onukii RNAi core genes EoSID-1、EoDCR-2 and EoAGO-2 was analyzed by real-time PCR.The results showed that:Empoasca onukii microinjection mechanical injury Significant effects on EoSID-1、EoDCR-2、EoAGO-2 gene expression.EoSID-1,EoDCR-2,and EoAGO-2 increased significantly after 24 hours of microinjection of dsRNA compared to the control,indicating that 397 bp of dsRNA was successfully introduced into Empoasca onukii by microinjection and 24 hours after injection.The RNAi pathway was activated,and the expression of the three genes was not significantly different with the blank control after 48 h,suggesting that the RNAi process may have been completed.In summary,extreme temperature,extreme photoperiod,starvation and other stress treatments may affect the activity of RNAi pathway in Empoasca onukii,which may affect the efficiency of RNAi.The results of this test suggest that the RNAi test can be performed on the Empoasca onukii at a temperature of 25℃ and a photoperiod of 14 L-10 D by microinjection.The above research results are intended to provide a theoretical reference for the subsequent RNAi microinjection test to eliminate interference factors in Empoasca onukii,but the specific effect on silencing efficiency still needs to find the Empoasca onukii silencing target gene and further experiments to verify.