Analysis Of Lethal Effect Of RNAi Targeting On RXR1 Gene Of Tetranychus Cinnabarinus During Egg

Tetranychus cinnabarinus is a worldwide pest mite with rapidly expanding populations and often cause serious damage to many crops.The control of agricultural mites,including T.cinnabarinus,is mainly based on chemical control,but at present,the pest mites represented by Tetranychidae have become the most prominent arthropods with resistance problems to chemical pesticides,so it is of great significance to explore safe and efficient new control targets and methods for mite prevention and control.Based on RNAi technology,key genes during egg development were used as candidate targets to screen suitable target genes for RNAi control in this paper,and its lethal mechanism was preliminarily analyzed.The main research results are as follows:1.Screening of key genes for egg developmentThe transcriptome sequencing was conducted to analyze gene expressing during the egg stage.It was found that the expression of hormone receptor gene TcRXR1 were relatively high.TcRXR1 was highly expressed on the second and third days of egg stage,and then decreased gradually.As molecular chaperone of receptor protein,a heat shock protein gene,TcHsp70-E also had the highest expression level in 2-day-old and3-day-old eggs.The expression of chitinase gene was important for hatching of eggs,and the expression of TcCHT-E was higher in the late stage of egg development,and its expression peak appeared immediately after the peak of TcRXR1.Based on the above results,the follow-up research was focused on TcRXR1 to evaluate its potential as a specific target gene for control of mite.2.Analysis of molecular characteristics and expression mode of TcRXR1Sequence analysis showed that TcRXR1 was 1419 bp,encoding 496 aa.The predicted molecular weight of protein encoded by TcRXR1 was 55.64 k D,and its isoelectric point was 7.39.TcRXR1 had two conserved domains,DNA Binding Domain(DBD)with two C4-type zinc fingers and Ligand Binding Domain(LBD).In addition,it also had RNA Recognition Motif(RRM).Its protein structure indicated that TcRXR1 can activate and exercise its function through other receptors or ligands.The phylogenetic tree of TcRXR1 showed that TcRXR1 had the highest homology with RXR of P.citri.After detecting the expression level of TcRXR1 in the all stages of mite,it was found that the expression peak was 2-day-old eggs,consistent with transcriptome data.3.Research on RNAi lethal effect of TcRXR1qPCR results showed that the expression of TcRXR1 had significant decrease of42.20% after RNAi.The newly hatched larvae of T.cinnabarinus was feed continuously on ds TcRXR1 and still normally developed into adult mites.Results showed that ds TcRXR1 made no difference to mites after egg hatchin.Subsequently,eggs that were treated with ds TcRXR1 had the significant lethal effect,and the unsuccessful hatching rate of eggs was as high as 70.94%.Dead eggs showed malformation and shrinking,and incomplete hatching.The results indicating that TcRXR1 expression was reduced to produce lethal effect on egg,and it could be a specific target of nucleic acid pesticide for egg control.In order to further analyze the lethal mechanism of ds TcRXR1,transcriptome sequencing analysis was performed to analyze gene expressing under the effect of ds TcRXR1.Totally 173 differentially expressed genes were got(105 up-regulated genes and 68 down-regulated genes).Among the down-regulated genes,three genes were found to be related to chitin metabolism,they were annotated as homologous genes of tetur17g02930,tetur24g01740,tetur32g00060,respectively.Among them,TcCHT-E(tetur17g02930)was found expressed high in egg in previous sequencing.Since the lethal phenotype in egg stage was related to molting of egg shell,TcCHT-E was thought to be a direct lethal factor to be evaluated in follow-up research of this study.4.Molecular characterization and lethal effect analysis of TcCHT-EAccording to sequence analysis,the coding region of TcCHT-E was 1593 bp,TcCHT-E consisted of 530 aa,with 59.74 k Da molecular weight and 6.41 isoelectric point.TcCHT-E had a typical GH18 chitolectin chitotriosidase domain,as well as Chitin-binding domain type 2(Cht BD2)The phylogenetic tree of TcCHT-E showed high homology with the chitinase of mite.The expression pattern of TcCHT-E was high during late egg development and peaked at 3-day-old egg,which was just follow the expression peak of TcRXR1.With RNAi,the expression of TcCHT-E significantly decreased by 63.83%.More than 40% of eggs could not hatch,and the mortality of eggs was observed under the effect of ds TcCHT-E as the same as ds TcRXR1,such as egg malformation and atrophy.This indicated that the function of TcCHT-E was related to egg growth and development,and expression of TcCHT-E was regulated by TcRXR1.The interference of TcRXR1 also reduced the expression of TcCHT-E,resulting in a lethal effect on eggs and a high rate of unsuccessful hatching.5.Identification of TcRXR1 ligand proteinsTcRXR1 with GST tag was obtained by prokaryotic expression method,and 40 different interacting proteins from total protein of mite were obtained by the GST-pull down.According to protein function,eight functional proteins were screened,among which TcHsp70-E(tetur08g01320),as a ligand protein,was the most highly expressed gene of the heat shock protein in previous analysis.On the basis,the specific binding among TcRXR1 and TcHsp70-E was further proved by GST-pull down,which confirmed that TcHsp70-E was one of the ligand proteins of TcRXR1.RNAi experiment results of TcHsp70-E showed that TcHsp70-E was involved in egg growth and development,but its gene expression was not significantly related to the expression of TcRXR1 at room temperature.This study proved that the interference of TcRXR1 reduced the expression of its downstream gene TcCHT-E,which was involved in egg growth and development,hindered the chitin metabolism of egg shell during egg hatching,thus producing a specific lethal effect on eggs.It was also found that that TcHsp70-E was the ligand protein of TcRXR1.The results showed that TcRXR1 and TcCHT-E can be used as potential targets of nucleic acid pesticides for the control of T.cinnabarinus during egg.