Functional Analysis Of TSSKs Genes Of Cydia Pomonella

The codling moth,Cydia pomonell,is a major agricultural invasive pest in China,which seriously threatens the safety of fruit production in my country’s Loess Plateau and the Bohai Bay.It has been included in the List of Class I Crop Diseases and Pests by the Ministry of Agriculture and Rural Affairs.The control of C.pomonella in almost all apple producing areas in the world mainly relies on chemical pesticides.However,the overuse of pesticides has resulted in developing resistance to more than 60pesticides.Sterile insect technique（SIT）is an environmentally friendly pest control technology.It mainly made target pests to sterilize through physical irradiation,chemical treatment,genetic modification,and so on.Then it is released in large quantities and continuously into the pest occurrence area to produce sterile offspring through mating with wild females,so as to reduce the population and control the pests.Based on RNA interference（RNAi）and gene editing to regulate testis specific reproductive related genes is a new idea of insect infertility technology.In this study,transcriptome sequencing was used to identify the testis-specific expression genes（TSSKs）in the male adult of C.pomonella,and the gene functions were determined by RNAi and CRISPR/Cas9 technologies.It provides target genes for the control of C.pomonella using genetically modified SIT technology.The main results are:1.Dynamic observation of testis development of C.pomonellaWe found that the 5th instar larvae of C.pomonella had two kidney-shaped testes through dissecting the testes,which are symmetrically distributed on both sides of the body,and the testis was divided into 4 chambers.In the pre-pupal stage,the two independent testes were close to each other and gradually adhered.But it can also be clearly seen that there are two testes.During the pupal stage,the two testes fused into a nearly round testis,which was yellow in color.With the development of the testis,the testis matures in the adult stage and was reddish-brown.After mated,the testis became deflated and the color becomes lighter.2.Testis transcriptome analysis of C.pomonellaTranscriptome analysis was selected on the second and fifth day testis of the codling moth adult.7824 genes were up-regulated in the testis of the codling moth adult on the second day,and 6989 genes were up-regulated in the testis of the adult codling moth on the fifth day.We found that serine/threonine kinases,cyclin genes,and Tektin genes were all highly expressed in the testis tissue of C.pomonella.At the same time,testis-specific serine/threonine kinases were identified（TSSK1,TSSK1a,TSSK2,TSSK2a and TSSK4,log₂Fold Change>7.57）.RT-q PCR was used to analyze the expression pattern of TSSKs family.The results showed that the expression levels in the 5th instar larvae were significantly higher than in other instar larvae,and TSSKs family genes were all highly exoression in adults.They reached the highest level in males（pupa,adults）and were significantly higher than that in females.Among five genes,the expression level of TSSK4 was significantly higher than that of other genes,while the expression levels of TSSK1 and TSSK2a in the adult testis after mating were significantly lower than those before mating.3.RNAi of TSSKsBased on the above results,ds TSSK1a,ds TSSK2 and ds TSSK4 were injected into C.pomonella.Pupa-adult injection was used for this study.After 48 hours of injection into adults of C.pomonella.Compared with the ds GFP,the interference efficiencies of ds TSSK2、ds TSSK1a and ds TSSK4 were 86.09%,42.67%and 75.62%,respectively.72 hours after the injection of the adult beetles,the interference efficiency of ds TSSK4 was 24.57%in 72h,but there was no significant difference ds TSSK1a and ds TSSK2 treatment groups compared with the ds GFP.After injection of ds TSSK2,ds TSSK1a and ds TSSK4,the egg hatching rates were 0%,24.12%and0%,respectively（84.15%in the ds GFP-treated group）.4.Construction of C.pomonella gene editing systemWe Knocked out TSSK1 gene in C.pomonella based on CRISPR/Cas9technology.The results showed that we have successfully verified a homozygous mutant with a deletion of 13 bases,with a gene editing efficiency of 4.35%.But,we can’t obtain the date of reproduction through CRISPR/Cas9.But our results have shown that CRISPR/Cas9 technology is suitable for C.pomonella.But no fertile offspring were obtained,and the function of TSSK1 in codling moth needs to be further determined.