| Euseius nicholsi is an important species of Phytoseidae,which can prey on agricultural pest insects and mites such as Panonychus citri and thrips.As a dominant species in China,E.nicholsi is a predatory natural enemy with various characteristics.At present,it has not achieved low-cost propagation,only can rely on natural prey and various pollen.Nutrient addidents(e.g.,protein and sugar)are considered to be important regulatory factors in growth,development,and reproduction of insects,and they obtain enough nutrition from foods to maintain their own reproduction.As a critical gene in the nutritional signaling pathway,target of rapamycin(TOR)is vital for insect growth,development,and reproduction.In this study,the full length of TOR gene was firstly cloned by RT-PCR combined with RACE technology,and its sequences were analyzed by bioinformatics.Then,the RT-qPCR technology was used to determine the mRNA expression levels of EnTOR in different developmental stages from E.nicholsi,as well as EnTOR and EnVg after adding different nutrients.Furthermore,RNA interference was used to explore the biological function of EnTOR from E.nicholsi.The findings will lay a foundation for elucidating molecular mechanism of nutrition-mediated TOR signaling pathway in E.nicholsi,and providing a theoretical basis for nutritional regulation of reproduction and biological control application of E.nicholsi.The main results are as follows:1.Bioinformatics analysis of TOR gene sequence from E.nicholsi.In this research,the full length of TOR gene(GenBank accession number: OK665840)from E.nicholsi was firstly cloned.It contained 7999 bp,including an open reading frame(ORF)of 7491 bp,encoded by 2496 amino acids which had 283.69 k Da molecular weight and 7.21 theoretical PI.The protein encoded by EnTOR contained five conserved domains: DUF3385 domain,FAT domain,FRB domain,PI3 Kc domain and FATC domain.The results of deduced amino acid sequence indicated that EnTOR was91.95% similar to the TOR gene of Galendromus occidentalis.Both of them are belonging to Phytoseiidae family.In addition,the phylogenetic tree consists of three branches: Insecta,Arachnida and Malacostraca.In the Arachnid branches,EnTOR and Go TOR were clustered together and related the most.2.Analysis of gene expression levels of TOR and Vg from E.nicholsi.RT-qPCR was used to analyze the expression pattern of the TOR gene in E.nicholsi at various developmental stages.According to the results,the expression of EnTOR was detectedin in different developmental stages of E.nicholsi.The expression level of the TOR gene in adult females was 6.87,1.34,and 2.16 times higher than in egg,larva,and duetonymph stages(P<0.05),respectively.However,there was no distinct difference in expression levels between adult female and protonymph(P>0.05).Yeast supplementation significantly promoted the mRNA expression of TOR gene in adult females of E.nicholsi,while sugar supplementation significantly inhibited the mRNA expression of TOR gene in adult females(P<0.05).After adding yeast,the transcription levels of the TOR gene in protonymphs,duetonymphs,and adult females were 1.37,21.55,and 18.25 times higher than control groups,respectively.Compared with control groups,the mRNA expression of EnVg gene in the experimental groups(pollen+sugar group and pollen+yeast group)increased significantly in the period of protonymph(P<0.05).The transcription level of adult female of E.nicholsi was significantly increased(P<0.05)with the additional yeast treatment,and was 2.82 and2.32 times greater than the control and sugar added groups,respectively.3.Study on the biological function of TOR gene from E.nicholsi.The biological function of EnTOR was firstly explored by RNAi with oral delivery of dsRNA in E.nicholsi in this study.The biological parameters related to reproduction(i.e.,spawning time,fecundity,lifespan of adult female,egg hatching rate,and the mRNA expression of TOR and Vg genes after interference)were calculated after oral delivery ds TOR for 48 hours to 2-day-old adult females.Compared with the control group,when the TOR gene was disturbed,the period of oviposition,fecundity and egg hatching rate of E.nicholsi were significantly reduced(P<0.05),which were 52.40%,52.70%,and 86.80% of the control group,respectively.Although the lifespan of adult female of E.nicholsi in ds TOR group was shorter than that of ds GFP group,the difference was not significant(P>0.05).After interfering with the TOR gene of female adult of E.nicholsi,the mRNA expression of TOR gene and Vg genes were detected by RT-qPCR.It was found that the mRNA expression of TOR gene was significantly inhibited,with about 47.74% reduction in expression.The transcript level of Vg gene was also significantly reduced compared with the control group(P<0.05). |
PDF Full Text Request