| Adelphocoris suturalis is an omnivorous agricultural pest with a wide range of hosts,and its production mainly depends on chemical control.However,the ensuing problems such as increasing resistance and the potential impact of chemical insecticides on the environment have seriously hindered the sustainable management of A.suturalis.Nutrition is essential for the reproduction of insects.Clarifying the reproductive regulation mechanism of nutritional signaling pathways in A.suturalis can provide new targets for the development of nucleic acid biological pesticides and transgenic insect-resistant crops.Therefore,this study used gene cloning,RNA interference(RNAi),real-time quantitative PCR(RT-q PCR)and other techniques.To explore the key genes of TOR signaling pathway TOR(Target of rapamycin),S6k1(S6 kinase 1),S6k2(S6 kinase 2),Rheb(Ras homolog enriched in brain)and 4E-bp(4E-binding protein)on the fecundity of A.suturalis and its function in reproductive regulation.Finally,the nanomaterial star polycation(SPc)was used to encapsulate ds TOR to spray A.suturalis females,and its effect on fecundity was determined.Analysis of nano nucleic acid pesticide research and development potential.The main results are as follows :1.Effects of nutrition on the fecundity of A.suturalis and the response of TOR pathway to itDuring the growth and development of A.suturalis,the emergence of newly hatched nymphs into adults was significantly shortened by about 0.33 days after feeding animalderived food.The lifetime fecundity of female adults fed with Acyrthosiphon pisum and adults fed with A.pisum significantly increased by about 50% and 52%,respectively.The pre-oviposition period of female adults significantly shortened by about 1.69 d and 1.26 d,respectively.The egg hatching rate significantly increased by about 39%,but there was no significant difference in female longevity.The pre-oviposition period of the female was significantly shortened by 0.43 days when the nymphs were fed with animal-derived food compared with the adult stage.After feeding animal-derived food,the expression levels of TOR,S6k1,S6k2,Rheb and 4E-bp genes in the TOR signaling pathway were significantly up-regulated by about 2.31 times,1.25 times,1.54 times,1.79 times and 1.54 times,respectively,it shows that the TOR signaling pathway of A.suturalis can regulate fecundity by responding to nutritional signals.2.Cloning and sequence analysis of TOR signaling pathway genesThe open reading frame(ORF)of TOR gene was 7344 bp in length,encoding 2447 amino acids(AA)with a molecular weight of 278.71 k Da and an isoelectric point(p I)value of 6.88.Through sequence alignment,it was found that the TOR protein sequence of A.suturalis had the highest similarity with the TOR protein sequence of Cimex lectularius.The results of spatio-temporal expression showed that the expression of TOR was the highest in the 1st instar newly hatched nymphs,and the highest in the chest of 8-day-old female.The ORF sequence of S6k1 gene was 1377 bp in length,encoding 458 AA,with a molecular weight of 51.99 k Da and a p I of 5.77.The ORF sequence of S6k2 gene was 2223 bp in length,encoding 740 AA,with a molecular weight of 83.21 k Da and a p I of 7.92.Through sequence alignment,it was found that the S6K1 and S6K2 protein sequences of A.suturalis had the highest similarity with the S6K1 and S6K2 protein sequences of Apolygus lucorum,respectively.The temporal and spatial expression results showed that the expression of S6k1 was the highest in the male adults of 1 day old,the expression of S6k2 was the highest in the newly hatched nymphs of 1 day old,and the expression of S6k1 and S6k2 was the highest in the ovaries of different tissues of 8 day old females.The ORF sequence of Rheb gene was 549 bp in length,encoding 182 AA,with a molecular weight of 20.63 k Da and a p I value of 5.81.Sequence alignment showed that the RHEB protein sequence of A.suturalis had the highest similarity with the RHEB protein sequence of A.lucorum.The results of spatio-temporal expression showed that Rheb had the highest expression in 1-day-old female and the highest expression in ovary of 8-day-old female.The ORF sequence of 4E-bp gene was 351 bp in length,encoding 116 AA,with a molecular weight of 12.59 k Da and a p I value of 9.34.Through sequence alignment,it was found that the 4E-BP protein sequence of A.suturalis had the highest similarity with the 4E-BP protein sequence of Nesidiocoris tenuis.The results of spatio-temporal expression showed that the expression of 4E-bp was the highest in the first instar nymphs and the highest in the chest of 8-day-old female.3.The effect of TOR pathway genes on fertility and the screening of nucleic acid pesticide target genesAfter injection of TOR inhibitors Rapamycin,ds TOR,ds S6k1,ds S6k2,ds Rheb and ds4E-bp,the lifetime fecundity of female A.suturalis significantly decreased by about 58%,81%,62%,35%,72% and 58%,respectively,and the preoviposition period was significantly prolonged by about 1.08 d,0.72 d,0.51 d,0.72 d,0.59 d and 0.62 d,respectively.The female longevity was significantly shortened by about 1.72 d and 1.98 d after interference with TOR and Rheb,respectively.There was no significant difference in the longevity of females injected with Rapamycin and interfering with the remaining genes,and there was no significant difference in egg hatching rate.In addition,our previous studies have found that Vg(Vitellogenin),Far(Fatty acyl-coenzyme a reductase)and Ap(Aspartic protease)genes are involved in the reproductive regulation of A.suturalis.In this study,we found that the expression of Vg gene in female A.suturalis was significantly down-regulated by about 66%,31%,44% and 22% after RNAi interference with TOR,S6k1,Rheb and 4E-bp genes.The expression of Far gene was significantly down-regulated by about 32%,30%,16% and 50%,respectively,and the expression of Ap gene was significantly down-regulated by about 46%,42%,39% and 21%,respectively.After RNAi interference of S6k2 gene,the expression of Vg gene was significantly down-regulated by about 31%,while the expression of Far and Ap genes was not significantly different.The above results indicate that the TOR signaling pathway may regulate reproduction by regulating the expression of reproduction-related genes,and the TOR gene has the greatest effect on fertility.Therefore,the TOR gene is selected as the target gene of nanonucleic acid pesticides.4.The inhibitory effect of spraying ds TOR/SPc/detergent on the fecundity of A.suturalisThe TOR gene with the greatest influence on the fecundity of A.suturalis was selected,and ds TOR was encapsulated by SPc to prepare ds TOR/SPc/detergent,which was sprayed on the first-day-old female adults.After 3 days of spraying,the expression of female TOR gene was significantly down-regulated by about 46%.After 10 days of spraying,the number of female eggs was significantly decreased by about 54%.In summary,the TOR,S6k1,S6k2,Rheb and 4E-bp genes in the TOR signaling pathway of A.suturalis may affect the expression of reproduction-related genes Vg,Far and Ap,thereby ultimately regulating the fecundity of A.suturalis.Further spraying female insects with SPc-encapsulated ds TOR significantly inhibited fecundity.Therefore,TOR gene can be used as a target gene for RNAi-mediated pest control strategies.The above results provide important theoretical basis and data support for the development of new nano-nucleic acid pesticides for A.suturalis,and provide new strategies for integrated pest management. |
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