| Mythimna separata is a Lepidoptera Noctuidae insect,which is one of the agricultural pests that need to be controlled in China.It has a variety of feeding habits and harms many plant species,which can cause devastating damage in the outbreak.At present,the main prevention and control methods are still based on chemical pesticides.Due to the pollution of chemical pesticides to the environment and the harm to human health,it can not be ignored.Therefore,it is of great significance to explore and develop new green prevention and control methods that are pollution-free to the environment,harmless to agricultural products,and harmless to human health.Trehalase is involved in the hydrolysis of trehalose to glucose,providing energy sources for various life activities of insects,and playing a role in various physiological processes and coping with abiotic stresses.In this paper,we used Mythimna separata as the experimental object,and the trehalase genes Treh1 and Treh2 as the target genes.The target gene silencing was mediated by RNAi technology to explore the mortality of Mythimna separata larvae,the expression changes of target genes and their effects on cold tolerance.The main results are as follows :(1)The relative expression levels in different developmental stages and different tissues were detected by fluorescence quantitative PCR.The results showed that Ms Treh1 and Ms Treh2 genes were expressed in all developmental stages of M.separata.The relative expression level of Ms Treh1 gene was the highest on the first day of egg,followed by the first day of pupa and the first instar larvae.The relative expression of Ms Treh2 gene reached the highest value on the third day of the 6th instar,which was significantly higher than that of other instars,followed by the first day of the 5th instar and the first day of the pupa.There was no significant difference between other instars.Ms Treh1 and Ms Treh2 were expressed in eight tissues,including head,muscle,epidermis,fat body,Malpighian tubule,midgut,testis and ovary.Among them,there was no significant difference in the relative expression of Ms Treh1 in various tissues,but the relative expression of muscle was the highest,while the relative expression of malpighian tube and ovary was higher,and the relative expression of testis was the lowest.The relative expression of Ms Treh2 in muscle was the highest,which was significantly higher than that in the other seven tissues,while the relative expression in head was the lowest.(2)The mortality rate of larvae injected with ds Ms Treh1 reached 53%after 72 h,and the mortality rate of larvae injected with ds Ms Treh2 reached 57% after 72 h,while the control group injected with ds GFP was only 16%.The above results indicate that silencing the target gene by RNAi technology has a certain lethal effect on M.separata.(3)After the synthetic ds Treh1 and ds Treh2 were injected into the 5th instar larvae of M.separata,it was found by fluorescence quantitative PCR that the gene expression levels decreased by 46.43% and 76.79%after 12 hours of injection of ds Ms Treh1 and ds Ms Treh2,respectively.After 24 hours,Treh1 decreased by 34.48%,and Treh2 increased by 14.46%.After 48 hours and 72 hours,it was also lower than the control group,but the interference efficiency was relatively low.(4)The analysis of the supercooling point and freezing point of ds Ms Treh1,ds Ms Treh2 and ds GFP 12 h after injection showed that the supercooling point and freezing point of the larvae injected with ds Ms Treh1 were significantly different from the control group injected with ds GFP(p< 0.01),while the supercooling point of the larvae injected with ds Ms Treh2 was also significantly different from the control group(p<0.01),and the freezing point was not significantly different from the control group.Compared with the control group,the distribution range of supercooling point and freezing point of M.separata injected with ds Ms Treh1 and ds Ms Treh2 interference decreased significantly,and the minimum value increased significantly.After 24 hours of injection of ds Treh1,ds Treh2 and ds GFP,the supercooling point and freezing point of M.separata injected with ds Ms Treh1 and ds Ms Treh2 were higher than those of the control group,especially the freezing point of M.separata injected with ds Treh2 was significantly different from that of the control group(p=0.034).There was no significant difference in the distribution range of supercooling point and freezing point of M.separata injected with ds Treh1 and ds Treh2,but the distribution range was smaller than that of the control group.After 48 hours of injection of ds Ms Treh1,ds Ms Treh2 and ds GFP,compared with the control group,there was no significant difference in the supercooling point of M.separata injected with ds Ms Treh1 and ds Ms Treh2,but the freezing point was significantly different from that of the control group(p=0.009;p=0.022).There was no significant difference in the distribution range of supercooling point and freezing point of M.separata injected with ds Ms Treh1 and ds Ms Treh2.(5)The trehalose content of M.separata injected with ds Ms Treh1 was slightly higher than that of the control group at 12 h,but lower than that of the control group at other stages.The trehalose content of M.separata injected with ds Ms Treh2 was also lower than that of the control group at each time period.The glycogen content was also lower in the post-treatment group than in the control group at 12 h,24h,and 48 h after injection.After 72 h,there may be no difference between the treatment group and the control group with the degradation of ds RNA.In summary,the statistical analysis of the mortality of larvae injected with ds RNA showed the lethal effects of ds Ms Treh1 and ds Ms Treh2 on target larvae.The relative expression levels of Ms Treh1 and Ms Treh2 genes at different time periods after injection of ds RNA were analyzed by RT-q PCR.The results showed that after injection of ds Ms Treh1 and ds Ms Treh2,except that the Ms Treh2 gene showed higher than the control group at 24 h,the rest showed silencing efficiency;the supercooling point and freezing point of larvae injected with ds RNA were detected by thermocouple method and the trehalose content and glycogen content of larvae injected with ds RNA were detected by anthrone colorimetry.The results showed that silencing Treh1 and Treh2 genes could reduce the cold tolerance of M.separata. |
