Effects Of Temperature Tolerance In Neoseiulus Barkeri Based On DNA Methyltransferase

During the 21 st century,biological control has been playing a more and more important role in agricultural system.Neoseiulus barkeri,due to its advantages of short developmental period,many predatory species,high fecility and strong diffusion ability,has become an important biocontrol factor for controlling mites and small agricultural succulent pests.With the global climate warming,mites,as a kind of thermotropic animal,are extremely sensitive to temperature.Therefore,high temperature has also become an abiotic factor restricting predatory mites to continuously control mites in agricultural ecosystems and exert their biological control efficiency.To improve the performance of N.barkeri under field conditions,our research team selected and obtained a high temperature-adapted strain(HTAS)from its conventional strain(CS)via long-term heat hardening,which showed significant temperature plasticity and adaptability when exposed to high temperature stress,previous studies have shown that its tolerance to high temperature stress was significantly enhanced.This study focused on the ecological adaptation mechanism of the mite,and studied the differences of DNA methylation levels between two strains of N.barkeri and each generation under high temperature stress from the perspective of epigenetics.On this basis,Nb Dnmt1 gene was screened and cloned,and its m RNA expression pattern was analyzed.Finally,the temperature tolerance function of the gene was verified by RNAi technology.The main research results obtained in this study are as follows:1.The whole genome methylation level of two strains of mite were determined by high temperature stress treatment of 42°C.The results showed that the whole genome methylation level of the HTAS was higher than that of the CS.The genome-wide methylation level of the female adult mites of two strains of N.barkeri reached the maximum of 0.065% and 0.213% respectively after treatment for 1h.However,with the increase of treatment time,the whole genome methylation level of female adult mites of the two strains decreased,and the whole genome methylation level of the two strains slightly increased after treatment for 3 h.In addition,the whole genome methylation level of the female adult mites of different generations of mites showed a decreasing trend with the prolongation of generations,and reached the lowest level in F10 generation.The results showed that the tolerance to high temperature might be closely related to DNA methylation.2.cDNA sequence of Nb Dnmt1 gene was cloned by molecular biology experiment.The results showed that the number of amino acids encoded by Nb Dnmt1 was 458.Phylogenetic analysis showed that the cloned Nb Dnmt1 was on the same evolutionary branch with the mite of the same order,the Dnmt1 gene cloned in this study has the closest relationship with the Dnmt1 gene of Galendromus occidentalis.Using q PCR technology for Nb Dnmt1 gene m RNA expression in different development stages were analyzed,and the results showed that Dnmt1 gene in the two strains of N.barkeri was the most highly expressed in the female adult stage and showed a trend of gradually upregulation with growth and development.Dnmt1 gene may be involved in the growth and development of N.barkeri and the regulation of female reproductive development;Analysis of the Dnmt1 gene expression pattern of N.barkeri under high temperature stress showed that the Dnmt1 gene expression pattern of the two strains could be induced by high temperature,but the expression pattern of the two strains was varied with the treated stress temperature and time: High temperature of 42°C can significantly induce the expression of Nb Dnmt1 gene,especially in the HTAS,which indicates that HTAS is more sensitive to extreme high temperature response.The expression of Nb Dnmt1 gene in the CS was rapidly induced in the medium high temperature environment,and the expression of Nb Dnmt1 gene in the CS at different time nodes was higher than that in the HTAS.The expression trend of Dnmt1 gene in the CS under the medium high temperature environment of 35°C was similar to that under the high temperature environment of 38°C,indicating that both the medium temperature environment of 35°C and the high temperature environment of 38°C could exert stress on the CS.On the other hand,the expression trend of Nb Dnmt1 gene at 42°C high temperature treatment is basically consistent with the trend of methylation level measured at the same temperature,it also suggests that change in the expression level of Dnmt1 gene is one of the mechanisms leading to the change of DNA methylation level in N.barkeri.3.The Dnmt1 gene of both strains was effectively silenced by feeding the ds RNA of the Dnmt1 gene to N.barkeri,and compared with feeding ds GFP,the silencing efficiency was 38% and 52%,respectively.After interference,it was found that the two strains of N.barkeri to high temperature tolerance were decreased,which showed a significant increased mortality and a shortened heat knockdown time.Further illustrated that Dnmt1 gene plays an important role in high temperature tolerance of N.barkeri.In summary,this study enriched the theoretical research on the epigenetic mechanism of temperature adaptation of N.barkeri,and provide a foundation for improving the efficacy of N.barkeri in the continuous control of mites in the field.