Cloning And Study On Detoxification Function Of Carboxylesterase Genes From Mythimna Separata

Carboxylesterase is one of the three major detoxifying enzymes of insects,and plays an important role in the production and development of insect resistance.Mythimna separata is an important agricultural pest in agricultural production.Once they happen on a large scale,they will cause serious economic losses.In this study,12 Car Es gene c DNA sequences were screened from M.separata transcriptome library,the complete and correct gene sequences were obtained through verification.Through insecticides treatment,genes which expression significantly increased were screened by q RT-PCR for further detailed research.We studied the spatiotemporal expression of the selected gene sequences and the effects of the insecticides.Then we analyzed the function of the screened gene sequences by RNA interference,detected the efficiency of gene silencing at different times,measured changes of total Car Es activity in M.separata after interference and the insecticidal activity of the insecticide against M.separata after interference.The specific results are as follows:?1?In this study,12 Car Es gene c DNA sequences were obtained.They both have two conserved domains of the carboxylesterase genes:the carboxylesterase serine active center F/Y-G-G-D/N-x?3?-V/I-T/V-L/I/V-x-G-x-S-A/G-G;the active site of the disulfide bond formation E-D-C-L-x-A/L/I/V-N-V/I-Y-x-P,which were registered on NCBI and named as Ms Car E1-12?Genbank accession:MK440541-MK440552?.The full-length c DNA sequences of Ms Car Es were1623,2683,1865,1811,2200,2281,2299,1851,1882,1961,1782,and 1824 bp,and they contained open reading frames of lengths 1365,2250,1740,1665,1644,1767,1665,1608,1665,1623,1665 and 1689 bp,respectively,which encoded 454,749,579,554,547,588,554,535,554,540,554 and 562 amino acids with isoelectric points of 5.41,4.72,6.24,4.95,4.70,6.06,5.69,5.73,5.22,7.96,5.13,5.15 and molecular weights of 51.196,83.919,65.435,62.918,61.713,63.241,62.113,59.816,62.706,60.417,61.603,62.837 k Da.?2?After treatment with LD₅₀ of two insecticides,the expression of each gene was significantly different.After induction with LD₅₀ of 20%chlorantraniliprole,Ms Car E1,Ms Car E3,Ms Car E4,Ms Car E7,Ms Car E8 and Ms Car E12 six genes were significantly up-regulated,and the up-regulation of Ms Car E3 and Ms Car E4 were most significant.After treatment with LD₅₀ of 5%lambda-cyhalothrin,the expression levels of the three genes Ms Car E5,Ms Car E8 and Ms Car E10were significantly increased,Ms Car E5 and Ms Car E10 expression levels were most significantly up-regulated.Based on the above results,four gene sequences Ms Car E3,Ms Car E4,Ms Car E5 and Ms Car E10 were selected for subsequent research.?3?Ms Car E3,Ms Car E4,Ms Car E5 and Ms Car E10 were expressed at different developmental stages and tissues,but the expression levels were significantly different.The expression level of Ms Car E3 in adults was significantly higher than in larval and pupal stages,the expression level of Ms Car E3 in 3^rd instar larvae was significantly higher than those in other larval stages,and it was lowest in 5^th instar larvae.The expression levels of Ms Car E4 increased gradually from 1^st instar to 4^th instar larvae.The relative expression was highest in 4^th instar,which was about 78.2-fold higher than it in 1^st instar larvae.There was no significant difference in the relative expression between 4^th and 6^th instar.The relative expression was the lowest in the adult stage.The relative expression level of Ms Car E5 was the lowest in the prepupal stage,but it was not significantly different from the expression levels in the adult and pupal stages.The expression level of Ms Car E5 was highest in 5^th instar larvae.The expression level of Ms Car E10 in 5^th instar larvae was highest and the expression level of Ms Car E10 during the prepupal stage was relatively lowest.The highest expression of Ms Car E3 was found in the integuments,and the lowest expression was found in the hindguts.Ms Car E4 expression was highest in the salivary glands,while there was no significant difference in the integuments.And Ms Car E4 expression was relatively lowest in the midguts.The expression level of Ms Car E5 in the salivary glands was relatively lowest,and the highest expression of Ms Car E5 was found in the midguts,which was223.0-fold higher than it in the salivary glands.The highest expression of Ms Car E10 was found in the integuments,which was 75.8-fold higher than it in the salivary glands with the lowest expression,and there was no significant difference in the expression level of the foreguts.?4?Ms Car Es expression levels were different significant by using different doses of insecticides to treat larvae on the first day of 4^th instar at different times.LD₁₀ of 20%chlorantraniliprole induced,Ms Car E3 was induced at 48 h after treatment,however,Ms Car E4 was induced at 24 h after treatment,and the induction effect continued to 48 h.Ms Car E3 expression was invariant then induced after treatment with LD₃₀,that was inducted in 24-48 h.However,Ms Car E4 expression was only induced at 24 h.LD₅₀ treatment was the same as LD₃₀treatment that Ms Car E3 expression was induced in 24-48 h,and Ms Car E4 was induced only in 24h.After LD₇₀treatment,Ms Car E3 expression invariant then induced,which was inducted at 6 h and continued to48 h,while Ms Car E4 expression was always suppressed.The results demonstrate that Ms Car E3was induced more obvious by high-dose insecticide treatment,while Ms Car E4 was the opposite.LD₁₀ of 5%lambda-cyhalothrin treatment,the Ms Car E5 expression level was induced after 24 h treatment,and continued to 48 h,however,the Ms Car E10 expression level was only induced at 24h.LD₃₀ treatment,Ms Car E5 expression was invariant then induced,and was inducted at 24 and 48h.The situation for Ms Car E10 was the same as that of LD₁₀ treatment,which only was induced at24h.Ms Car E5 and Ms Car E10 expression by treating with LD₅₀ was same as treating with LD₃₀.Ms Car E5 expression level was always suppressed after treatment with LD ₇₀,while Ms Car E10expression was induced in 3-12h then invariant.The results demonstrate that Ms Car E5 was induced more obvious by low-dose insecticide treatment,while Ms Car E10 was the opposite.?5?Ms Car Es expression levels were successfully suppressed after RNA interference.When interfered with single gene,the expression level of Ms Car E3 in treatment group was significantly inhibited at 12 h and 24 h after injection.The inhibition effect was best at 12 h,and the inhibition rate reached 82.8%.The expression of Ms Car E4 gene was significantly suppressed at 3 h and 6 h after injection,and the highest inhibition rate was 90.2%at 6 h after injection.The expression of Ms Car E5 showed a trend of inhibition within 48 h after injection,but the inhibition effect was best at 3 h after injection.The expression of Ms Car E10 was inhibited at 6 h and 12 h after injection,and the inhibition rate was highest at 6 h.When interference with mixed genes,the expression of Ms Car E3 was only inhibited at 3 h after interference,and the inhibition rate was 50.0%.Ms Car E4expression was inhibited within 12 h after injection,but the inhibition effect was best at 3 h after injection.The expression of Ms Car E5 gene was suppressed within 12 h after injection.In addition,the interference effect was best at 3 h,which was the same as it interfered with single gene.Ms Car E10 expression was inhibited at 6 h after injection.?6?After Ms Car E5 was interfered,the insecticidal activity of lambda-cyhalothrin against M.separata increased by about 25-30%.Larvae that injected with si RNA3,si RNA4 and their mixture,the Car Es activities did not decrease but increased.And the LD₅₀ of chlorantraniliprole treated the interfered larvae,it was found that the mortality rate of larvae injected with si RNA3 and si RNA4was reduced compared with the control,and the mortality rate of larvae injected with the mixture of both was also lower than the control after 24 h.The activities of Car Es in larvae injected with si RNA5 and mixture were significantly reduced after 3-12 h treatment,while the activity of Car Es was significantly reduced in larvae injected with si RNA10 only at 12 h.Treat ing the interfered larvae with LD₅₀ of lambda-cyhalothrin showed that the mortality of larvae injected with si RNA5and the mixture increased significantly,and the mixed int erference effect was better at 24 h after treatment and the mortality rate increased by about 30%.This experiment provides a theoretical basis for further understanding of M.separata carboxylesterase and lays a foundation for analyzing the function of ca rboxylesterase and understanding the resistance mechanism of M.separata.