| Plutella xylostella(Diamondback moth,DBM)is an oligophagous insect pest,which is seriously harmful to cruciferous crops.In our previous study,significantly greater variation of wing shape and injury of wing vein showed in chlorpyrifos-resistant(Rc)DBM was found than chlorpyrifos-susceptible(Sm)DBM under heat stress.To investigate the toxicological mechanism of this difference,the cloning and heat shock expression of wing development genes in DBM were studied in this paper.The main results are as follows:1.Based on RT-PCR and RACE technology,two full-length cDNA sequences(dpp,srf)and two partial cDNA sequences(salm,ser)were isolated and characterized in DBM.The length of dpp sequence is 1891 bp,encoding 423 amino acids,containing DWA and DWB domain.The length of srf sequence is 949 bp,encoding 229 amino acids,containing MADS domain.The length of salm sequence is 1885 bp,encoding 614 amino acids,containing ZnF C2H2 domain.The length of ser sequence is 1196 bp,encoding 369 amino acids,containing the Coiled coil domain.According to the results of multiple sequence alignment and phylogenetic analysis showed that these genes had higher homology with other Lepidoptera insects.It is indicate that these genes are highly conserved in the development and evolution of Lepidoptera insect wings,which provide a theoretical foundation for further study on the difference of mRNA expression between Rc and Sm DBM under heat stress.2.The pupae of Rc and Sm DBM was treated with heat shock at(44?-1h,25?-1h),(42?-4h,42?-8h,25?-8h),(40?-8h,40?-16h,25?-16h),(38?-48h,25?-48h)respectively.The relative mRNA expression of these genes was determined by using RT-qPCR technique.The results showed that the mRNA expression of the four genes in the pupae of the Sm DBM was generally higher than Rc DBM under heat stress.In the 44?-1h,25?-1h temperature treatment group,compared with normal temperature(25?),the mRNA expression of dpp was significantly up-regulated,the mRNA expression of srf was significantly down-regulated,the mRNA expression of salm and ser was no significant changed in the Sm DBM pupae.The mRNA expression of dpp and ser showed no significant change,the mRNA expression of srf and salm was significantly down-regulated in the Rc DBM pupae.In the 42?-4h,42?-8h,25?-8h temperature treatment group,compared with normal temperature(25?),the mRNA expression of dpp,srf,salm,ser was all significantly down-regulated in the Sm DBM and Rc DBM pupae after 42?-8h treatment.In the 40?-8h,40?-16h,25?-16h temperature treatment group,compared with normal temperature(25?),the mRNA expression of dpp was no significant changed and the mRNA expression of srf,salm,ser was significantly down-regulated in the Sm DBM pupae.The mRNA expression of dpp,srf,salm,ser was all significantly down-regulated in the Rc DBM pupae.In the 38?-48h,25?-48h temperature treatment group,compared with normal temperature(25?),the mRNA expression of dpp,srf,ser was no significant changed and the mRNA expression of salm was significantly down-regulated in the Sm DBM pupae.The mRNA expression of dpp and ser showed no significant change,the mRNA expression of srf and salm was significantly down-regulated in the Rc DBM pupae.The overall results showed that the mRNA expression of these wing development-related genes in the Rc DBM pupae was lower and decreased significantly under heat stress.We speculate that the higher shape variation probability and significantly greater injury of wing vein showed in Rc DBM under heat stress might be associated with the stronger down regulation of wing development-related genes. |
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