Effects Of Heat Stress On Apoptosis In The Insecticide-resistant And-susceptible Plutella Xylostella And Coloning And Expression Of Px-caspase-1b

Caspase is an important gene in the process of cell apoptosis, and its over-expression can induce cell apoptosis, even death. In this study, cell apoptosis of Plutella xylostella is researched, which not only has high commercial value, but also is significant for science research.In this experiment, Plutella xylostella were treated at different time (24h,48h,72h) and temperature (25℃,35℃,40℃). By using the comet electrophoresis, cell apoptosis of egg cells of Plutella xylostella were observed. Because cell apoptosis is closely related to the expression of caspase, so a complete sequence was gained using degenerate primer, RACE and nest PCR. It belonged to caspase gene families through compared with other genes and was named Px-caspase-1b. Finally, the relations among temperature and time and expression were researched using Q-PCR. In this experiment, the typical temperature were33.5℃and40℃, the dealed times were9h and3d. The temperature of control group was25℃and reference gene was β-actin.we find that with increasing of temperature and time, cell apoptosis of insecticide-resistant and-susceptible Plutella xylostella increased gradually and reached the highest level at40℃and72h. But the level cell apoptosis of insecticide-resistant was far higher than that of insecticide-susceptible Plutella xylostella.In the experiment, a middle sequence with length645bp was gained using degenerate primer, and it belonged to caspase gene families through compared with other genes. Then by using RACE technology and designing a pair of specific primers in3’end and5’end respectively, two fragments were cloned with length of1119bp and637bp respectively. According to sequence overlap of three fragments, I got a full-length gene with length2284bp by gene splicing. Finally, according to the results, I designed a pair of special primers and got a gene sequence with length990bp afer PCR. This sequence named Px-casapse-1b included a complete open reading frame (ORF) and belonged to caspase-1gene families. The results of Real time PCR showed that the expression level of Px-casapse-1b mRNA of secticide-resistant Plutella xylostella became higher with the temperature and time rising. The expression level of Px-casapse-1b mRNA of secticide-sensitive Plutella xylostella increased at33.5℃, but the scale was light and did not rise at40℃.The results in dicated that heat tress can induce cell apoptosis, the level of cell apoptosis of secticide-sensitive Plutella xylostella is lower than that of secticide-resistant Plutella xylostella under the circumstance of high temperature. This proves a physiological character that sensitive Plutella xylostella are more resistant to high temperature in a view of molecular biology, which has a important value for basic science study.