黑腹果蝇保幼激素受体Met泛素化修饰位点预测及功能验证

Prediction and functional analysis of ubiquitin sites of juvenile hormone receptor Met in Drosophila melanogaster

目的预测黑腹果蝇保幼激素(juvenile hormone,JH)受体Methoprene tolerant(Met)的泛素化修饰位点,并验证其功能。方法利用UbPred和CKSAAP_UbSite在线预测工具对Met的泛素化位点进行预测;通过重叠PCR技术将预测的各泛素化修饰位点赖氨酸分别突变为丙氨酸,同时将第46和526位赖氨酸突变为丙氨酸;将突变后的Met插入至表达载体p Ac5. 1/V5-His B中,构建各位点突变的表达质粒;转染果蝇Kc细胞,并经格尔德霉素(geldanamycin,GA)处理,Western blot法检测各位点突变后Met的表达。结果根据UbPred和CKSAAP_UbSite在线网站预测结果,选取7个赖氨酸位点作为Met的潜在泛素化修饰位点,CKSAAP_UbSite及UbPred预测分值最高的分别为第46和526位赖氨酸。构建的Met各位点突变表达质粒转染果蝇Kc细胞后,于相对分子质量约82 000处均可见Met目的蛋白的表达,经GA处理后,Met表达下降。结论成功构建了Met的7个预测泛素化修饰位点的单突变表达质粒及1个双突变表达质粒,这些位点的突变均不足以抑制GA诱导Met的降解。

Objective To predict the ubiquitin sites of juvenile hormone(JH)receptor Methoprene tolerant(Met)in Drosophila melanogaster and analyze their functions.Methods The ubiquitin sites of Met were predicted by UbPred and CKSAAP_UbSite online prediction tools.The lysine in predicted sites was mutated into alanine by over-lapping PCR technique.Meanwhile,the lysine at sites 46 and 526 were mutated into alanine simultaneously.The site mutants of Met were inserted into expression vector pAc5.1/V5-His Bex,and the constructed recombinant plasmids were transfected to D.melanogaster Kc cells and treated with GA.The expressions of mutated Met at various sites were analyzed by Western blot.Results Seven lysine sites were selected as potential ubiquitin sites of Met according to the prediction result by UbPred and CKSAAP_UbSite websites.The lysine with the highest score predicted by CKSAAP_UbSite was at site 46,while that by UbPred at site 526.Met with a relative molecular mass of about 82 000 was expressed in Kc cells transfected with recombinant plasmids containing various site mutants,of which the expression level decreased after treatment with GA.Conclusion The expression vectors containing a single point mutant of seven predicted ubiquitin sites of Met and that containing the indicated double point mutants were constructed successfully.However,these site mutations were not sufficient to inhibit GA-induced Met degradation.