表皮蛋白BmCPR41在2龄不眠蚕突变体nm2中的表达和功能分析

Expression and Functional Analysis of Silkworm Cuticular Protein BmCPR41 in nm2 Mutant

家蚕(Bombyx mori)2龄不眠蚕(non-molting in the 2nd instar,nm2)突变体是从家蚕品种C603中发现的一种致死突变体,发育至2龄将眠期后不能正常入眠蜕皮,持续5~7 d后陆续死亡,图位克隆结果表明该突变体是由表皮蛋白BmCPG10突变引起的,且血淋巴中的蜕皮激素滴度显著低于野生型。蜕皮激素主要在前胸腺合成,因此本研究以nm2突变体和野生型家蚕2龄将眠蚕的前胸腺(包含第一对气管丛)为材料进行双向电泳分析,从中检测到一个表皮蛋白BmCPR41在突变体中的表达量高于野生型,组织表达谱表明BmCPR41在前胸腺的表达量远高于其他组织。通过RNAi敲低BmCPR41的表达,发现有部分家蚕推迟入眠或不入眠,蚕体重也显著低于对照组,且相关基因基于RNA-seq的表达情况与nm2突变体中的基本一致。基于上述实验结果推测BmCPR41基因在nm2突变体形成,尤其蜕皮激素信号通路上发挥重要作用。本研究结果为进一步研究nm2突变体的形成机制提供了实验证据,并可为研究蜕皮激素介导的信号通路提供理论依据。

The Bombyx mori mutant nm2(non-molting in the 2 nd instar),a lethal mutant,was found from silkworm variety C603.The nm2 mutant could not molt in the 2 nd instar,lasted for about 5 to 7 d and died successively.The map-based cloning results indicated that a cuticular protein BmCPG10(cuticular protein glycine-rich 10)was responsible to the mutation,and the titer of ecdysone in hemolymph of nm2 mutant was significantly lower than that of the wild type.In this study,owing to the synthesis of ecdysone in prothoracic gland(PG),the PG(including the first pair of tracheal plexus)of nm2 mutant on pre-molting of the 2 nd instar were used for two-dimensional electrophoresis(2-DE)analysis and the wild type serving as the control.As the results,a cuticular protein BmCPR41(cuticular protein RR-1 motif 41)was detected to be significantly upregulated in nm2 mutant compared to the wild type.The tissue expression profile exhibited that the expression of BmCPR41 in PG was much higher than that of other tissues.Then,to explore the function of BmCPR41,it was knocked down by RNAi technology,and the results showed several deferred-molting and non-molting individuals.Besides,the body weight was lower than that of the control,and the expression levels of related genes were also consistent to that of nm2 mutant(based on the RNA-seq results).Based on these results above,we conjectured that the BmCPR41 gene played important roles in nm2 mutant especially in ecdysone signal pathway.The results of this study could provide experimental evidence for further study of the formation mechanism of nm2 mutant and also theoretical basis for the study of ecdysone-mediated signaling pathways.