四种钙结合蛋白基因在沙葱萤叶甲幼虫生长发育中的作用

Effects of four calcium-binding protein genes on the growth and development of Galeruca daurica larvae(Coleoptera:Chrysomelidae)

为明确沙葱萤叶甲Galeruca daurica钙结合蛋白基因的功能,利用本实验室组装的沙葱萤叶甲转录组数据库鉴定钙结合蛋白基因序列,采用RNA干扰(RNA interference,RNAi)技术沉默沙葱萤叶甲3龄幼虫体内钙调蛋白(calmodulin,CaM)、钙网蛋白(calreticulin,CRT)、类钙磷蛋白(calcyphosine-like,CAPSL)和类肌钙蛋白C(troponin C-like,TnCl)共4种钙结合蛋白基因,观察幼虫发育历期、存活率、体重及蛹重的变化;应用实时荧光定量PCR测定干扰效率。结果表明,共筛选到41条编码钙结合蛋白的基因序列,选取4条具有完整开放阅读框的钙结合蛋白基因序列进行后续研究。4种钙结合蛋白基因的干扰效率从高到低依次为CAPSL(94.4%)、TnCl(76.2%)、CRT(70.5%)和CaM(54.5%);干扰效率最高的时间分别为干扰后第4、2、6和2天。沉默CAPSL后,沙葱萤叶甲幼虫体重显著降低了21.0%~34.9%,存活率显著下降了53.5%,发育历期显著缩短了15.1%,蛹重显著降低了15.8%。沉默TnCl后,沙葱萤叶甲幼虫体重显著降低了10.5%~25.0%,存活率显著下降了19.1%,蛹重显著降低了11.0%,而对发育历期无显著影响。沉默CaM后,沙葱萤叶甲幼虫体重显著降低了5.9%~6.6%,存活率显著降低了8.7%,而幼虫发育历期和蛹重无显著变化。沉默CRT后,沙葱萤叶甲幼虫体重、蛹重、存活率和发育历期均无显著变化。表明CAPSL、TnCl和CaM在沙葱萤叶甲幼虫生长发育过程中发挥着重要作用,而CRT可能未参与沙葱萤叶甲幼虫生长发育的调控过程。

In order to reveal the functions of calcium-binding protein(CBP)genes in Galeruca daurica,the sequences encoding CBPs were identified from the transcriptome databases of G.daurica.RNAi was used to silence four CBP genes,calmodulin(CaM),calreticulin(CRT),calcyphosine-like(CAPSL),and troponin C-like(TnCl),in the 3 rd-instar larvae of G.daurica,and the changes of larval developmental duration,survival rate,body weight and pupal weight after RNAi were investigated.Meanwhile,RNAi efficiency was measured by quantitative real-time PCR(qRT-PCR).The results showed that 42 CBP genes were identified,and four CBP genes with complete open reading frames were chosen for further study.RNAi efficiencies for these four CBP genes were different,and the ranking from high to low was CAPSL(94.4%),TnCl(76.2%),CRT(70.5%),and CaM(54.5%)at 4,2,6,and 2 d after RNAi,respectively.After silencing CAPSL,the body weight of the 3 rd-instar larvae of G.daurica were significantly decreased by 21.0%-34.9%,the survival rate by 53.5%,the developmental duration by 15.1%,and the pupal weight by 15.8%.After silencing TnCl,the larval weights were significantly decreased by 10.5%-25.0%,the survival rate by 19.1%,the pupal weight by 11.0%,whereas the developmental duration did not significantly change.After silencing CaM,the larval weight were significantly reduced by 5.9%-6.6%,the survival rate by 8.7%,but the larval developmental duration and pupal weight did not change significantly.After silencing CRT,all above parameters did not significantly change.These results indicated that CAPSL,TnCl and CaM might play important roles in the growth and development of G.daurica larvae,whereas CRT might not be involved in regulating the growth and development of G.daurica larvae.