棉铃虫V-ATP酶亚基A基因克隆及表达谱分析

为探索棉铃虫Helicoverpa armigera液泡型ATP酶(vacuolar-type proton ATPase,V-ATP酶)亚基A对棉铃虫生长发育的影响及其在Bt杀虫机制中的作用,采用PCR结合RACE技术克隆了棉铃虫V-ATP酶亚基A基因序列,通过实时荧光定量PCR技术测定了其在棉铃虫不同发育历期和幼虫肠道不同组织中的表达量;并比较了4龄幼虫取食含Cry1Ac蛋白饲料后中肠中该基因表达量的变化。结果显示,棉铃虫V-ATP酶亚基A基因全长2 578 bp(Gen Bank登录号KP090287),开放阅读框1 863 bp,编码621个氨基酸。V-ATP酶亚基A高度保守,不同物种间氨基酸序列同源性大于90%。V-ATP酶亚基A在棉铃虫整个生育期都有表达,在4龄幼虫体内表达量最高,是卵期的3.00倍;在幼虫肠道不同组织中,中肠中表达量最高,是后肠中的2.65倍。4龄棉铃虫幼虫取食含Cry1Ac蛋白的人工饲料后,中肠V-ATP酶亚基A的表达受到抑制,表达量为对照的0.39~0.81倍。表明V-ATP酶亚基A基因可能参与棉铃虫的生长发育和代谢过程,并可能与抵御Cry1Ac的毒杀作用有一定关系。

Cloning and expression analysis of V-ATPase subunit A gene in the cotton bollworm Helicoverpa armigera (Hübner)

In order to explore the influence of vacuolar-type ATPase (V-ATPase) subunit A on the growth and development of the cotton bollworm Helicoverpa armigera and its role in the insecticidal mechanism of Bacillus thuringiensis (Bt), the full-length sequence of the gene encoding V-ATPase subunit A was cloned from H. armigera by using PCR (polymerase chain reaction) and RACE (rapid amplification of cDNA ends) technology. The expression levels of V-ATPase subunit A in different developmental stages and different tissues of larval intestinal tract were analyzed by real-time quantitative PCR. The expression levels of V-ATPase subunit A were also checked in larval midgut after 4th-instar larvae fed on artificial diet containing Bt insecticide protein-Cry1Ac. The results indicated that the full-length of the cDNA was 2 578 bp (GenBank accession no. KP090287) with an open reading frame of 1 863 bp in length, encoding 621 amino acid residues. The V-ATPase subunit A was highly conserved, and the amino acid sequence homology was more than 90% among different insect species. The real-time quantitative PCR results showed that V-ATPase subunit A of H. armigera was expressed in all growth stages, highest in 4th-instar larvae and 3.00 times as in the eggs. Further, qPCR results for different tissues of larval intestinal tract showed that highest expression was found in the midgut, 2.65 times higher than that in the hindgut. After larvae fed on Cry1Ac toxin, the expression of V-ATPase subunit A gene was suppressed dramatically and declined to 0.39~0.81 times as the control. These results indicated that V-ATPase subunit A gene might participate in the growth and metabolic process of H. armigera and might be involved in the defensive effects of Cry1Ac.