烟夜蛾精氨酸激酶基因的克隆及mRNA表达分析

Cloning and mRNA expression analysis of arginine kinase gene from Helicoverpa assulta (Guenée) (Lepidoptera: Noctuidae)

为了深入了解精氨酸激酶基因的作用和寻求害虫防治新的分子靶标,本研究采用RT-PCR和RACE技术,从烟夜蛾Helicoverpa assulta脂肪体中克隆了精氨酸激酶cDNA序列,命名为HassAK(GenBank登录号:HQ336337),并采用荧光定量PCR测定了HassAK基因在不同发育阶段(4龄幼虫第1天到化蛹第1天)、不同组织(头部、中肠、脂肪体、体壁和腹足)和不同温度条件下的表达情况。测序和序列分析结果表明,HassAK基因阅读框架全长1068bp,编码355个氨基酸残基,预测蛋白质分子量和等电点分别为40.0kD和5.76。氨基酸序列分析表明,该序列具有精氨酸激酶典型的酶活性部位、酶活性中心位点和能形成离子偶结构的保守区。序列比对结果表明,HassAK与其他昆虫AK的氨基酸序列具有70%以上的一致性。荧光定量分析结果显示,HassAK基因在幼虫头部、中肠、脂肪体、体壁和腹足均可表达,其中以腹足和中肠内的表达水平较高。时序表达分析表明,预蛹期HassAK基因的表达量达到高峰。此外,高温和低温均诱导HassAK基因的表达,说明该基因可能参与昆虫抵御外界不良环境。

In order to better understand the role of arginine kinas （AK） gene and seek new molecular targets for insect pest control, we cloned AK cDNA sequence from fat body of Helicoverpa assulta by RT-PCR and RACE methods. The AK cDNA was named HassAK （GenBank accession no. HQ336337）. The expression pattern of HassAK at different developmental stages （from 1st day of 4th instar larva to 1-d-old pupa）, in different tissues （head, midgut, fat body, cuticle and abdominal legs） and after treatment with different temperatures was further determined by RT-PCR. The results of sequencing and sequence analysis showed that the full-length open reading frame of HassAK is 1 068 bp, encoding 355 amino acid residues with the predicted molecular weight and isoelectric point of 40.0 kD and 5.76, respectively. Amino acid sequence analysis showed that the HassAK sequence has the typical characteristics of arginine kinase, which contains the active sequence, the active site and a pair of highly conserved amino acids that form an ion pair of AK. Sequence comparison results showed that HassAK has more than 70% amino acid sequence identity with AKs from other insects . The results of fluorescent quantitative analysis revealed that HassAK was expressed in the head, midgut, fat body, cuticle and abdominal legs of larva. Among them, the expression level of HassAK was higher in abdominal legs and the midgut. Temporal analysis indicated that the expression level of HassAK reached the peak in the pre-pupation stage. In addition, high and low temperatures led to increased expression of HassAK, suggesting that HassAK gene may be involved in insect resistance to adverse external environments.