关键词: 烟夜蛾, 精氨酸激酶, 基因克隆, 荧光定量PCR, 表达谱分析

Abstract: 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.

Key words: Helicoverpa assulta, arginine kinase, gene cloning, fluorescent quantitative PCR, expression pattern analysis