棉铃虫脂肪酸结合蛋白的克隆与表达

Cloning and expression of fatty acid-binding protein from cotton bollworm Helicoverpa armigera

为深入了解棉铃虫Helicoverpa armigera脂肪酸结合蛋白HaFABP1在参与细胞内脂肪酸代谢过程中的功能,基于酵母单杂交结果从幼虫中肠cDNA中扩增得到HaFABP1的开发阅读框,构建重组菌株BL21-pET28a-HaFABP1,进行融合蛋白His-HaFABP1的诱导和纯化,并利用实时荧光定量PCR检测棉铃虫不同时期和组织中HaFABP1的表达规律,最后检测2-十三烷酮处理下棉铃虫6龄幼虫中肠组织内HaFABP1的变化情况。结果显示,克隆获得的棉铃虫HaFABP1为405 bp,编码134个氨基酸,相对分子量和等电点分别为15.08 kD和5.54;在37℃下用0.5 mmol/L异丙基硫代半乳糖苷(IPTG)诱导重组菌株4 h后,得到约18.4 kD的可溶性蛋白His-HaFABP1,纯化后的融合蛋白条带单一且大小符合预期;HaFABP1在棉铃虫的脂肪体、中肠、体壁和头部中都有表达,在头部的表达量最低,在中肠中的表达量最高;HaFABP1在棉铃虫幼虫的每个发育时期都有表达,表达量总体呈现先降低后升高的趋势,4龄时最低而1龄时最高;10 mg/g 2-十三烷酮处理饲料饲喂棉铃虫6龄幼虫6 h和15 h后,中肠内HaFABP1的表达量与对照相比显著提高,且随着时间的延长逐渐增加。表明HaFABP1与棉铃虫的生长发育有关,并且有可能参与了昆虫的解毒过程。

In order to better understand the function of HaFABP1, a fatty acid-binding protein of cotton bollworm Helicoverpa armigera, in the process of fatty acid metabolism in cells, the open reading frame (ORF) of HaFABP1 was amplified from the larval midgut cDNA based on the result of yeast one-hybrid technology. The recombinant Escherichia coli strain BL21-pET28a-HaFABP1 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein His-HaFABP1 was purified. Then the relative expression levels of HaFABP1 in four tissues (fat body, midgut, integument and head), at different larval stages (the 1st to the 6th instar and pre-pupal stage) and 10 mg/g 2-tridecanone-treated 6th instar were detected with real-time fluorescence quantitative PCR (qPCR). The results showed that the ORF of HaFABP1 was 405 bp, encoding 134 amino acids, with a relative molecular mass and theoretical isoelectric point of 15.08 kD and 5.54, respectively. After the recombinant strain was induced with 0.5 mmol/L IPTG for 4 h at 37℃, the soluble protein His-HaFABP1 (about 18.4 kD) was obtained. The purified fusion protein was single-banded and the size was in line with expectation. The results of qPCR showed that HaFABP1 was expressed in the fat body, midgut, integument and head of 6th instar larvae. The expression of HaFABP1 was the highest in midgut, the lowest in head, and in all larval stages. The expression profile of HaFABP1 firstly decreased and then increased, highest in the 1st instar larvae and lowest in the 4th instar larvae. The expression level of HaFABP1 gradually increased from 6 h to 15 h when the 6th instar larvae were treated with 10 mg/g 2-tridecanone. These results indicated that HaFABP1 was related with the growth and development of H. armigera and might be involved in the detoxification process of insects.