美国白蛾碱性磷酸酶HcALP3基因的克隆、表达及功能分析  

Cloning, Expression and Functional Analysis of Alkaline Phosphatase HcALP3 Gene in Hyphantria cunea

作　　者：赵丹[1] 常梦颖 乔英翠 王哲 刘子欢 陆秀君[1] 郭巍[1,2] ZHAO Dan;CHANG Meng-Ying;QIAO Ying-Cui;WANG Zhe;LIU Zi-Huan;LU Xiu-Jun;GUO Wei(College of Plant Protection,Hebei Agricultural University,Baoding 071001,China;Graduate School of Chinese Academy of Agricultural Sciences,Beijing 100081,China;Huanghua Port Customs,Cangzhou 061113,China)

机构地区：[1]河北农业大学植物保护学院,保定071001 [2]中国农业科学院研究生院,北京100081 [3]黄骅港海关,沧州061113

出　　处：《农业生物技术学报》2021年第7期1354-1363,共10页Journal of Agricultural Biotechnology

基　　金：河北省自然科学基金(C2021204106);河北省重点研发计划项目(20326812D);财政部和农业农村部:国家现代农业产业技术体系建设专项(CARS-13);中国农业科学院基本科研业务费(1610042020002)。

摘　　要：美国白蛾(Hyphantria cunea)是一种重要的世界性检疫害虫,也是我国重大入侵害虫之一。苏云金杆菌(Bacillus thuringiensis,Bt)作为目前研究应用最广泛的杀虫微生物之一,对美国白蛾的作用机制尚不清楚。为了明确美国白蛾中肠受体碱性磷酸酶(alkaline phosphatase,ALP)与Bt Cry毒素的结合特性,探索Bt Cry毒素对其作用机理,本研究利用PCR方法扩增获得美国白蛾中肠碱性磷酸酶HcALP3基因(GenBank No.MH796759),该基因的开放阅读框1557 bp,编码518个氨基酸。构建原核重组表达载体pET30a-HcALP3,转化大肠杆菌(Escherichia coli)BL21,经IPTG诱导表达56.65 kD的HcALP3蛋白。q RTPCR方法进行组织和龄期表达分析,结果显示HcALP3在美国白蛾的各龄期均有表达,5龄幼虫表达量最高,在不同组织中,中肠的表达量最高;Ligand blot分析结果发现HcALP3重组蛋白仅可以与Bt Cry1Ab35活化毒素特异结合,不与Bt Cry1Ac和Cry9Ea10结合。利用RNAi技术沉默HcALP3基因,HcALP3表达水平下降了75.32%;注射dsRNA-HcALP3后的美国白蛾幼虫对Bt Cry1Ab35、Cry1Ac和Cry9Ea10的校正死亡率分别为45.93%、73.33%和70%,对Bt Cry1Ab35的敏感性显著低于注射dsRNA-egfp的美国白蛾幼虫,推测HcALP3蛋白为Bt Cry1Ab35毒素的潜在功能受体。本研究通过对美国白蛾碱性磷酸酶HcALP3的鉴定及功能验证,为深入探讨Bt制剂防治美国白蛾的机制提供了科学依据。The fall webworm(Hyphantria cunea) is an important quarantine pest worldwide and a major invasive pest in China. Bacillus thuringiensis(Bt) is widely used insecticidal microorganisms, however the mechanism of Bt on H. cunea is still unclear. To analyze the binding characteristics of alkaline phosphatase(ALP) of H. cunea with Bt toxin and explore the insecticidal mechanism, the HcALP3 gene(GenBank No.MH796759) was cloned by PCR method, contained a 1 557 bp ORF which encoded 518 amino acids. The recombinant HcALP3 protein induced by isopropyl-β-D-thiogalactoside(IPTG) was expressed as a 56.65 kD recombinant protein in E. coli BL21. The transcription analysis of HcALP3 gene in different tissue and at different development stages were analyzed by qRT-PCR, the results showed that HcALP3 gene was expressed in every instar and was highly abundant in the fourth-instar larvae and midgut. Ligand blot assay showed that HcALP3 protein only bound to Bt cry1 Ab35 toxin specifically, could not bind to Bt Cry1 Ac and Cry9 Ea10.The transcript levels of HcALP3 were reduced by 75.32% using RNAi technology for gene silencing of HcALP3. The dsRNA-HcALP3 injected larvae were reared on Bt Cry1 Ab35, Cry1 Ac and Cry9 Ea10 protein,the corrected mortalities were 45.93%, 73.33% and 70%, respectively. The results revealed that the corrected mortalities induced by Bt Cry1 Ab35 to dsRNA-HcALP3 injected larvae were significantly lower than those injected with dsRNA-egfp. In this study, the HcALP3 of H. cunea was identified and the function of HcALP3 was analysed, which provides basis for further study on the mechanism of Bt against H. cunea.

关 键 词：美国白蛾 碱性磷酸酶 Bt Cry毒素 Ligand blot RNAI 

分 类 号：S-4[农业科学]