Cloning, Expression and Functional Analysis of Alkaline Phosphatase HcALP3 Gene in Hyphantria cunea
ZHAO Dan1,*, CHANG Meng-Ying1,*, QIAO Ying-Cui1, WANG Zhe1, LIU Zi-Huan3, LU Xiu-Jun1,**, GUO Wei1,2,**
1 College of Plant Protection, Hebei Agricultural University, Baoding 071001, China; 2 Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China; 3 Huanghua Port Customs, Cangzhou 061113, China
Abstract：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 cry1Ab35 toxin specifically, could not bind to Bt Cry1Ac and Cry9Ea10. 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 Cry1Ab35, Cry1Ac and Cry9Ea10 protein, the corrected mortalities were 45.93%, 73.33% and 70%, respectively. The results revealed that the corrected mortalities induced by Bt Cry1Ab35 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.
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