美国白蛾中肠碱性磷酸酶HcALP1与苏云金杆菌3种Bt蛋白的体外结合特性分析

In vitro Binding Characteristics of Hyphantria cunea Midgut HcALP1 with Three Kinds of Bacillus thuringiensis Cry Toxins

美国白蛾(Hyphantria cunea)是危害桑树的主要害虫之一。为明确苏云金杆菌(Bacillus thuringiensis,Bt)杀虫晶体蛋白Cry对其作用机制,克隆美国白蛾中肠碱性磷酸酶基因,并对其编码蛋白的时空表达特异性及与Bt Cry1Ab35、Cry1Ac和Cry9Ea10的结合特性进行分析。根据美国白蛾中肠转录组数据获得碱性磷酸酶基因序列,设计特异引物,PCR扩增得到美国白蛾中肠碱性磷酸酶基因全长,命名为hcalp1(GenBank登录号为MH796758)。该基因开放阅读框为1 629 bp,编码542个氨基酸,预测蛋白质分子质量和等电点分别为60413 kD和583。实时荧光定量PCR和Western blot结果表明,hcalp1在美国白蛾4龄幼虫表达量最高,HcALP1蛋白在4龄幼虫中肠中的表达量也最高。构建原核重组表达载体pET30a-hcalp1,IPTG诱导表达获得分子质量约为70 kD的重组HcALP1蛋白;将Bt Cry1Ab35、Cry1Ac和Cry9Ea10原毒素分别经胰蛋白酶消化后与HcALP1重组蛋白进行体外配体印迹试验,发现HcALP1重组蛋白可与Bt Cry1Ab35、Cry1Ac和Cry9Ea10发生特异结合,推测HcALP1可能为3种Bt蛋白的中肠受体蛋白。

Hyphantria cunea is one of the main pests that endange mulberry trees. In order to clarify the functional mechanism of insecticidal crystal proteins from Bacillus thuringiensis(Bt)on Hyphantria cunea,alkaline phosphatase gene was cloned and identified from midgut of Hyphantria cunea. Expression characteristics of target protein as well as its binding characteristics with Bt Cry1Ab35,Cry1Ac and Cry9Ea10 were also analyzed. A alkaline phosphatase gene sequence was obtained based on the transcriptome data of Hyphantria cunea. By using the designed specific primers,the full-length gene of alkaline phosphatase was identified by PCR amplification,which was named as hcalp1(GenBank accession No.:MH796758). This gene had an open reading frame of 1 629 bp,encoding a protein of 542 amino acids with predicted molecular weight of 60 413 kD and isoelectric point of 5 83. According to real-time fluorescent quantitative PCR and Western blot analysis,hcalp1 was highly expressed in 4 th instar larvae,and HcALP1 protein was highly expressed in midgut of 4th instar larvae. Moreover,recombinant prokaryotic expression vector pET30a-hcalp1 was also constructed,and recombinant HcALP1 protein with molecular weight of 70 kD was expressed after induced by IPTG. After protoxins of Bt Cry1Ab35,Cry1Ac,Cry9Ea10 digested by trypsin,in vitro ligand blot assay showed that Bt Cry1Ab35,Cry1Ac,Cry9Ea10 could bind to HcALP1 recombinant protein specifically. The result suggested that HcALP1 might be the midgut receptor of 3 Bt proteins.