摘要
【目的】明确苏云金芽胞杆菌Bacillus thuringiensis(Bt)晶体蛋白对暗黑鳃金龟Holotrichia parallela幼虫的杀虫机制。【方法】基于暗黑鳃金龟转录组数据,PCR克隆暗黑鳃金龟碱性磷酸酶(alkaline phosphatase,ALP)基因HpALP全长cDNA序列;利用原核表达系统在体外表达HpALP,并进行Western blot检测;利用qRT-PCR测定HpALP在暗黑鳃金龟3龄第2天幼虫不同组织(前肠、中肠、直肠、回肠、马氏管、脂肪体和体壁)中的表达水平;利用配体印记实验及ELISA技术对HpALP蛋白与苏云金芽胞杆菌晶体蛋白Cry8Ea3体外结合特性进行分析。【结果】得到了暗黑鳃金龟HpALP全长cDNA(GenBank登录号:MZ004964),长1536 bp,编码512个氨基酸,预测蛋白质分子量为57.32 kD,等电点为4.70;HpALP与食粪金龟Onthophagus taurus ALP的氨基酸序列一致性最高;原核表达获得重组蛋白HpALP,经Western blot检测HpALP重组蛋白在IPTG诱导8 h时的表达量最高。qRT-PCR分析的组织表达谱结果表明,HpALP在幼虫前肠中丰度最高。HpALP重组蛋白可以与Cry8Ea3特异结合,解离常数Kd值为76.21±26.44 nmol/L,最大亲合力Bmax值为0.59±0.068;而HpALP重组蛋白与对照Cry1Ab35不结合,Kd值为142.50±137.30 nmol/L,Bmax值为0.013±0.005。【结论】分离鉴定了暗黑鳃金龟HpALP蛋白,它与Bt Cry8Ea3亲和力强,推断其为Cry8Ea3的受体蛋白。研究结果为明确Cry8Ea3蛋白对暗黑鳃金龟的作用机制提供了理论依据。
【Aim】To ascertain the insecticidal mechanism of Bacillus thuringiensis(Bt)crystal protein against the larvae of the dark black chafer,Holotrichia parallela.【Methods】The full-length cDNA sequence of alkaline phosphatase gene HpALP of H.parallela was cloned by PCR based on the transcriptome data of H.parallela.HpALP was expressed in vitro by prokaryotic expression system and detected by Western blot.The expression levels of HpALP in different tissues(foregut,midgut,rectum,ileum,Malpighian tubules,fat body and integument)of the day-23rd instar larvae of H.parallela were detected using qRT-PCR.The in vitro binding characteristics of HpALP with the Bt crystal protein Cry8Ea3 was analyzed by ligand blot and ELISA.【Results】The full-length cDNA of HpALP(GenBank accession no.:MZ004964)of H.parallela was obtained.It is 1536 bp in length,encoding 512 amino acids with the predicted molecular weight of 57.32 kD and the pI of 4.70.HpALP has the highest amino acid sequence identity with ALP of Onthophagus taurus.The recombinant HpALP was obtained by prokaryotic expression,and it had the highest expression level at 8 h after IPTG induction detected by Western blot.Tissue expression profiles revealed that HpALP showed the highest abundance in the larval foregut analyzed by qRT-PCR.The recombinant HpALP could specifically bind to Cry8Ea3 with the dissociation constant Kd value of 76.21±26.44 nmol/L and the maximum affinity Bmax value of 0.59±0.068,but could not bind to the control Cry1Ab35 with the Kd value of 142.50±137.30 nmol/L and the Bmax value of 0.013±0.005.【Conclusion】Based on the isolation and identification of HpALP with high affinity to Bt Cry8Ea3,we inferred that HpALP might be the receptor protein of Cry8Ea3.The results provide a theoretic basis for clarifying the action mechanism of Cry8Ea3 protein on H.parallela.
作者
乔英翠
赵丹
王哲
陆秀君
郭巍
李瑞军
QIAO Ying-Cui;ZHAO Dan;WANG Zhe;LU Xiu-Jun;GUO Wei;LI Rui-Jun(College of Plant Protection, Hebei Agricultural University, Baoding, Hebei 071001, China;Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China;Hebei Walnut Technical Engineering Research Center, Lincheng, Hebei 054300, China)
出处
《昆虫学报》
CAS
CSCD
北大核心
2022年第4期409-416,共8页
Acta Entomologica Sinica
基金
财政部和农业农村部国家现代农业产业技术体系资助
河北省重点研发计划项目(20326812D)
中国农业科学院基本科研业务费(1610042020002)。
