|本期目录/Table of Contents|

[1]许 炼,刘 波,潘志针,等.小菜蛾钙黏蛋白PxCR10-11结构域的克隆、原核表达及功能分析[J].厦门大学学报(自然科学版),2017,56(01):59-63.[doi:10.6043/j.issn.0438-0479.201603014]
 XU Lian,LIU Bo,PAN Zhizhen,et al.Cloning,Prokaryotic Expression and Functional Analysis of PxCR10-11 Domain in Plutella xylostella Cadherin[J].Journal of Xiamen University(Natural Science),2017,56(01):59-63.[doi:10.6043/j.issn.0438-0479.201603014]
点击复制

小菜蛾钙黏蛋白PxCR10-11结构域的克隆、原核表达及功能分析(PDF)
分享到:

《厦门大学学报(自然科学版)》[ISSN:0438-0479/CN:35-1070/N]

卷:
56卷
期数:
2017年01期
页码:
59-63
栏目:
研究论文
出版日期:
2017-01-23

文章信息/Info

Title:
Cloning,Prokaryotic Expression and Functional Analysis of PxCR10-11 Domain in Plutella xylostella Cadherin
文章编号:
0438-0479(2017)01-0059-05
作者:
许 炼1刘 波2潘志针2朱育菁2高焕娟1陈清西1*
1.厦门大学生命科学学院,福建 厦门 361102; 2.福建省农业科学院农业生物资源研究所,福建 福州 350003
Author(s):
XU Lian1LIU Bo2PAN Zhizhen2ZHU Yujing2GAO Huanjuan1CHEN Qingxi1*
1.School of Life Sciences,Xiamen University,Xiamen 361102,China; 2.Agricultural Bio-resources Research Institute,Fujian Academy of Agriculture Sciences,Fuzhou 350003,China
关键词:
钙黏蛋白 CR结构域 同源建模 克隆 原核表达 Cry2Ab 配体印迹
Keywords:
cadherin CR domain homologous modeling clone prokaryotic expression Cry2Ab ligand blot
分类号:
S 433.4
DOI:
10.6043/j.issn.0438-0479.201603014
文献标志码:
A
摘要:
钙黏蛋白(cadherin)是一类跨膜糖蛋白,因其在苏云金芽胞杆菌(Bacillus thuringiensis,Bt)的杀虫过程中作为主要的受体而受到广泛研究.钙黏蛋白的特征之一是由若干钙黏蛋白重复单元(cadherin repeats,CR)组成的长链状蛋白,其中靠近细胞膜的CR结构域被认为是钙黏蛋白与Bt毒素发生互作的区域.小菜蛾(Plutella xylostella)钙黏蛋白由11个CR结构域和1个跨膜结构域组成,其中第10和11个CR结构域PxCR10-11被认为是钙黏蛋白与Bt毒素的互作区域.本研究从小菜蛾中肠cDNA中克隆得到小菜蛾钙黏蛋白PxCR10-11结构域的DNA片段,并通过原核表达系统对PxCR10-11结构域进行表达.配体印迹结果表明PxCR10-11可以特异性和Cry2Ab结合; 杀虫实验结果表明PxCR10-11能提高Cry2Ab对小菜蛾的毒力.此外,利用蛋白质同源建模和糖基化位点预测网站对PxCR10-11蛋白质结构进行了分析.上述结果表明PxCR10-11可能参与了Cry2Ab毒素对小菜蛾的毒杀过程,为后续研究小菜蛾钙黏蛋白和Cry2Ab的相互作用机制奠定了基础.
Abstract:
Cadherin,formed by a number of characteristic cadherin repeats(CR)domains,is a chain-like membrane glycoprotein.It was widely reported that cadherin in insect midgut played an essential role in the insecticidal activitives of Bacillus thuringiensis(Bt)toxins.The CR domain near the cell membrane was documented as an interaction region with Bt toxins.Plutella xylostella cadherin PxCR was formed by 11 CR domains and a transmembrane domain.The 10th and 11th CR domains(PxCR10-11)in PxCR were regarded as a potential interaction region with Bt toxins.In this study,PxCR10-11 domain was cloned from Plutella xylostella midgut cDNA,and then a prokaryotic expression vector pGEX4t-PxCR10-11 was constructed and the fusion protein GST-PxCR10-11 was induced to express.Ligand blot indicated that PxCR10-11 could specially bind to Cry2Ab and the bioassay revealed that PxCR10-11 had synergistic effect on Cry2Ab against Plutella xylostella larvae.Furthermore,homologous modeling of PxCR10-11 was conducted and the glycosylation sites in PxCR10-11 were predicted.These results indicate that PxCR10-11 is involved in the processing of Cry2Ab against Plutella xylostella larvae,which lays the foundation for the interaction mechanism of PxCR10-11 with Cry2Ab toxins.

参考文献/References:

[1] PIGOTT C R,ELLAR D J.Role of receptors in Bacillus thuringiensis crystal toxin activity[J].Microbiology and Molecular Biology Reviews,2007,71(2):255-281.
[2] VADLAMUDI R K,JI T H,BULLA L A.A specific binding protein from Manduca sexta for the insecticidal toxin of Bacillus thuringiensis subsp. berliner[J].Journal of Biological Chemistry,1993,268(17):12334-12340.
[3] SOBERóN M,PARDO-LóPEZ L,LóPEZ I,et al.Engineering modified Bt toxins to counter insect resistance[J].Science,2007,318(5856):1640-1642.
[4] GAHAN LJ,GOULD F,HECKEL D G.Identification of a gene associated with Bt resistance in Heliothis virescens[J].Science,2001,293(5531):857-860.
[5] XIE R,ZHUANG M,ROSS L S,et al.Single amino acid mutations in the cadherin receptor from Heliothis virescens affect its toxin binding ability to Cry1A toxins[J].Journal of Biological Chemistry,2005,280(9):8416-8425.
[6] QIU L,HOU L,ZHANG B,et al.Cadherin is involved in the action of Bacillus thuringiensis toxins Cry1Ac and Cry2Aa in the beet armyworm,Spodoptera exigua[J].Journal of Invertebrate Pathology,2015,127:47-53.
[7] FABRICK J,OPPERT C,LORENZEN M D,et al.A novel Tenebrio molitor cadherin is a functional receptor for Bacillus thuringiensis Cry3Aa toxin[J].Journal of Biological Chemistry,2009,284(27):18401-18410.
[8] SAMBROOK J,RUSSELL D W.分子克隆实验指南[M].黄培堂,王栋墚,周晓薇,等译.3版.北京:科学出版社,2002:1217-1255.
[9] CONTRERAS E,SCHOPPMEIER M,REAL M D,et al.Sodium solute symporter and cadherin proteins act as Bacillus thuringiensis Cry3Ba toxin functional receptors in Tribolium castaneum[J].Journal of Biological Chemistry,2013,288(25):18013-18021.
[10] LIU C,WU K,WU Y,et al.Reduction of Bacillus thuringiensis Cry1Ac toxicity against Helicoverpa armigera by a soluble toxin-binding cadherin fragment[J].Journal of Insect Physiology,2009,55(8):686-693.
[11] HUI F,SCHEIB U,HU Y,et al.Structure and glycolipid binding properties of the nematicidal protein Cry5B[J].Biochemistry,2012,51(49):9911-9921.
[12] ZU?IGA-NAVARRETE F,GóMEZ I,PE?A G,et al.A Tenebriomolitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles[J].Peptides,2013,41:81-86.Cloning,Prokaryotic Expression and Functional Analysis of PxCR10-11 Domain in Plutella xylostella CadherinXU Lian1,LIU Bo2,PAN Zhizhen2,ZHU Yujing2,GAO Huanjuan1,CHEN Qingxi1* (1.School of Life Sciences,Xiamen University,Xiamen 361102,China; 2.Agricultural Bio-resources Research Institute,Fujian Academy of Agriculture Sciences,Fuzhou 350003,China)Abstract:Cadherin,formed by a number of characteristic cadherin repeats(CR)domains,is a chain-like membrane glycoprotein.It was widely reported that cadherin in insect midgut played an essential role in the insecticidal activitives of Bacillus thuringiensis(Bt)toxins.The CR domain near the cell membrane was documented as an interaction region with Bt toxins.Plutella xylostella cadherin PxCR was formed by 11 CR domains and a transmembrane domain.The 10th and 11th CR domains(PxCR10-11)in PxCR were regarded as a potential interaction region with Bt toxins.In this study,PxCR10-11 domain was cloned from Plutella xylostella midgut cDNA,and then a prokaryotic expression vector pGEX4t-PxCR10-11 was constructed and the fusion protein GST-PxCR10-11 was induced to express.Ligand blot indicated that PxCR10-11 could specially bind to Cry2Ab and the bioassay revealed that PxCR10-11 had synergistic effect on Cry2Ab against Plutella xylostella larvae.Furthermore,homologous modeling of PxCR10-11 was conducted and the glycosylation sites in PxCR10-11 were predicted.These results indicate that PxCR10-11 is involved in the processing of Cry2Ab against Plutella xylostella larvae,which lays the foundation for the interaction mechanism of PxCR10-11 with Cry2Ab toxins.

备注/Memo

备注/Memo:
收稿日期:2016-03-07 录用日期:2016-06-02
基金项目:国家自然科学基金(31371999,31370059)
*通信作者:chenqx@xmu.edu.cn
更新日期/Last Update: 1900-01-01