| 少孢节丛孢菌胞外几丁质酶AO-379基因克隆及其表达分析 |
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| 引用本文： | 钟文强,孟庆玲,乔军,陈英,贡莎莎,王熙凤,黄运福,才学鹏. 少孢节丛孢菌胞外几丁质酶AO-379基因克隆及其表达分析[J]. 南方农业学报, 2018, 49(1): 148-154. DOI: 10.3969/j.issn.2095-1191.2018.01.24 |
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| 作者姓名： | 钟文强 孟庆玲 乔军 陈英 贡莎莎 王熙凤 黄运福 才学鹏 |
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| 作者单位： | 石河子大学 动物科技学院,新疆 石河子,832003中国农业科学院 兰州兽医研究所,兰州,730046 |
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| 基金项目： | 新疆研究生科研创新项目国家自然科学基金项目 |
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| 摘 要： | [目的]深入了解捕食线虫性真菌几丁质酶的生物学功能,为今后以少孢节丛孢菌重组几丁质酶AO-379研发线虫病生物防控制剂打下基础.[方法]克隆少孢节丛孢菌XJ-A1几丁质酶AO-379基因的全长序列,利用Signal P4.1 Server、InterPro、ExPASy等在线分析软件对其进行生物信息学分析;构建重组表达载体pET32a-AO-379,转化大肠杆菌BL21(DE3)感受态细胞后用IPTG进行诱导表达,并以SDS-PAGE和Western blotting检测分析表达获得的融合蛋白.[结果]少孢节丛孢菌几丁质酶AO-379基因全长1475bp,含有1个1203bp的开放阅读框(ORF),编码400个氨基酸.几丁质酶AO-379基因序列与少孢节丛孢菌标准株(ATCC 24927)几丁质酶基因序列的同源性为97.08%,其推导氨基酸的同源性为98.75%.几丁质酶A0-379属于糖苷水解酶18家族,具有SXGG和VDGFDLDFE两个催化区保守序列.其中,SLGGS位于第127~131位,为底物结合位点;VDGFDLDFE位于第173~181位,为水解酶催化活性位点.经大肠杆菌诱导表达获得的融合蛋白AO-379相对分子质量为60.0kD,且能与抗少孢节丛孢菌多克隆抗体发生反应.[结论]少孢节丛孢菌几丁质酶AO-379可在大肠杆菌中成功诱导表达,且获得的融合蛋白AO-379能与抗少孢节丛孢菌多克隆抗体发生反应,可用于研发线虫病生物防控制剂.
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| 关 键 词： | 少孢节丛孢菌 几丁质酶AO-379 克隆 分子特征 原核表达 |
Cloning of extracellular chitinase gene AO-379 in Arthrobotrys oligospora and its expression |
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| ZHONG Wen-qiang,MENG Qing-ling,QIAO Jun,CHEN Ying,GONG Sha-sha,WANG Xi-feng,HUANG Yun-fu,CAI Xue-peng. Cloning of extracellular chitinase gene AO-379 in Arthrobotrys oligospora and its expression[J]. Journal of Southern Agriculture, 2018, 49(1): 148-154. DOI: 10.3969/j.issn.2095-1191.2018.01.24 |
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| Authors: | ZHONG Wen-qiang MENG Qing-ling QIAO Jun CHEN Ying GONG Sha-sha WANG Xi-feng HUANG Yun-fu CAI Xue-peng |
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| Abstract: | [Objective]The biological function of nematode-trapping fungus chitinase was researched thoroughly to lay a foundation for developing nematodosis biological control agents by Arthrobotrys oligospora recombinant chitinase AO-379.[Method]The full-length cDNA sequence of A.oligospora XJ-A1 chitinase gene AO-379 was cloned,and bioin-formatics analysis was carried out using on-line analysis softwares such as SignalP 4.1 Server,InterPro and ExPASy.The recombinant plasmid vector pET32a-AO-379 was constructed and transformed into Escherichia coli BL21(DE3)compe-tent cells.The recombinant protein was induced by IPTG and the obtained fusion proteins were analyzed by SDS-PAGE and Western blotting.[Result]The full length of A.oligospora chitinase gene AO-379 was 1475 bp,contained a 1203 bp open reading frame(ORF)and encoded 400 amino acids.The homology between chitinase AO-379 gene sequence with that of standard A.oligospora strain(ATCC 24927)was 97.08%,and the deduced amino acid homology was 98.75%.Chi-tinase AO-379 belonged to the family of glycoside hydrolase 18 with two catalytic region conserved sequences: SXGG and VDGFDLDFE.Located in the 127thto 131stposition,SLGGS was substrate binding point.VDGFDLDFE was located in 173rdto 181stposition as hydrolase catalytic active site.The relative molecular mass of fusion protein AO-379 induced by E.coli was 60.0 kD,and the protein could react with polyclonal antibodies against A.oligospora.[Conclusion]A.oli-gospora chitinase AO-379 can be induced successfully in E.coli.The obtained fusion protein AO-379 can react with poly-clonal antibodies against A.oligospora,and therefore it can be used in developing nematodosis biological control agents. |
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