| 费氏中华根瘤菌15142的cysDN基因克隆及其相关功能 |
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| 引用本文： | 张武,田润,申佩弘,蒋承建,许兢,李俊芳,武波.费氏中华根瘤菌15142的cysDN基因克隆及其相关功能[J].应用与环境生物学报,2011(6):864-868. |
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| 作者姓名： | 张武 田润 申佩弘 蒋承建 许兢 李俊芳 武波 |
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| 作者单位： | 广西大学生命科学与技术学院;微生物及植物遗传工程教育部重点实验室;广西亚热带生物资源保护利用重点实验室;广西民族大学化学与生态工程学院; |
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| 基金项目： | 国家重点基础研究发展计划项目(No.2010CB126502)资助~~ |
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| 摘 要： | 为确定cysDN操纵子的功能及对根瘤菌结瘤的影响,通过三亲本接合,将质粒转座子pTnMod-RKm′随机插入费氏中华根瘤菌15142中,建成随机插入突变体库,随后通过含有不同硫源的MM培养基的筛选得到一株不能利用硫酸盐但能够利用半胱氨酸的突变体.进一步克隆和测序分析后发现该操纵子与已报道的Sinorhizobium sp.strain BR816的cysDN在核苷酸水平上有92%的相似性,在氨基酸水平上有96%的相似性.用自杀质粒pK18mob分别构建含有cysD部分片段和cysN部分片段的重组质粒,通过三亲本接合导入出发菌株15142中,经过同源单交换,分别获得cysD的pK18mob正反向插入突变株cysDF/15142以及cysDR/15142和cysN的pK18mob正反向插入突变株cysNF/15142与cysNR/15142.用广谱宿主质粒pLAFRJ载体连接完整操纵子cysDN构建互补质粒cysDN+pLAFRJ,将该质粒通过三亲本接合导入突变株中,获得互补菌株.用不同硫源的液体MM培养基培养,发现互补菌株能够补回突变菌株不能利用硫酸盐作为唯一硫源的缺陷,说明cysDN操纵子确实与硫酸盐同化途径有关;植株试验表明突变株比出发菌株推迟结瘤1~2 d,固氮酶活也比出发菌株稍低;竞争结瘤试验表明突变菌株占瘤率较差,但在平均瘤数、平均瘤重、平均植株干重上则无差异.
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| 关 键 词： | 费氏中华根瘤菌 硫代谢 cysDN 基因克隆 同源单交换 植株试验 |
Cloning and Related Function of cysDN in Sinorhizobium fredii 15142 |
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| ZHANG Wu,,TIAN Run,SHEN Peihong,JIANG Chengjian,XU Jing,LI Junfang, & WU Bo.Cloning and Related Function of cysDN in Sinorhizobium fredii 15142[J].Chinese Journal of Applied and Environmental Biology,2011(6):864-868. |
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| Authors: | ZHANG Wu TIAN Run SHEN Peihong JIANG Chengjian XU Jing LI Junfang & WU Bo |
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| Institution: | ZHANG Wu1,2,3,TIAN Run1,SHEN Peihong1,JIANG Chengjian1,XU Jing1,LI Junfang1,3 & WU Bo2,4(1College of Life Science and Technology,Guangxi University,Nanning 530005,China)(2Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering,China)(3Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization,China)(4College of Chemistry and Ecology Engineering,Guangxi University for Nationalities,Nanning 530007,China) |
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| Abstract: | In order to know function of cysDN and its effect on plant nodulation,a random insertion mutant library of Sinorhizobium fredii 15142 was constructed by three parental hybridization with the plasmid pTnMod-RKm' as transposon vector.A mutant strain which could not metabolize sulfate while could use cysteine as sulfur source,was selected by MM medium with different sulfur sources from the library.Molecular cloning and sequence analysis showed that this operon had 92% and 96% similarity with the cysDN gene seq... |
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| Keywords: | Sinorhizobium fredii sulfate assimilation cysDN gene clone homologous single crossover plant test |
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