肺炎克雷伯氏菌S001草甘膦靶标酶基因的克隆及其抗性验证

doi:10.3864/j.issn.0578-1752.2014.01.009

摘要/Abstract

摘要： 【目的】由于草甘膦在农业生产中的广泛使用，抗草甘膦转基因作物的研究一直是转基因作物研究的热点，其中草甘膦抗性功能新基因的挖掘是核心问题。自然界中微生物种类繁多，基因资源丰富，论文拟从广东地区农田土壤中筛选和鉴定出高抗草甘膦菌株，克隆其草甘膦靶标酶基因并进行抗性水平验证，以期获得高抗草甘膦新基因资源用于抗草甘膦转基因作物的研究。【方法】用含有浓度梯度草甘膦的选择培养基从备选土壤中筛选出具有高抗草甘膦特性的菌株；通过显微观察、革兰氏染色以及16S rDNA序列分析结果对菌株种类进行鉴定；利用RT-PCR技术克隆该菌株的草甘膦靶标酶基因aroAS001，并通过序列比对和系统发育树分析aroAS001序列的基本特征；利用重叠 PCR法对aroAS001变异位点进行定点突变获得aroAS001-mut序列后，将aroAS001和aroAS001-mut基因片段转入aroA基因缺陷型菌株DH5α/△aroA中进行基因功能互补和草甘膦抗性水平验证。【结果】分离出一株高抗草甘膦菌株，经形态学和分子生物学鉴定该菌株为肺炎克雷伯氏菌（Klebsiella pneumoniae），命名为kpS001；克隆该菌株草甘膦靶标酶5-烯醇式丙酮酰莽草酸-3-磷酸合成酶（EPSPS）基因aroAS001，序列分析结果显示由该基因所编码的氨基酸序列具有典型的Class I EPSPS特征，但与已报道肺炎克雷伯氏菌的aroA相比其第227位碱基发生突变，致使对应的氨基酸发生变异。对该基因差异位点进行核酸定点突变获得aroAS001-mut基因片段后，将aroAS001和aroAS001-mut基因片段分别转入缺陷型大肠杆菌菌株DH5α/△aroA进行功能互补验证，与对照菌株相比，转入aroAS001和aroAS001-mut的重组菌株均能在含200 mmol?L-1以下浓度草甘膦的培养基中能够正常生长，表现出良好的抗性，之后随着草甘膦浓度增加其生长状态开始受到抑制，当草甘膦浓度达到350 mmol?L-1时，菌株生长基本完全被抑制。【结论】肺炎克雷伯氏菌kpS001菌株是一株高抗草甘膦菌株，由aroAS001编码的草甘膦靶标酶EPSPS属于Class I EPSP合成酶，aroAS001对草甘膦具有优良抗性，能在含350 mmol?L-1以下浓度草甘膦的培养基中生长，该基因可以作为备选基因资源用于抗草甘膦转基因作物的研究。

关键词: 肺炎克雷伯氏菌 , 5-烯醇式丙酮酰莽草酸-3-磷酸合成酶（EPSPS） , aroAS001 , 草甘膦抗性

Abstract: 【Objective】Due to the widespread use of glyphosate in agricultural production, glyphosate has become the preferred object on transgenic crops resistant to herbicides. Finding glyphosate resistance genes is a primary issue in the study of transgenic crops resistant to glyphosate. There are various kinds of microorganisms in nature with rich genetic resources, so this study intends to screen and identify high glyphosate-resistance bacterium strains from field soil samples of Guangdong area, then clone the target gene of glyphosate from the strain and test its glyphosate-resistance level, in order to obtain high glyphosate resistance gene resources for research of glyphosate resistant transgenic crops.【Method】The gradient dilution glyphosate selected culture method in the isolation of high glyphosate-resistance bacterium from field soil samples of Guangdong area was applied. Identification of the strain species according to the microscopic observation, gram staining and 16 S rDNA sequence analysis results. Using RT-PCR method to clone the strain’s target gene of glyphosate, and analyse the basic characteristics of aroAS001 sequences by sequence alignment and phylogenetic tree. The aroAS001 variant performed site directed mutagenesis by overlapping PCR method to obtain aroAS001-mut gene fragments. The aroAS001 and aroAS001-mut fragments were transferred into defective Escherichia coli DH5α/△aroA strains, respectively, to detect the resistant levels of glyphosate. 【Result】 A high glyphosate-resistance bacterium strain was isolated, and it was identified as Klebsiella pneumoniae by morphological and molecular biology methods, named kpS001 strain. The target gene of glyphosate from kpS001 strain named aroAS001 was cloned. Sequence analysis showed that the amino acid sequence of the gene encoded had a typical Class I EPSPS features, and there was a single amino acid different from with another K. pneumoniae strain’s aroA. After obtained the aroAS001-mut fragments by overlap PCR, aroAS001 and aroAS001-mut fragments were transferred into the defective E. coli DH5α/△aroA strains, respectively, to detect the resistant levels of glyphosate. Compared with the control strains, the recombinant strains containing aroAS001 and aroAS001-mut were able to grow normally in the culture medium containing less than 200 mmol?L-1 glyphosate, however, as glyphosate concentration increasing, the growth state of the recombinat strains gradually suppressed, while the concentration of glyphosate increased to 350 mmol?L-1, the growth was completely suppressed. 【Conclusion】The K. pneumoniae S001 is a high glyphosate-resistance bacterium, the target gene of glyphosate aroAS001 belongs to the Class I EPSPS and shows a significant glyphosate-resistance characteristics, it could be used as potential gene materials in transgenic glyphosate-resistant crop studies.

Key words: Klebsiella pneumoniae , 5-enolpyruvoylshikimate-3-phosphate synthase , aroAS001 , glyphosate-resistance

张纯1, 吴丹丹1, 2, 冯莉1, 田兴山1, 郭爱玲2. 肺炎克雷伯氏菌S001草甘膦靶标酶基因的克隆及其抗性验证[J]. 中国农业科学, 2014, 47(1): 80-89.

ZHANG Chun-1, WU Dan-Dan-1, 2 , FENG Li-1, TIAN Xing-Shan-1, GUO Ai-Ling-2. Cloning and Resistance Verification of a Target Gene of Glyphosate from Klebsiella pneumoniae S001[J]. Scientia Agricultura Sinica, 2014, 47(1): 80-89.

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