草甘膦抗性菌株的筛

发布时间：2018-07-07 19:38

本文选题：aroA基因 + 草甘膦抗性　；参考：《华中农业大学》2013年硕士论文

【摘要】：5-磷酸烯醇式丙酮酸-3-磷酸莽草酸合成酶(EPSPS)是草甘膦的靶标酶,转基因抗草甘膦作物主要通过转入外源EPSPS基因来培育。抗草甘膦基因(EPSPS)的克隆、表达及其功能验证等是现代植物分子育种研究的重点。因此,加大挖掘相关基因资源和基因改造力度,对进一步获取具有独立知识产权的抗草甘膦新基因有重要意义。本研究从华南地区的农田和水体中采样,并对样品进行草甘膦处理,用含不同浓度梯度的草甘膦选择性培养基分离得到1株草甘膦抗性细菌和3株抗性真菌。通过对抗性菌株的形态观察和基于16S rDNA序列、ITS序列分析的系统发育分类方法,鉴定出该抗性细菌为肺炎克雷伯氏菌(Klebsiella pneumoniae),暂定名为kpS001；鉴定出3株抗性真菌分别为淡紫拟青霉(Paeciloomyces lilacinus)、多变根毛霉(Rhizomucor variabilis)和黄曲霉(Aspergillus flavus),分别命名为F35,F37,F62。克隆出肺炎克雷伯氏菌kpS001的aroA基因,命名为aroAs001对其序列进行分析,发现aroAs001基因片段长度为1284bp,在氨基酸序列的91-98和第175-183位有EPSP酶功能保守序列-L-G-N-A-G-T-A-和-A-L-L-M-T-A-P-L-A-。将该氨基酸序列与NCBI中Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044全长aroA基因编码的氨基酸序列相比,其编码的氨基酸序列的第76位发生变异,由苏氨酸突变为异亮氨酸,推测该位点的变化可能是导致肺炎克雷伯氏菌kpS001菌株对草甘膦抗性增强的原因之一。用pProA载体构建重组质粒pProA-aroAs001,转入aroA缺陷性大肠杆菌菌株DH5a (DH5a/△aroA)得到目标重组菌。通过诱导该菌的蛋白表达,发现目的蛋白在重组菌中有特异表达,大小约为48kDa,与预期蛋白大小相符；然后将重组菌株接入到含不同浓度草甘膦的M9基础盐培养基中培养,进行功能互补实验。实验结果显示,转入aroAs001基因的重组菌株的生长状态明显优于对照组菌株；在含200mmol/L以下草甘膦的培养基中,重组菌的生长基本没有受到影响,随着草甘膦浓度增加,重组菌的生长逐渐受到抑制,但仍明显高于对照菌株,重组菌能够在350mmol/L草甘膦浓度的选择性培养基上生长,表明克隆出来的aroA基因是kpS001对草甘膦产生抗性的主要原因。
[Abstract]:5-phosphoenolpyruvate-3-shikimic acid synthetase (EPSPS) is the target enzyme of glyphosate. The cloning, expression and functional verification of glyphosate resistant gene (EPSPS) are the focus of molecular breeding in modern plants. Therefore, it is of great significance to excavate related genetic resources and genetic modification to obtain new glyphosate resistant genes with independent intellectual property rights. In this study, glyphosate resistant bacteria and 3 resistant fungi were isolated from farmland and water in South China by glyphosate treatment. The resistant strain was identified as Klebsiella pneumoniae), as kpS001 by morphological observation and phylogenetic classification based on its sequence analysis of 16s rDNA sequence. Three strains of resistant fungi were identified as Paeciloomyces lilacinus), Rhizomucor variabilis and Aspergillus flavus (Aspergillus flavus), as F35, F37, F62, respectively. The aroA gene of Klebsiella pneumoniae kpS001 was cloned and named as aroAs001 to analyze its sequence. It was found that the length of aroAs001 gene was 1284 bp. there were EPSP functional conserved sequences -L-G-N-A-G-T-A- and -A-L-L-M-T-A-P-A-at the 91-98 and 175-183 amino acid sequences of Klebsiella pneumoniae. Compared with the amino acid sequence encoded by Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044 full-length aroA gene, the amino acid sequence was mutated from threonine to isoleucine. It is speculated that the change of this site may be one of the reasons for the increase of resistance to glyphosate of Klebsiella pneumoniae kpS001. The recombinant plasmid pProA-aroAs001 was constructed by using pProA vector and transferred into aroA deficient Escherichia coli strain DH 5a (DH 5a / aroA) to obtain the target recombinant strain. By inducing the protein expression of the strain, we found that the target protein was specifically expressed in the recombinant strain, the size of which was about 48 kDa, which was in line with the expected protein size, and then the recombinant strain was transferred into the M9 basic salt medium containing different concentrations of glyphosate, and was cultured in the M9 basic salt medium containing different concentrations of glyphosate. The functional complementary experiment was carried out. The results showed that the growth state of the recombinant strain transferred to aroAs001 gene was significantly better than that of the control strain, and the growth of the recombinant strain was almost unaffected in the medium containing glyphosate below 200 mmol / L, with the increase of glyphosate concentration. The growth of the recombinant strain was inhibited gradually, but still significantly higher than that of the control strain. The recombinant strain could grow on the selective medium with 350 mmol / L glyphosate concentration, indicating that the cloned aroA gene was the main cause of the resistance of kpS001 to glyphosate.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：Q78

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