| 甘蓝黑腐病菌XC_3374基因功能的初步探究 |
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| 引用本文： | 梁伟,白先放,徐玉婷,李磊,安世琦,陆光涛.甘蓝黑腐病菌XC_3374基因功能的初步探究[J].广西农业科学,2013(1):1-5. |
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| 作者姓名： | 梁伟 白先放 徐玉婷 李磊 安世琦 陆光涛 |
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| 作者单位： | [1]广西大学生命科学与技术学院,南宁530005 [2]广西医科大学生物靶向诊治研究中心,南宁530021 [3]亚热带衣业生物资源保护与利用国家重点实验室,南宁530005 |
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| 基金项目： | 国家自然科学基金项目（30771177）；广西亚热带生物资源保护与利用重点实验室项目（SB0705） |
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| 摘 要： | 【目的】初步探究XC_3374基因在甘蓝黑腐病菌Xcc8004中的功能，为深入研究甘蓝黑腐病菌Xcc8004中L-天冬酰胺酶的功能奠定基础。【方法】从KEGG数据库内获取XC_3374基因的旁侧序列，通过常规PCR对其进行克隆，同时利用自杀质粒pK18mobSacB通过同源双交换的方法构建了其缺失突变体DM3374并检测其表型。【结果】以引物D3374L-F/D3374R-R扩增，野生型菌株能够扩增出1789 bp的DNA片段，而候选突变体菌株能扩增出1367 bp的片段；以内部引物3374F/3374R扩增，野生型菌株能扩增出332 bp的片段，而候选突变体菌株不能扩增出任何片段，表明目标基因已缺失，缺失突变体DM3374构建成功。平板检测法结果表明，XC_3374基因突变后不影响胞外多糖及胞外酶的合成与分泌。在MMX基本培养基上，突变体菌株能够正常生长，形成的菌落大小与野生型菌株基本相同，表明缺失突变体不是营养缺陷性。游动平板检测结果表明， XC_3374缺失突变体的运动能力比野生型稍强，但差别不明显。采用剪叶法检测缺失突变体的致病性，结果表明，野生型Xcc8004和缺失突变体DM3374 病斑平均长度分别为13.000和11.983 mm，两者致病性无显著差异。【结论】XC_3374基因突变体在胞外多糖、几种胞外酶的合成与分泌、致病性等各种表型与野生型基本一致。
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| 关 键 词： | 甘蓝黑腐病菌 Xcc8004 XC_3374基因 突变体 表型分析 L-天冬酰胺酶 |
Preliminary study on the function of Xanthomone campestris pv. campestris XC3374 gene |
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| LIANG Wei,BAI Xian-fang,XU Yu-ting,LI Lei,AN Shi-qi,LU Guang-tao.Preliminary study on the function of Xanthomone campestris pv. campestris XC3374 gene[J].Guangxi Agricultural Sciences,2013(1):1-5. |
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| Authors: | LIANG Wei BAI Xian-fang XU Yu-ting LI Lei AN Shi-qi LU Guang-tao |
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| Institution: | 1College of Life Scienee and Technology, Guangxi University, Nanning 530005, China; 2Baologlcal Targeting Diagnosis ＆ Therapy Research Center of Guangxi Medical University, Nanning 530021, China; 3State Key Laboratory of Conservation and Utilization of Subtropical Agro-bioresources, Nanning 530005, China) |
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| Abstract: | 【Objective】The functions of XC_3374 gene in Xanthomonas campestris pv. campestris Xcc8004 strain were preliminarily studied in order to lay the foundation for future studies of L-asparaginase function in Xcc8004. 【Method】From the flanking sequence of XC_3374 gene obtained from the KEGG database， through conventional PCR methods， the genes were cloned， and at the same time， using the Dutch act plasmid pK18mobSacB through the homologous double exchange method， the mutant DM3374 was constructed and its phenotype was detected. 【Result】By the D3374L-F/D3374R-R primer amplification， wild strains could amplify 1789 bp DNA fragments， while the mutant strains could amplify 1367 bp fragments. Using the amplified internal primers 3374F/3374R， wild strains could amplify 332 bp fragments， while the mutant strains could amplify fragments of any length， which showed that the target gene has been deleted， so the DM3374 deletion mutant was constructed successfully. The results showed that through the plate detection method， mutated XC_3374 gene did not affect the exopolysaccharides and extracellular enzyme synthesis or secretion. On top of the MMX basic medium culture， the mutant strains could grow normally and the colony size and form were basically the same as the wild strains， which indicated that the deletion mutant was not auxotrophic. The moving plate test results showed that the exercise capacity of XC_3374 deletion mutant was better than that of the wild strains， but the difference was not significant. Other test results showed that by using the clipping method for detecting deletion mutant＆rsquo;s pathogenicity， the average length of the scabs in wild Xcc8004 and deletion mutant DM3374 were respectively 13.000 and 11.983 mm， and there was no significant difference between the two strains. 【Conclusion】In the synthesis of extracellular polysaccharide and several extracellular enzymes， the secretion and pathogenic phenotype of XC_3374 mutant strains were consistent with those of the wild strains. |
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| Keywords: | Xanthomonas campestris pv eampestris Xcc8004 XC_3374 gene mutant phenotypie analysis L-asparaginase |
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