藤县药用野生稻内生固氮菌分离鉴定及系统发育分析

doi:10.13560/j.cnki.biotech.bull.1985.2016.06.016

摘要/Abstract

摘要： 从广西梧州市藤县药用野生稻中筛选内生固氮菌，为微生物肥料生产收集菌种资源。采用3种无氮培养基经平板划线法筛选内生固氮菌；通过乙炔还原法测定其固氮酶活性；利用IS-PCR(Insertion sequence-based PCR)指纹图谱和全细胞蛋白电泳图谱对获得的内生固氮菌进行聚类分析；根据16S rRNA基因序列分析确定其进化关系；使用分光光度计比色法检测其产生长素能力、产铁载体能力、溶磷能力和ACC脱氨酶活性。结果显示，从野生稻中筛选获得34株内生固氮菌分为5个类群，类群I和II各有8株，类群III、IV、V分别有2株、5株和11株，其固氮酶活性介于5.0和1 036.7 nmol/(mL·h)之间。类群I代表菌株HU33与无乳固氮螺菌(Azospirillum amazonense ATCC 35119^T)相似性为97.13%；类群II代表菌株HU31与变栖克雷伯氏菌(Klebsiella variicola DSM 15968^T)相似性为99.71%；类群III代表菌株HU17与蒙氏假单胞菌(Pseudomonas monteilii ATCC 700476^T)相似性为99.86%；类群IV代表菌株HU13与黄黄色杆菌(Xanthobacter flavus ATCC 35867^T)相似性为100%；类群V代表菌株CM05与越南伯克霍尔德菌(Burkholderia vietnamiensis LMG 10929^T)相似性为99.86%。药用野生稻内生固氮菌资源具有遗传多样性；有些菌株有较强的产生长素、产铁载体、ACC脱氨酶活性和溶磷能力，在农业生产上具有一定的应用潜力；类群I可能是一个新类群。

关键词: 药用野生稻, 内生固氮菌, 系统发育分析, 全细胞蛋白电泳, IS-PCR

Abstract: This research aims to select the endophytic diazotrophs from wild medical-use Oryza officinalis growing on Teng County of Guangxi for collecting the strain resources contributing to the production of microbial fertilizer. Endophytic diazotrophs were isolated from O. officinalis using three selective mediums of free nitrogen and plate streaking method. Acetylene reduction assay was used to determine the activity of nitrogenase in the endophytic diazotrophs. The SDS-PAGE(dodecyl sulfate sodium salt-polyacrylamide gel electrophoresis)patterns of whole-cell proteins and IS-PCR DNA fingerprinting patterns were employed for the clustering analysis of the isolated endophytic diazotrophs. The 16S rRNA gene sequences of the representative strains of each group were analyzed to construct the phylogenetic tree. The abilities of producing auxin，producing siderophores，dissolving phosphorus and the activity of ACC deaminase were tested by colorimetry of spectrophotometer method. Thirty-four strains of endophytic diazotrophs were isolated from O. officinalis and assigned to 5 groups and their nitrogenase activity was between 5.0 to 1 036.7 nmol/(mL·h). Group I and II each had 8 strains. Group III，IV，V had 2，5，11 strains respectively. The homology of 16S rRNA gene was 97.13% between Group I and Azospirillum amazonense ATCC 35119^T，99.71% between Group II and Klebsiella variicola DSM 15968^T，99.86% between Group III and Pseudomonas monteilii ATCC 700476^T，100% between Group IV and Xanthobacter flavus ATCC 35867^T，and 99.86% between Group V and Burkholderia vietnamiensis LMG 10929^T. In conclusion，some strains have the ability of producing auxin，producing siderophores，ACC deaminase activity，and dissolving phosphorus. The results suggest that endophytic diazotrophs in medical-use Oryza officinalis present genetic diversity and potential applications in agricultural practices；and group I probably is new.

Key words: medical-use Oryza officinalis, endophytic diazotrophs, phylogenetic analysis, whole-cell protein electrophoresis, insertion sequence-based PCR

胡文哲, 谭泽文, 王勇, 徐羡微, 谭志远. 藤县药用野生稻内生固氮菌分离鉴定及系统发育分析[J]. 生物技术通报, 2016, 32(6): 111-119.

HU Wen-zhe, TAN Ze-wen, WANG Yong, XU Xian-wei, TAN Zhi-yuan. Isolation，Identification and Phylogenetic Analysis of Endophytic Diazotrophs in Tengxian Medical-use Oryza officinalis[J]. Biotechnology Bulletin, 2016, 32(6): 111-119.

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