【作者】 张俊杰；

【导师】 陈文新； 陈文峰；

【作者基本信息】 中国农业大学 ， 微生物学， 2013， 博士

【摘要】 鹰嘴豆是药膳兼用的豆科植物,具有非常好的结瘤固氮能力。但分布在中国境内的能与鹰嘴豆共生固氮的根瘤菌至今未对其做过任何研究。鉴于此,本论文首次针对分布在我国主产区——新疆木垒县和奇台县的鹰嘴豆根瘤菌进行系统的生物学特征研究,以填补此方面研究的空白。论文先对我国新疆地区的鹰嘴豆根瘤菌进行了遗传多样性分析。从鹰嘴豆主产区新疆木垒和奇台两个县采集根瘤,经过分离纯化得到95株根瘤菌,采用16S rRNA基因和IGS-PCR RFLP、16S rRNA基因、三个持家基因(atpD、recA和gln11)和两个共生基因(nodC和nifH)的系统发育和分子进化分析。结果表明：1)供试根瘤菌全部属于中慢生根瘤菌属,但不同于该属的已知种,为潜在的新基因种；2)这些根瘤菌的共生基因与国外已报道的鹰嘴豆根瘤菌——Mesorhizobium mediterraneum和M. ciceri的相似性高达98.6%(nodC)和97.9%(nifH)以上,交叉结瘤试验也证明其与宿主植物严格的专一性和对应性；3)采集地的土壤pH值呈碱性(8.24-8.45),这与中慢生根瘤菌在生长时产酸,以中和并适应土壤的碱性特征相关；4)新疆鹰嘴豆根瘤菌种群与M. mediterraneum、M. ciceri和M. temperatum之间的分化系数(Kst*)分别为0.44227、0.48364和0.31383,而基因交流系数(Nm)分别为0.03、0.02和0.04,另外,M.mediterraneum、M. ciceri和M. temperatum相互间的分化系数(Kst*)均为1.0000,而基因交流系数(Nm)均为0.00。说明新疆鹰嘴豆根瘤菌种群与三个已知种之间遗传分化较多,基因交流较少,这表明新疆鹰嘴豆根瘤菌种群既与国外己知的两个鹰嘴豆根瘤菌种群不同,也与遗传距离较近的M. temperatum不同,由此推测,新疆特殊鹰嘴豆根瘤菌种群可能是在长期地理隔离及鹰嘴豆对根瘤菌共生基因选择的共同作用下形成的。在以上研究基础上,论文又对新疆鹰嘴豆根瘤菌的潜在新基因种进行多相分类鉴定。经过多序列基因比对、系统发育分析、全细胞蛋白电泳、脂肪酸种类和含量、极性脂种类分析、DNA同源性、数值分类和交叉结瘤试验等综合分析,结果表明：新疆鹰嘴豆根瘤菌种群属于中慢生根瘤菌属的一个新种,命名为木垒中慢生根瘤菌(Mesorhizobium muleiense sp. nov.)。为研究根瘤菌在自然条件下的进化规律和生态适应性,在最初采样研究的四年后,又重新采集了原采样点的鹰嘴豆根瘤和土壤样品,开展了鹰嘴豆根瘤菌的持家基因recA分析、生态适应性和竞争结瘤试验等工作。研究发现,田间重采集根瘤菌的持家基因recA较好地保持了木垒中慢生根瘤菌种群recA基因型的多样性和稳定性,与四年前的结果一致；而经过温室条件下鹰嘴豆从土壤中捕捉的方法得到的根瘤菌,其recA序列与四年前相比,缺少了CCBAU83939所代表的recA基因型。但是,两种条件下得到的鹰嘴豆根瘤菌都归属于唯一的种,即M. muleiense。说明种群M. muleiense在新疆土壤中保持着相对的遗传稳定性。使用四年前分离的M. muleiense与M. mediterraneum和M. ciceri进行的土壤适应性和竞争结瘤分析表明,灭菌的蛭石和土壤内,M. ciceri的占瘤率最高,M. mediterraneum次之；而未灭菌土壤内,M. muleiense占瘤率最高,表现出土著菌M. muleiense的生态适应性和高竞争性。更多还原

【Abstract】 Chickpea is a kind of legumes whose seeds can both be used for medicine and food, also can form nodules with rhizobia and fix nitrogen. While rhizobia associated with chickpea grown in China had never been surveyed until now. Therefore it is meaningful to explore the biological characterization of chickpea rhizobia from the cities of Mulei and Qitai in Xinjiang-the main planting area in China. And it will be a necessary supplementary for research of chickpea rhizobia all over the world.In this study, the phylogeny and diversity of chickpea rhizobia from Xinjiang were analyzed. The nodules were collected and total of95chickpea rhizobial strains were obtained from Mulei and Qitai in Xinjiang. These strains were analyzed by RFLP of genes of16S rRNA and1GS, the phylogenetic analysis of16S rRNA, three housekeeping genes (atpD, recA and glnⅡ), and symbiotic genes (nodC and nifH), and molecular evolution analysis. It showed that,1) all of the chickpea rhizobia in Xinjiang belonged to the genus of Mesorhizobium which were different from the known species from Mesorhizobium genus and were proposed as a distinctive genomic species;2) there was strong host-specificity of the symbiotic genes among the tested chickpea rhizobia which were above98.6%of nodC and above97.9%of nifH compared with the well studied chickpea rhizob’ia-Mesorhizobium mediterraneum and Mesorhizobium ciceri;3) the soil samples were all with the alkaline characteristic and pH values ranging from8.24to8.45, while the strains from Mesorhizobium can produce acids through the metabolism which may be used for neutralizing and adapting to the alkaline soils;4) The genetic differentiation index Kst*between the chickpea rhizobial group from China and the known species of M. mediterraneum, M. ciceri or M. temperatum was0.44227,0.48364or0.31383respectively, and the gene flow index Nm was0.03,0.02or0.04respectively while Kst*was1.00000and Nm was0.00between each couple of M. mediterraneum, M. ciceri and M. temperatum. It suggested that the tested species were significantly and geneticly differentiated from each other and that the distinct chickpea rhizobial group from China may result from the long history of geogrophical seperation and the selection of symbiotic genes from their host chickpea.Based on the research above, the distinctive genomic species of chickpea rhizobia from Xinjiang was further identified by polyphasic taxonomy including the multi locus sequence analysis (MLSA), the analysis of whole cell protein profiles, fatty acids profiles, polar lipids profiles, DNA-DNA hybridization, numerical taxonomy and cross nodulation. It confirmed the chickpea rhizobial group from Xinjiang as a novel species which was named as Mesorhizobium muleiense sp. nov..In order to find the evolution principle and ecological adaptation of rhizobia in natural conditions, four years after the original research, the chickpea nodules and soil samples from the original sites were collected again. Then housekeeping gene (recA), ecological adaptation and competitive nodulation tests were analyzed. It showed that recA sequences of the resampling chickpea rhizobia still kept well of the diversity and stability of recA gene types when compared with representative strains from M, muleiense four years ago; while recA sequences from the chickpea rhizobia trapped from soils in the condition of greenhouse were lack of the recA gene type related with CCBAU83939. However, no matter resampling or trapping, the chickpea rhizobia isolated both belonged to the single species of M. muleiense. It suggested that M. muleiense was the relatively stable species associated with chickpea in Xinjiang.Furthermore M. muleiense, isolated four years ago. M. mediterraneum and M. ciceri were tested for their soil adaptation and competitive nodulation. No matter in sterilized vermiculate or sterilized soil samples, M. ciceri showed higher nodule occupancy rates and M. mediterraneum followed behind. While when it comes to the unsterilized soil samples, M. muleiense both in inoculation and uninoculation treatments was the more competitive species and with higher nodule occupancy rates than the other two species.更多还原