【作者】 谷慧琳；

【导师】 李友国；

【作者基本信息】 华中农业大学 ， 微生物学， 2011， 硕士

【摘要】 ftsZ基因是一个重要的细胞分裂相关基因,在真细菌中保守存在,在部分古菌和真核生物中也有发现。FtsZ蛋白在原核细胞及叶绿体、线粒体等质体分裂过程中发挥重要作用。当染色质复制并完成拟核分离,FtsZ即最早出现在分裂位点,直接参与并起始细胞分裂环的形成。大多数物种一般只含有一个ftsZ基因,有些物种基因组含有两个或更多的ftsZ基因。研究发现多种根瘤菌基因组中含有两个ftsZ基因：ftsZ1和ftsZ2。FtsZ1和FtsZ2虽具有一定的同源性但存在结构上的差异。华癸中慢生根瘤菌7653R与紫云英共生形成不定型根瘤,具有不定型根瘤类菌体的典型分化特征,且其基因组中含有两个细胞分裂相关基因ftsZ。本研究旨在探讨其细胞分裂相关基因ftsZ1在类菌体分化发育过程中的功能。首先通过构建同源重组置换载体,利用三亲本接合转移和选择培养基,筛选获得双交换的ftsZ1缺失突变株Z5-17。对突变株的培养特征和共生表型进行了进一步的检测分析等实验研究。结果表明：培养条件下的Z5-17形态、大小和生长速度与野生菌株7653R没有明显差异；在共生条件下,与突变株z5-17共生的宿主紫云英植株表现出明显的氮饥饿状态,形成白色小瘤,仅检测到极微弱的固氮酶活性,但结瘤数量明显增多。根瘤组织石蜡切片和电镜切片观察发现,突变株z5-17类菌体细胞分化异常,功能性共生体形成受阻,根瘤菌持续性侵染-结瘤固氮的过程中断,根瘤表现出明显的提前衰老特征。最后,采用荧光定量PCR检测了ftsZ1基因的表达水平和特性,发现其白生培养条件下的表达水平明显高于共生条件。综合以上结果,不难看出ftsZ1基因突变虽不影响华癸中慢生根瘤菌7653R培养条件下的生理特性,但显著影响了其与紫云英的共生固氮功能,ftsZ1的正常表达为类菌体分化、并形成功能性共生体所必需。更多还原

【Abstract】 ftsZ is an important cell-division gene, which ubiquitously exists in eubacteria, also in some Archaeas and eukaryotes. FtsZ protein is crucial for the division processes of prokaryotic cell and plastids, such as chloroplast and mitochondria. Once chromosome replication and separation are completed, FtsZ protein appears at the division site earliest, then participate in the initiation of division ring formation. Normally there is only one ftsZ gene in most species, but some particular ones have more than one. Recent studies reveal that some rhizobia species have two ftsZs:namely ftsZl and ftsZ2, which encoded two FtsZs with significant sequence similarity but difference in ultrastructure.Mesorhizobium huakuii 7653R, which could form indeterminate nodules on legume plant-Astragalus sinicus, has the typical characteristics of bacteroid differentiation in indeterminate nodules and two ftsZs gene in genome. This work aimed at investigating the function of M. huakuii 7653R cell-division gene ftsZl during the process of bacteroid differentiation. Firstly, a replacement vector, with which partial deletion in ftsZl gene, was constructed for the homologous recombination. Followed by tri-parental transconjugation and screening on selective medium, a mutant strain Z5-17 undergone double crossover was obtained. The phenotypes of mutant strain Z5-17 under both free-living and symbiotic conditions were further examined and analyzed in details. The results showed that Z5-17 displayed similar morphology, size and growth speed as compared with wild type strain 7653R under free-living condition. However, when host plant A. sinicus was in symbiosis with Z5-17, it showed obvious nitrogen-deficiency features and formed small-white-nodules with hardly nitrogenase activity, but the nodule number was dramatically increased. Furthermore, paraffin section and ultrastructure observations were carried out for the 25-dpi (day post inoculation) nodules formed by wild type strain 7653R and mutant Z5-17, respectively. It was found that the mutant strain Z5-17 displayed abnormal bacteroid differentiation, and the functional symbiosome development was blocked, consequently resulting in the interruption of rhizobial chronic infection programme and the lost of symbiotic nitrogen fixation ability. Paraffin section of nodules induced by mutant Z5-17 exhibited prematurely senescence features. Finally, by using the real time PCR, it was revealed that the expression level of ftsZl under free-living condition was much higher than that in symbiotic root nodules. In summary, although the mutation of ftsZl had no significant influences on the physiological attributes of free-living M. huakuii 7653R, it led to the impairment of symbiotic nitrogen fixation ability with A. sinicus. The results suggest that the normal expression of ftsZl is indispensable for bacteroid differentiation and functional symbiosome formation.更多还原