【作者】 姜静；

【导师】 冯永君；

【作者基本信息】 北京理工大学 ， 生物学， 2015， 硕士

【摘要】 为适应环境，细菌进化出多种信号调控机制，其中吲哚介导的信号调控是近年来备受关注的一种。吲哚信号调控可以涉及种内和种间，影响菌体的细胞聚集等诸多重要生理行为，但其调节机制尚属未知。细胞聚集是菌体生存适应性的典型体现，其形成机制是目前细菌学的研究热点。因此获得吲哚调控通路的关键蛋白，对于认识吲哚的调控机制和菌体聚集体结构的形成机制具有重要意义。本研究选择能形成共质体这一独特菌体聚集结构的植物内生菌成团泛菌YS19为研究对象，利用Tn5转座子插入突变技术构建突变文库，从中筛选得到了一株受吲哚信号负调控的阳性突变子，报告基因的表达被下调32.6%。利用热不对称交错PCR技术鉴定突变基因为聚腺苷酸化酶基因pcnB，其序列全长1332bp，系统发育学分析显示该基因在成团泛菌种内高度保守。利用RT-PCR技术检测发现，吲哚信号显著抑制了该基因的表达。当pcnB突变时，菌株的多种生理行为都发生了变化。在LB培养基中，pcnB的突变并未影响菌株在指数期的生长，但加快了菌体稳定后期的衰亡速度，36h时菌体生物量较野生株下降了24.6%。同时，突变株培养至稳定期后期(40h)时共质体结构的形成受到显著影响，成团率和成团平均大小仅为野生株的19.6%和37.9%。可见，虽然聚腺苷酸化酶并不是菌体保持正常生长所必需的蛋白，但其在菌体的抗胁迫(营养不足)生长和共质体的形成过程中发挥重要的调控作用。运动性作为与细菌聚集密切相关的生理行为在pcnB突变时也受到抑制，在添加1mM吲哚时，与野生株相比，突变株的泳动性和丛动性分别被抑制了29.5%和11.1%。为了进一步探索其在吲哚信号调控通路中的调节机制，本研究测定了与细菌吲哚信号调控和聚集行为密切相关的碳存储因子(CsrA)的表达与聚腺苷酸化酶表达之关系。发现吲哚显著抑制csrA的表达，在菌体培养至12h时表达量较比对照组下降了74.8%。当pcnB基因突变时，csrA的转录也被抑制，尤其在稳定期极为显著，40h时其表达量仅为野生株的41.3%，但pcnB的突变并没有影响吲哚对csrA的调节作用，吲哚可能与聚腺苷酸化酶通过独立的通路作用于csrA或吲哚通过包括聚腺苷酸化酶在内的多调通路实现对csrA的调控作用，但具体是前者还是后者仍需进一步研究。利用SDS-PAGE技术检测了野生株和突变株的全细胞蛋白表达情况，结果显示突变株中有一大小约30kD左右的蛋白的表达量被上调。为了研究pcnB基因表达对菌株与水稻共生体系的影响，在EPA培养基上悉生培养水稻幼苗21d后，接种野生株和突变株的水稻整株鲜重分别增加了36.1%和20.1%，即pcnB突变减弱了菌体的促生能力。吲哚信号可增强菌体对植株地上部分的促生效果，当添加吲哚时，野生株侵染组和突变株侵染组水稻的地上部分鲜重分别增加了27.2%和21.6%。同时，pcnB突变也显著抑制了菌体的定殖能力，突变株的定殖量较比野生株下降了16.6%。但不论突变该基因突变与否，吲哚对菌体的定殖能力都有增强效果，添加吲哚后，突变株和野生株的定殖量分别增加34.7%和25%。本研究从RNA转录后修饰的角度探讨了吲哚信号调控通路，获得了吲哚调控通路的作用模式，同时丰富了对细胞内聚腺苷酸化调节及生物学功能的的认识。更多还原

【Abstract】 Bacteria evolve a lot of signal regulatory mechanisms among individuals inconfronting diverse environmental stresses, of which indole-based regulation attractsmore attention. As a kind of signal molecule which works in both intraspecies andinterspecies, indole regulates a variety of bacterial behaviors such as cell aggregation,etc., but the regulatory mechanism is still under investigation. The formation ofbacterial multicellular aggregate structure is a typical performance of adaptability andsurvivability, whose formation mechanism is a hot research area of the bacteriology.Obtaining the key proteins in indole regulatory pathway is significant forunderstanding its regulatory mechanism and also the formation mechanisms ofbacterial aggregation.This study selected Pantoea agglomerans YS19, a rice edophyte beingcharactered to form a special aggregate structure called symplasmata, as the modelstrain to construct a mutant library using the mTn5transposon mutagenesis method.From the mutant library, a positive mutant that is inhibited by indole was obtained,where the expression of the reportor gene decreased by32.6%when indole was added.By TAIL-PCR, it is demonstrated that the mutational site is pcnB, which codes thepoly(A) polymerase to catalyze the polyadenylation of RNAs. The full-length of thepcnB sequence is1332bp and phylogenetic analysis showed that pcnB is highconservative among strains in the species of P. agglomerans. It is revealed that indolesignificantly inhibited (by36.6%) the expression of pcnB by RT-PCR.Many physiological behaviors were changed in the pcnB mutant. In LB medium,the mutation of pcnB does not affect the growth of the bacterium inexponential growth phase, but increases the rate of decay in the late stationary growthphase. The cell concentration of the mutant decreased by24.6%in comparation to thewild type in36h. Meanwhile, the symplasmata formation ratio and average size wereonly19.6%and37.9%of that in the wild type at40h of cultivation, repectively.These data suggested that poly(A) polymerase played an important regulatory role inconfronting the environmental stresses and the symplasmata formation, although it isnot essential for the normal growth.The swimming and swarming abilities decreased by29.5%and11.1%comparedwith the wild strain, respectively, which might be one of the reasons that affect thecellular aggregation behaviors. The relationship between pcnB and the gene transcription of CsrA, a carbon storage regulator that is closely related to indoleregulation and symplasmata formation was further explored. It was revealed thatindole inhibites the expression of csrA, whose relative expression was inhibited by74.8%in comparison with the control at12h of cultivation. The deletion of pcnB alsoinhibited the transcription of csrA, especially in stationary phase. The relativeexpression of csrA was only41.3%of the control at40h. However, indole alsoinhibited the transcription of csrA by73.3%at12h. In other words, the deletion ofpcnB does not affect the the regulation pathway of indole on csrA expression,indicating that indole may cooperate with poly(A) polymerase by independentpathways to inhibit the csrA expression or regulate the csrA expression by severalpathways with poly(A) polymerase being involved in, which however need to befurther studied. Besides, SDS-PAGE examination on the whole cellular protein of thewild type and the mutant revealed that the expression of30-kD in the mutant wasupregulated.To explore the role of poly(A) polymerase in the plant-endophytes associationsystem, gnotobiotic cultivation of rice seedling was carried out on EPA medium.Inoculation of the wild strain and△pcnB improved the fresh weight of the21-d-cultivated host rice seedlings by36.1%and20.1%, respectively, suggesting thatthe deletion of pcnB inhibited the growth promoting effect of YS19. Meanwhile,indole improved the promoting effect of YS19. The fresh weight of the overgroundrice seedlings in the exogenous indole supplementary group increased by27.2%and21.6%, respectively, in comparison with the none supplemented groups. Thecolonization ability of YS19was also inhibited by the deletion of pcnB. Comparedwith the wild type, the total colonization number of△pcnB decrased by16.6%.Regardless of the mutation of pcnB, indole improved the of colonization ability YS19.The colonization number of the wild type and△pcnB mutant were increased by34.7%and25%, respectively, when indole was added.This study discussed the indole regulatory pathways from the point of RNApost-transcriptional modification, which enriched our knowledges of polyadenylationand expanded the research ideas of indole regulation.更多还原