【作者】 石磊；

【导师】 张承才；

【作者基本信息】 华中农业大学 ， 微生物学， 2008， 博士

【摘要】 鱼腥蓝细菌PCC 7120是一种丝状蓝细菌,在培养基中缺乏化合态氮源的条件下,其菌丝上约5-10%的细胞会分化成异形胞。异形胞为生物固氮提供场所,并将固定的氮源传递给临近的营养细胞,以维持整条菌丝的生长。由于氧气可以使固氮酶不可逆失活,异形胞采取了3种措施来创造无氧环境,以保护固氮酶:首先,其胞外覆盖有由外层的多糖层和内层的糖脂层组成的包被,以限制氧气的进入;其次,异形胞不具有完整的PSⅡ,丧失了光合放氧的能力;此外,异形胞内的呼吸作用大大加强。异形胞的分化过程分为不同的阶段并且经历了复杂的形态变化,大量信号传导相关的蛋白质参与其中。在鱼腥蓝细菌PCC 7120中,存在一类命名为hstK的基因家族,拥有13个成员。该家族的特点是,在其编码蛋白质的氨基端具有一个丝氨酸/苏氨酸激酶结构域,而羧基端具有一个组蛋白激酶结构域。在本研究中,对hstK基因家族的2个成员pkn44和pkn30的功能进行了探讨。pkn44和pkn30的双突变体D4.3不能在缺乏化合态氮源的条件下维持生长,在缺氮诱导24hr时,观察不到异形胞的分化,而在缺氮诱导48hr时,可以观察到一些以异形胞模式分布的细胞,这些细胞可以被使异形胞特异性多糖着色的阿尔新蓝染色（Het⁺表型）。这表明,D4.3的异形胞发育出现了延迟并停滞在了较早的阶段。乙炔还原法测定固氮酶活性结果表明,在环境中存在氧气时,D4.3丧失了固定N₂的能力;但是,当环境处于微氧状态时,D4.3能够表现出微弱的固氮酶活性（Fix⁺表型）。Western Blotting结果与固氮酶活性测定结果一致,在有氧条件下,D4.3中检测不到NifH的存在,只有在微氧条件下,才能在D4.3中检测到少量的NifH。RT-PCR和实时定量RT-PCR结果也显示,D4.3中只有在微氧条件下才能检测到微弱的nifH的转录。这些结果暗示,D4.3仍具有转录并表达NifH的能力,它在有氧条件下表现出来的种种异常很有可能是受到高水平细胞内氧气浓度的侵害,换而言之,D4.3分化出的不成熟的异形胞可能并不具备有效隔离氧气的能力。对D4.3的电子显微镜观察结果支持这一推论,在D4.3的异形胞外,没有观察到完整的异形胞包被,本应位于内层的糖脂层并不存在,而外层的多糖层也较野生型稀薄。薄层层析分析表明,D4.3只能合成异形胞特异性糖脂中的1-（α-D-葡萄糖）-3,25-二十六烷基二醇而不能合成1-（α-D-葡萄糖）-3-酮基-25-二十六烷醇。随后通过RT-PCR和实时定量RT-PCR对D4.3中部分糖脂相关基因的转录进行了检测,结果表明,这些基因的转录在D4.3中受到了不同程度的影响。在鱼腥蓝细菌PCC 7120中,prpJ也是一个只参与一种异形胞特异性糖脂合成的基因,它编码PP2C类蛋白磷酸酶。其突变体可以合成1-（α-D-葡萄糖）-3-酮基-25-二十六烷醇,而不能合成1-（α-D-葡萄糖）-3,25-二十六烷基二醇。prpJ2是prpJ的同源基因,同样编码一个PP2C类蛋白磷酸酶。prpJ2的突变并不影响菌体在缺氮条件下的生长和异形胞分化,但是,prpJ2和prpJ同时突变的双突变体S19/S20丧失了分化异形胞的能力。实时定量RT-PCR结果显示,prpJ2在缺氮后上调表达,而这种上调表达在ntcA突变体中观察不到,这暗示,prpJ2可能处于NtcA的调控之下。EMSA实验结果支持这一推论,在prpJ2编码区上游发现的NtcA结合位点可以在体外与NtcA相结合。此外,实时定量RT-PCR结果表明,突变体S19/S20中hetR的表达量在缺氮后不能维持上调表达,这有可能是其不能分化异形胞的原因。因此,PrpJ2可能参与了异形胞的早期发育。hanA编码组蛋白类似蛋白,其突变体不能分化异形胞。在本研究中发现,hanA的异位表达会导致异形胞的异常分化。研究还发现,在缺氮诱导24hr后,hanA的表达量在野生型中上调,而在ntcA突变体中hanA的表达量仍和诱导前持平。EMSA实验也证实,hanA编码区上游的NtcA结合位点可以与NtcA结合。这些结果暗示hanA可能直接受到NtcA的调控。更多还原

【Abstract】 Anabaena sp.strain PCC 7120 is a filamentous cyanobacterium capable of differentiating heterocysts,which account for 5 to 10%of the cells along each filament, upon limitation of a combined nitrogen source in the growth medium.Heterocysts provide an environment for nitrogenase to fix N₂ and support nitrogen source to vegetative cells.For nitrogenase that can be irreversibly inactivated by oxygen,three mechanisms are known to contribute to the formation of a microoxic environment to protect nitrogenase in heterocysts:first,formation of a thick cell envelope consisting of two distinct layers,the inner glycolipid layer and the outer polysaccharide layer,to limit oxygen penetration,secondly,inactivation of the oxygen-producing photosystemⅡ,and thirdly,increased respiration rate.Heterocyst development proceeds in stages and constitutes a complex process of morphogenesis,many proteins related to signal transduction are involved in this process.The genome of Anabaena sp.strain PCC 7120 possesses a family of 13 genes which named the hstK family,all encoding proteins with a putative Ser/Thr kinase domain at their N termini and a His-kinase domain at their C termini.In this study,founction of two members of hstK gene family,pkn44（all1625） and pkn30（all3691）,were identified.The double mutant D4.3 strain,in which pkn44 and pkn30 were both inactivated,is unable to sustain its growth in medium lacking a source of combined nitrogen and no obvious morphological detection were identified for heterocysts 24 h after nitrogen deprivation,however,in 48 h,the cells with a pattern similar to heterocysts could be observed,and weak alcian blue stained revealed the presence of heterocyst polysaccharides layer（Het⁺）.These observations suggest that the heterocyst development in D4.3 was delayed and arrested at a relatively early stage.Nitrogenase activity of D4.3 after step-down of combined nitrogen was measured using an acetylene reduction assay, no acetylene reduction by D4.3 was detected under aerobic condition,and however,a low nitrogenase activity was detected under microaerobic condition（Fix⁺）.Consistence with the nitrogenase activity,the expression of nifH in D4.3 could not be detected by Western blotting under aerobic condition,and only low expressional level detected under microaerobic condition.The nifH transcript was also determined by RT-PCR and real time RT-PCR,showing that nearly no transcripts were detectable in D4.3 under conditions provided except microaerobic condition.These results indicated that the ability for transcription and expression of nifH might not be lost in D4.3 and the high intracellular oxygenic level might be the main factor resulting in the defective procedures in D4.3,that maybe because of the incomplete heterocyst structure which might not prevent oxygen penetration.It was supported by which the ultrastructure of heterocysts in D4.3 by electron microscopy revealed that envelop of heterocysts was incomplete,the inner laminated glycolipid layer was absent and only a thin polysaccharide layer of the outer envelop was deposited.By the thin-layer chromatography,the mutant strain D4.3 had only one HGL corresponding to 1-（O-α-D-glucopyranosyl）-3,25-hexacosanediol,the other one corresponding to 1-（O-α-D-glucopyranosyl）-3-keto-25-hexacosanol was undetectable.The transcripts of genes involved in heterocyst glycolipid biosynthesis were abnormal compared with wild type through RT-PCR and real time RT-PCR.In Anabaena sp.strain PCC 7120,prpJ which encoding a PP2C-type protein phosphatases is another gene involved in only one HGL biosynthesis,one heterocyst-specific glycolipid,corresponding to 1-（O-α-D-glucopyranosyl）-3,25-hexacosanediol was completely missing in its mutant,while the other one corresponding to 1-（O-α-D-glucopyranosyl）-3-keto-25-hexacosanol could be detectable,prpJ2 also encodes a PP2C-type protein phosphatases and its deduced amino acid sequence is highly similar to that of PrpJ.The growth and heterocyst development were not defective in prpJ mutant,however,the heterocyst development was not be detected in the double mutant strain S19/S20 in which prpJ and prpJ2 were both inactivated.By real time RT-PCR,it showed that the the expression of prpJ2 was enhanced after step-down of combined nitrogen and this up-regulation was not detected in a ntcA mutant strain.EMSA were carried out with purified NtcA protein and a fragment from the prpJ2 upstream region containing the NtcA box,the retarded fragments were observed,indicating binding of NtcA.These results indicated that prpJ2 was directly under the control of NtcA.Through real time RT-PCR,the high expression level could not sustain in mutant stain S19/S20 after nitrogen deprivation compared with wild type.The defect in initiating heterocysts in S19/S20 might be due to the abnormal expression of hetR.hanA encodes historic-like protein,HU,in Anabaena sp.strain PCC 7120.In this study,ectopic expression of hanA defected heterocyst differentiation.The expression of hanA was enhanced at 24 hours after nitrogen step-down in wild type while the expression was not up-regulated in ntcA mutant.Combined with the result of EMSA that purified NtcA protein was able to bind upon a fragment from the hanA upstream region containing the NtcA box,hanA was directly under the control of NtcA.更多还原