华癸中生根瘤菌2020三个内源质粒的功能及其与豌豆根瘤菌共生质粒之间的相互关系

2020是一株分离自中国南方水稻田里的华癸中生根瘤菌（Mesorhizobiumhuakuii）,有3个内源质粒,分别命名为p2020a,p2020b和p2020c．用Tn5-sacB插入突变的方法对2020进行质粒消除,得到了两株质粒缺失突变株20201）29和2020D8．缺失了第一大质粒p2020c的突变株2020D29的结瘤固氮能力有显著的提高;而缺失了第二大质粒p2020b的突变株2020D8失去了在紫云英（Astragalus sinicus）上结瘤的能力;第三大质粒很难被消除,原因可能是该质粒上含有菌株生长所必需的基因．然后将豌豆根瘤菌（Rhizobium leguminosarum）的共生质粒pJB5JI转入2020及其质粒缺失突变株中,盆栽结果显示,2020-137（pJB5JI）的竞争结瘤能力和固氮能力显著高于2020．但是pJB5JI不能恢复2020D8在紫云英上的结瘤能力．2020D8—8（pJB5JI）可以在豌豆（Pisum sativum Linn）上形成无效瘤,这说明pJB5JI的功能可以在2020的遗传背景下进行表达．对pJB5JI在受体菌中的稳定性进行检测,结果发现在人工传代的情况下pJB5JI可以稳定的存在,但经过与植物共生之后只能在部分根瘤分离物中检测到pJB5JI,对这些转移接合子和出发菌株及分离菌株进行Km基因的PCR扩增,除了出发的受体菌外其余的菌株都可以得到PCR产物．由此推断,在没有检测到pJB5JI的分离株中,pJB5JI可能部分或全部整合到了受体根瘤菌的染色体DNA中．     

华癸中生根瘤茵7653R共生质粒pMH7653Rb的不相容性行为

摘要华癸中生根瘤菌菌株7653R含有2个内源质粒（pMH7653Ra,pMH7653Rb）．用三亲本杂交法将7653R的共生质粒pMH7653Rb分别导入HN308SR（Sm^r,Rif^r;含pMHHN308a,pMHHN308b和pMHHN308c）和HN3015SR（Sm^r,Rif^r;含pMHHN3015a,pMHHN3015b和pMHHN3015c）．发现受体菌HN308SR的两个稳定内源质粒pMHHN308b和pMHHN308c随着pMH7653Rb的导入而同时被消除．该接合子命名为HN308SRN14．这一结果表明pMH7653Rb与pMHHN308b和pMHHN308c不相容,可以归于同一不相容群．另一转移接合子HN3015SRN14的质粒图谱显示其第二大质粒pMHHN3015b由于pMH7653Rb的导入而被消除．该结果表明,pMH7653Rb与pMHHN3015b不相容．植物结瘤实验结果表明,pMH7653Rb的导入能恢复HN308SRN14的结瘤能力,其结瘤数目超过HN308SR,但不能替代pMHHN308b和pMHHN308c的固氮作用,HN308SRN14失去了固氮能力．质粒消除突变株HN308SRNl4D只含有pMHHN308a,能形成少量无效根瘤,确认pMHHN308a与HN308的结瘤能力有关．HN3015SRN14（含pMH7653Rb,pMHHN3015a和pMHHN3015c）只能形成无效根瘤,而质粒消除突变株HN3015SRN14D（仅含pMHHN3015a和pMHHN3015c）则完全失去结瘤能力．通过PCR反应从7653R,HN308,HN3015,HN308SRN14,HN3015SRN14中均检测到质粒复制基因repC．供试菌株的repC基因序列相似性达到99％．     

华癸中生根瘤菌HN3015非共生质粒pMhHN3015a的不相容性行为

采用三亲本杂交将Tn5-mob-sacB标记华癸中生根瘤菌(Mesorhizobium huakuii)HN3015的非共生质粒pMhHN3015a分别导入HN308SR和7653R-1SR, 获得2个转移接合子HN308SRN29和7653R-1SRN29。HN308SRN29的质粒图谱显示HN308SR的pMhHN308b被消除, 该结果暗示pMhHN3015a和pMhHN308b不相容。然而, HN308SRN29的质粒消除实验未获得标记质粒消除突变株。pMhHN3015a和pMhHN308a的大小     

华癸中生根瘤菌共生质粒的定向标记、消除与结瘤功能的鉴定

采用Tn5-mob-sacB转座子对华癸中生根瘤菌(Mesorhizobium huakuii)菌株7653R的共生质粒进行定向标记,获得该质粒标记菌株7653RT14.利用sacB基因对蔗糖的敏感性,对标记质粒进行消除实验,获得7653R的共生质粒消除突变株7653R-1.测得Tn5-mob-sacB转座频率高于10-5.突变株的培养特征与出发菌株基本一致.采用琼脂管法对7653RT14和7653R-1进行回接实验,结果显示7653RT14能正常结瘤固氮,表明Tn5的插入并未影响其共生能力,但失去共生质粒的7653R-1则为不结瘤或只结个别小瘤.稳定性实验结果表明供试菌株的标记质粒在本实验条件下是稳定的,可以作为共生质粒转移的供体菌.     

华癸中生根瘤菌质粒复制基因repC的克隆与鉴定

摘要：【目的】repC为质粒复制必需的起始蛋白基因。本研究旨在对华癸中生根瘤菌菌株HN3015及其质粒消除突变株进行repC基因的克隆和鉴定。【方法】采用通用引物RC1和RC3进行repC基因的PCR扩增,扩增产物克隆到载体pMD-18T,然后测序。利用Southern 杂交对repC基因定位。利用在线软件分析基因的序列特征,BLAST 工具进行同源性搜索;ExPASy推断其氨基酸的序列;ClustalW进行同源核苷酸和氨基酸序列的多重比较分析;PredictProtein 进行蛋白二级结构分析。【结果】     

华癸中慢生根瘤菌7653R MCHK-3535基因在自生和与紫云英共生中的功能

【目的】探究Mesorhizobium huakuii 7653R MCHK-3535基因在共生固氮中的功能。【方法】构建MCHK-3535插入失活突变体、超表达和互补菌株,对其进行共生表型鉴定;并测定各菌株的生长曲线、运动性及生物膜形成;利用qRT-PCR方法和组织表达定位分析MCHK-3535在共生过程中的时空表达特征。【结果】与野生型7653R相比,突变体Δ3535达到稳定期的生物量增加,运动性下降,生物膜形成增加。此外,MCHK-3535基因的失活会显著提高紫云英固氮能力和植物地上部分鲜重,但不影响根瘤数量及重量;且互补菌株C3535能部分回补到野生型性状,超表达菌株OV3535均无显著性差异;qRT-PCR和启动子组织表达定位发现,MCHK-3535基因主要在根瘤的侵染区、过渡区及固氮区表达,并且表达持续整个固氮期。【结论】MCHK-3535基因在自生及与紫云英共生过程中发挥重要功能,参与根瘤的正常发育并负向调控固氮酶活性。     

华癸中慢生根瘤菌7653R 3个脂质转运蛋白基因的克隆、突变及共生表型北大核心CSCD

【目的】脂类转移家族蛋白基因编码一类参与脂类转运及代谢的蛋白。本研究旨在构建华癸中慢生根瘤菌3个脂质转运家族蛋白基因的突变株,检测及分析突变体与紫云英共生条件下的表型及功能。【方法】利用生物信息学分析与预测转脂蛋白的结构特征及功能,采用荧光定量技术检测目标基因在自生和共生条件下的表达特性,通过插入突变技术构建目标基因突变株,并进行植物盆栽实验考察其共生表型。【结果】MCHK-5577、MCHK-2172和MCHK-2779基因编码蛋白属于START/RHO alpha_C/PITP/Bet_v1/Cox G/Cal C(SRPBCC)超家族,包含脂类转移结构域,参与脂类转运或代谢,与百脉根等中慢生根瘤菌相应基因的序列相似性达95%以上。这3个基因在共生条件下的表达水平都增高。分别构建了MCHK-5577、MCHK-2172和MCHK-2779基因突变菌株,与野生型菌株7653R相比,接种突变株MCHK-2172mut、MCHK-2779mut和MCHK-5577mut后的植株地上部分生物量和根瘤固氮酶活性显著降低。【结论】华癸中慢生根瘤菌脂质转移家族蛋白基因在共生互作过程中发挥重要作用,突变后明显影响共生固氮表型。本文的实验结果为深入研究脂类转移蛋白在共生固氮作用中的功能机制奠定了基础。     

华癸根瘤菌7653R hfq基因突变株的构建及其生物学特性

【目的】研究hfq基因在Mesorhizobium huakuii 7653R抵抗外界不利环境和共生固氮中的功能特性。【方法】利用pK19mob同源重组方法构建7653R hfq基因的插入失活突变株7653RΔhfq,并构建互补菌株7653RΔhfq-C,对hfq在压力胁迫和共生固氮中的功能特性进行研究。【结果】与野生型7653R相比,突变株7653RΔhfq的生长速率降低,热激处理后致死率升高;hfq突变影响了7653R中部分sRNA的表达;在4.5%乙醇和50 mmol H_2O_2生长胁迫下,突变株适应性明显较野生型差。另外,接种突变株的紫云英结瘤能力和固氮酶活性都明显降低。【结论】hfq基因作为重要的转录后调控因子,在7653R抵御外界胁迫环境和与宿主紫云英的共生固氮中发挥了重要作用。     

华癸根瘤菌urtB基因突变株的构建与共生固氮表型分析

【目的】尿素ABC转运体透性酶亚基编码基因urtB可能参与尿素代谢及支链氨基酸转运;本文旨在获得实验证据阐明urtB基因对华癸根瘤菌结瘤和固氮的影响,为深入研究其功能机制提供一定的科学依据。【方法】利用生物信息学分析urtB基因的结构特征及生物学功能,通过荧光定量检测urtB基因在自生和共生条件下的时空表达特征和启动子原位表达技术检测urtB基因组织表达特征,采用插入突变构建urtB突变株,通过植物盆栽并结合添加氮素处理,检测与分析突变体的共生固氮表型变化。【结果】分析表明urtB基因对于氮素转运非常重要,在共生条件下的表达水平比自生培养条件下显著上调表达;在成熟根瘤的固氮区中大量表达;正确构建和筛选获得了根瘤菌urtB突变株;接种urtB突变株与野生型菌株7653R相比较,突变体根瘤发育异常;植株地上部分生物量和根瘤固氮酶活性显著降低;添加氮素可恢复其共生缺陷表型。【结论】华癸中慢生根瘤菌urtB基因可能通过影响根瘤中氮转运或同化,进而在根瘤发育与共生固氮中发挥重要作用。     

华癸根瘤菌外膜孔蛋白编码基因MCHK_1326突变株的构建与共生固氮表型分析

[目的]Mesorh izob ium huakuii 7653R的MCHK _1326基因编码一种外膜孔蛋白,可能参与根瘤菌侵染宿主植物以及结瘤固氮过程,本研究旨在探索该基因在共生固氮中的功能.[方法]生物信息学分析MCHK _1326蛋白的结构特征及生物学功能,启动子原位表达技术检测MCHK_1326共生时空表达特...     

The function of three indigenous plasmids in Mtesorhizobium huakuii 2020 and its symbiotic interaction with Sym pJB5JI of Rhizobium leguminosarum

A Mesorhizobium huakuii strain 2020, isolated from a rice-growing field in southern China, contains three indigenous plasmids named p2020a, p2020b and p2020c, respectively. The plasmids were deleted via Tn5-sacB insertion, and two cured derivatives were obtained. Interestingly, the mutant 2020D29 curing of p2020c could significantly enhance the capacity of symbiotic nitrogen fixation. But the mutant 2020D8 curing of p2020b lost the ability to nodulate Astragalus sinicus. Furthermore, the third plasmid p2020a could be hardly eliminated, suggesting that some house-keeping genes necessary for strain growth located on this plasmid. Then the Sym plasmid pJB5JI of R. leguminosarum bv. viciae was transferred into 2020 and its cured derivatives. The pot plant test showed that the ability of competition and symbiotic nitrogen fixation of transconjugant 2020-137 (pJB5JI) was increased evidently in con-trast to 2020. pJB5JI could not restore the ability of 2020D8 to nodulate Astragalus sinicus. 2020D8-8 (pJB5JI) could form ineffective nodules on peas, which implied that the symbiotic plasmid pJB5JI could express its function at the chromosomal background of Mesorhizobium huakuii 2020. The plas-mid stability was checked in transconjugants under free-living and during symbiosis. The results indi-cated that pJB5JI failed to be detected in some nodule isolates. That Km resistance gene could be am-plified from all transconjugants and nodule isolates suggested that pJB5JI was fully or partially inte-grated into the chromosome of recipients.     

Incompatibility behavior of a symbiotic plasmid pMH7653Rb in <Emphasis Type=Italic>Mesorhizobium huakuii</Emphasis> 7653R

Mesorhizobium huakuii strain 7653R harbored two indigenous plasmids named pMH7653Ra and pMH7653Rb. The larger plasmid pMH7653Rb (symbiotic plasmid) was transferred to M. huakuii HN308SR harboring three plasmids: pMHHN308a, pMHHN308b and pMHHN308c, and HN3015SR harboring three plasmids: pMHHN3015a, pMHHN3015b and pMHHN3015c by tri-parent mating. Two stable indigenous plasmids, pMHHN308b and pMHHN308c of HN308SR, were co-eliminated due to the introduction of pMH7653Rb, and the transconjugant was named HN308SRN14. The results implied that pMH7653Rb and pMHHN308b, pMHHN308c were incompatible and might have been ascribed to the same incompatible group. The plasmid profiles of transconjugant HN3015SRN14 showed that the second largest plasmid pMHHN3015b of HN3015SR was cured due to the introduction of pMH7653Rb. The results also implied that pMH7653Rb and pMHHN3015b were incompatible. Results from plant nodulation tests showed that pMH7653Rb could only maintain the nodulation ability in transconjugant HN308SRN14 and its nodule number was more than that of wild strain HN308SR, but could not replace the nitrogen fixation effect of pMHHN308b and pMHHN308c. The plasmid cured mutant HN308SRN14D harboring only pMHHN308a formed null nodules that demonstrated pMHHN308a was relevant to nodulation ability. HN3015SRN14 harboring pMH7653Rb, pMHHN3015a and pMHHN3015c formed null nodules while HN3015SRN14D containing pMHHN3015a and pMHHN3015c lost the nodulation ability. The plasmid replication repC-like gene sequences were detected by a polymerase chain reaction from 7653R, HN308, HN3015, HN308SRN14 and HN3015SRN14. The repC gene sequence similarities of the strains tested attained 99%.     

转座子Tn5-Mob对菜豆根瘤菌共生基因的诱变、转移和初步定位

转座子Tn5-Mob在质粒RP4-4配合下能诱动(Mobilization)菜豆根瘤菌RCR3622内源质粒的诱动转移。在种间根瘤菌杂交过程中,二个巨型质粒的转移频率均大于10~(-3);分子量约为285kb的psym3622是带有结瘤(nod)和产黑素(mel)基因的共生质粒(Symbiotic plasmid);这二个基因的最大距离不超过70kb左右。另一个分子量约为135kb的质粒在试验中为不具结瘤功能的隐蔽质粒。将psym3622共生质粒导入不结瘤(Nod-)的豌豆根瘤菌菌株B151,能够使后者在菜豆植物上表达结瘤的特性,形成无效根瘤。将psym3622共生质粒导入不结瘤的菜豆根瘤菌菌株JI8400,能够在菜豆植物上形成正常发育的有效根瘤。     

Plasmid profiles and restriction fragment length polymorphism of Rhizobium leguminosarum bv. viciae in field populations

Abstract 56 isolates of Rhizobium leguminosarum biovar viciae from one field were characterized by analysis of plasmid profile, total DNA restriction pattern and restriction fragment length polymorphism (RFLP) of 2 chromosomal regions and of symbiotic (Sym) plasmid. Different levels of similarity exist in patterns generated by the different techniques. At the level of partial similarity these techniques give comparable results for more than 80% of the isolates, with the exception of RFLP profiling with the Sym probe. Analysis at this level allows the grouping of the isolates that have most of their non-Sym genome similarly organized. At the level of total similarity, the techniques are no more equivalent and provide complementary information on possible evolution of the different elements of the genome identified by each specific technique. The non-Sym plasmids defining classes were strongly associated with specific chromosomal backgrounds. In contrast, variations in Sym plasmids were not related with variations in the remaining genome. Host range towards chromosomes was variable among the Sym plasmids, which may reflect plasmid transfer between strains.     

Incompatibility behavior of a megaplasmid pMhHN3015c in Mesorhizobium huakuii HN3015

The Tn5-sacB-labeled symbiotic megaplasmid pMhHN3015c of Mesorhizobium huakuii HN3015 was, respectively, transferred into M. huakuii HN308SR containing three large plasmids of pMhHN308a, pMhHN308b and pMhHN308c, and 7653R-1SR, a symbiotic plasmid pMh7653Rb deleted mutant from M. huakuii 7653R by tri-parent mating. The stable indigenous plasmid pMhHN308c of HN308SR was cured by the introduction of pMhHN3015c and the transconjugant was named as HN308SRN18. The results implied that pMhHN3015c and pMhHN308c were incompatible and might be ascribed to the same incompatibility group. Furthermore, the results from plasmid curing tests of HN308SRN18 containing pMhHN3015c, pMhHN308b, and pMhHN308a showed that not only was pMhHN3015c deleted, but that pMhHN308a was also cured simultaneously. The plasmid profiles of transconjugant 7653R-1SRN18 showed pMhHN3015c could coexist with pMh7563Ra. The plasmid replication repC-like gene sequences were detected by polymerase chain reaction from 7653R-1SRN18, HN308SRN18 and its plasmid-curing derivatives, but failed to detect from plasmid-curing derivatives of 7653R-1SRN18. The repC gene sequence similarities of strains tested were up to 99%. Results from plant nodulation tests showed that introduction of pMhHN3015c failed to restore the nitrogen fixation ability of HN308SRN18 and 7653R-1SRN18.     

Plasmids and saprophytic growth of Rhizobium leguminosarum bv. trifolii W14-2 in soil

Abstract: The role of plasmids in the saprophytic growth of Rhizobium is mostly unknown. Plasmid-cured and complemented derivatives of R. leguminosarum bv. trifolii strain W14-2 were used to investigate the role of plasmids in the growth of this strain in sterile soil incubated under favorable moisture and temperature conditions. Strain W14-2 contains four plasmids ( a,b,c,d ). Absence of single plasmids in plasmid-cured derivatives generally did not reduce growth in soil when compared to the wild-type but absence of plasmid a delayed growth. Derivatives were unable to grow in soil when only plasmids a or d were present in cells. When only plasmids b or c were present, growth was delayed and the final population in 7 days was approximately 10% of the wild-type population. When the wild-type was co-inoculated at equal population into soil with derivatives lacking plasmids a , c , or d, the population of the wild-type at 7 days incubation was approximately 10 times larger than those of the derivatives. Elimination of only plasmid b did not reduce the ability of the strain to grow in soil when competing with the wild-type. Plasmids were involved in saprophytic growth of strain W14-2 in soil and may be important to the ecology of Rhizobium .     

The sym plasmid pRP2JI and at least two other loci of Rhizobium leguminosarum biovar phaseoli can confer resistance to infection by the virulent bacteriophage RL38

Abstract The virulent Rhizobium bacteriophage RL38 did not form plaques on R.leguminosarum by phaseoli but did so at high efficiency on a derivative of that strain lacking its symbiotic plasmid pRP2JI. Other strains with large deletions in pRP2JI which removed many nod and nif genes retained resistance to RL38, showing that the gene which confers phage resistance lies elsewhere on the plasmid. Although the wild-type strain of R. leguminosarum bv. phaseoli failed to plate RL38, it was possible to transduce chromosomal markers into this strain, indicating that the 'block' was not at an early stage in the infection process. Two different recombinant plasmids obtained from a clone bank of genomic DNA of R. leguminosarum bv. phaseoli , which appeared to have no DNA in common, both conferred resistance to RL38. Surprisingly, the DNA cloned in each of these plasmids did not originate from pRP2JI. Therefore, several different loci both on the Sym plasmid and elsewhere on the bacterial genome can be involved in conferring resistance to this bacteriophage.