【作者】 胡国元；

【导师】 周俊初；

【作者基本信息】 华中农业大学 ， 微生物学， 2006， 博士

【摘要】 从华中农业大学水稻田采集的紫云英根瘤中分离到62株华癸中生根瘤菌，经培养观察、显微观察和回接实验确认其中57株为典型的华癸中生根瘤菌。供试菌株的最早现瘤时间为8d，最迟超过30d，多数菌株为10-22d。对其中的44株质粒的检测结果表明：13株含3个质粒、21株含2个质粒、7株含1个质粒、3株不含质粒，大小范围为150-428kb。采用Tn5-mob-sacB转座子对含有的3个大质粒的HN3015菌株进行定向标记，获得各供试质粒的标记菌株。利用sacB基因对蔗糖的敏感性进行标记质粒消除实验，获得HN3015的质粒消除或缺失突变株，其中HN3015-1为第一大质粒pMhHN3015c消除突变株，HN3015-2为为第二大质粒pMhHN3015b消除突变株，HN3015-3为第三大质粒pMhHN3015a消除突变株，HN3015-6为pMhHN3015a部分缺失突变株。对HN3015与突变株的培养特征、生长速度和共生结瘤能力进行的研究结果表明：质粒消除或缺失突变株的培养特征均与出发菌株一致，第一大质粒pMhHN3015c与固氮有关，第二大质粒pMhHN3015b的消除则失去结瘤能力，第三大质粒pMhHN3015a的消除或缺失不但未影响结瘤与固氮能力，其消除反而可增强突变株HN3015-3的竞争结瘤能力和共生效率。抗酸性实验结果表明3个内源质粒均对HN3015的抗酸性有正控制作用，并对其抗碱性起负控制作用。生长实验结果表明：3个内源质粒亦对生长速率有负控制作用。将pMhHN3015c：：Tn5-mob-sacB从供体菌HN3015T18导入受体菌HN308SR后，受体菌的第一大质粒pMhHN308c被消除，表明pMhHN3015c与pMhHN308c不相容，应归于同一不相容群。在对转移接合子HN308SRN18进行质粒消除时，发现其内源小质粒pMhHN308a与pMhHN3015c：：Tn5-mob-sacB同时消除，获得了仅含pMhHN308b的质粒消除突变株HN308SRN18D。盆栽结瘤试验结果表明：转移接合子HN308SRN18只能在紫云英上形成无效根瘤，证明其内源大质粒pMhHN308c与固氮有关，且不能为导入大质粒pMhHN3015c取代。质粒消除突变株HN308SRN18D依然保持结瘤能力的结果表明pMhHN308b与结瘤有关。将HN3015的另一标记小质粒pMhHN3015a：：Tn5-mob-sacB导入HN308SR时，发现受体菌的第二大质粒被消除，显然pMhHN3015a与pMhHN308b不相容，应属于同一不相容群，并与HN308SR的pMhHN308a和pMhHN308c属于不同的不相容群。但转移接合子HN308SRN29的质粒消除实验未获得质粒消除突变株。pMhHN3015a和pMhHN308a的大小相近，仅凭凝胶电泳位置难于区分。盆栽结瘤试验结果表明：pMhHN308a与HN308的结瘤有关，而含有质粒pMhHN3015a、pMhHN308a和pMhHN308c的转移接合子HN308SRN29却失去了结瘤能力。本结果表明：pMhHN308a可能因与pMhHN3015a发生了重组而丢失了结瘤基因，其Tn5亦可能因转座至染色体而不能获得质粒消除突变株。用三亲本杂交法将供体菌HN3015的2个标记质粒pMhHN3015a和pMhHN3015c分别导入受体菌7653R-1SR时，发现这2个质粒均可分别与7653R-1SR中的pMh7653Ra共存，它们应属于不同的不相容群。但转移接合子7653R-1SRN29的质粒消除突变株7653R-1SRN29D-B在消除外源质粒的同时却产生了一个新质粒p76H4。但在另一质粒消除突变株7653R-1SRN29D-A中却未发现这个新质粒。盆栽结瘤试验结果表明：7653R-1SRN18只能形成少数无效根瘤，其瘤数与7653R-1SR相近。前面已证实pMhHN3015c与固氮能力有关，但它的导入却未能恢复受体菌7653R-1SR的固氮能力。7653R-1SRN18的质粒消除突变株依然与7653R-1SR-样，只能形成少量无效根瘤。7653R-1SRN29在紫云英根上亦形成无效根瘤，但瘤数接近7653R。前面已证实pMhHN3015a能抑制HN3015的共生效应，但本研究将它导入7653R-1SR时却反而提高了转移接合子的结瘤能力。7653R-1SRN29的质粒消除突变株7653R-1SRN29D-A也只能形成少量无效根瘤，但另一质粒消除突变株7653R-1SRN29D-B却完全丧失了结瘤能力。将7653R的共生质粒pMh7653Rb导入受体菌HN308SR时，发现受体菌的两个稳定内源质粒pMhHN308b和pMhHN308c随着外源质粒pMh7653Rb的导入而同时被消除。盆栽结瘤试验结果表明：pMhHN308b与结瘤相关，由于pMh7653Rb的导入能维持HN308SRN14的结瘤能力，其瘤数也超过HN308SR，但不能替代pMhHN308b和pMhHN308c。质粒消除突变株HN308SRN14D只形成少量无效根瘤，确认pMhHN308a与HN308的结瘤能力有关。用三亲杂交法将7653R的共生质粒pMh7653Rb导入HN3015SR时发现所获转移接合子HN3015SRN14的第二大质粒为pMhHN7653Rb，受体菌HN3015SR的第二大质粒则由于外源质粒的不相容性而被消除。HN3015SRN14的质粒消除实验获得了消除第二大质粒的HN3015质粒消除突变株(仅含pMhHN3015a和pMhHN3015c)。盆栽结瘤试验结果表明：转移接合子HN3015SRN14只能形成无效根瘤，其标记质粒消除突变株HN3015SRN14D则完全失去结瘤能力。采用通用引物RC1和RC3对6个供试转移接合子及其质粒消除突变株进行repC基因的PCR扩增结果表明：除质粒消除突变株7653R-1SRN18D和7653R-1SRN29D未能扩出repC基因外，其它供试菌株均扩出了750bp左右的repC序列。对所有供试10个菌株的repC序列测定与同源性比较结果表明：供试华癸中生根瘤菌株的repC序列高度相似(99％)，但与其它根瘤菌的repC基因有明显差异。更多还原

【Abstract】 Sixty two strains of M huakuii were isolated from nodules of Astragalus sinicus collected from rice-growing field of Huazhong Agriculture University. 57 typical strains of M huakuii were identified by cultural characteristics, microscopic observation and plant nodulation tests. The nodule appearence times of strains tested were different from 8d to 30d and for most strains varied from 10d to 22d. 44 strains were chosen to detect indigenous plasmid profile and the results showed that 13 strains harbored 3 plasmids, 21 strains harbored 2 plasmids, 7 strains only harbored 1 plasmids, 3 strains harbored no plasmid. The size of indigenous plasmids tested was from 150kb to 428kb.M huakuii HN3015 containing three mega-plasmids were labeled by Tn5-mob-sacB insertion and plasmid labeled mutants were obtained. Plasmid curing experiment was carried out by using the sacB positive selection method and plasmid cured or deleted derivatives were obtained. Same cultural characteristics were identified among HN3015 and its derivatives. The mutant HN3015-1 cured with its largest plasmid pMhHN3015c formed only white small nodules and lost nitrogen fixation ability in plant nodulation tests. The mutant HN3015-2 cured with its second largest plasmid pMhHN3015b lost nodulation ability. Only the mutant HN3015-3 or HN3015-6 cured or deleted with its smallest plasmid pMhHN3015a, could form pink effective nodules. Furthermore, curing of pMhHN3015a could enhance competitive nodulation ability and symbiotic efficiency of HN3015-3. The results from acidity and alkali tolerance assays indicated that three indigenous plasmids of HN3015 played positive control effect on its acidity tolerance, but played negative control effect on alkali tolerance. Surprisingly, all indigenous plasmids showed also negative control effect on its growth rate of HN3015.When plasmid pMhHN3015c labeled by Tn5-mob-sacB was transferred from HN3015T18 into HN308SR by tri-parental mating, the first largest plasmid of HN308SR was cured. The results implied that plasmids of pMhHN3015c and pMhHN308c were incompatible and might be ascribed to the same incompatible group. However, a new co-elimination phenomenon of pMhHN3015c:: Tn5-mob-sacB and pMhHN308a was detected when the transconjugant HN308N18 (harboring pMhHN3015c::Tn5-mob-sacB, pMhHN308b and pMhHN308a) was used to do plasmid curing tests. The plasmid cured derivative of HN308 harboring only pMhHN308b was obtained. Results of pot plant nodulation tests showed that both of HN308SRN18 and HN308SRN18D could form only white small nodules. Our results indicated that the introduction of pMhHN3015c could not replace the effect of pMhHN308c and failed to restore the nitrogen fixation ability of HN308SRN18. The results also indicated that pMhHN308c was relevant to nitrogen fixation ability and pMhHN308b relevant to nodulation ability.The second largest plasmid pMhHN308b of HN308SR was eliminated when pMhHN3015a::Tn5-mob-sacB was introduced into HN308SR. Results demonstrated that both of pMhHN3015a and pMhHN308b should be belonged to the same incompatible group and pMhHN3015a, pMhHN308a and pMhHN308c ascribed to the different incompatible groups. But plasmid cured derivatives of transconjugant HN308SRN29 failed to be obtained on TY plate containing 7% sucrose. Since the sizes of pMhHN3015a and pMhHN308a were almost the same and their positions on agarose gel were difficult to distinguished, so that two plasmids might be recombined. Results of pot plant nodulation tests showed that pMhHN308a was relevant to nodulation ability and transconjugant HN308SRN29 harboring pMhHN3015a, pMhHN308a and pMhHN308c lost nodulation ability. These results also implied that pMhHN3015a and pMhHN308a might be recombined and transposon Tn5 transferred into the chromosome which resulted in the lose of nodulation ability.When the two labeled plasmid pMhHN3015a::Tn5-mob-sacB and pMhHN3015c:: Tn5-mob-sacB were respectively transferred into 7653R-1SR harboring pMh7653Ra by tri-parental mating, the result showed that the two plasmids could respectively coexist with pMh7653Ra. Certainly, they were belonged to different incompatible groups. Results from plasmid curing test of 7653R-1SRN29 showed that one plasmid cured derivative 7653R-1SRN29D-B lost its plasmid pMhHN3015a and a new plasmid p76H4 appeared. But p76H4 could not be observed in another plasmid cured derivative 7653R-1SRN29D-A. Results from plant pot nodulation tests showed that the introduction of pMhHN3015c::Tn5-mob-sacB in transconjugant 7653R-1SRN18 failed to restore nitrogen fixation ability. Similar to 7653R-1SR, it could only form a few small white. Although Transconjugant 7653R-1SRN29 harboring pMhHN3015a::Tn5-mob-sacB and pMh7653Ra could still form null nodules, but its nodule number was more than that of wild strain 7653RSR. It was demonstrated that pMhHN3015a could inhibit symbiotic efficiency of HN3015, but pMhHN3015a could enhance the nodulation ability of 7653R-1SRN29. Plasmid cured mutant 7653R-1SRN29D-A could only form null nodules. But mutant 7653R-1SRN29D-B with a novel plasmid p76H4 and cured of pMhHN3015a::Tn5-mob-sacB lost its nodulation ability.When symbiotic plasmid pMh7653Rb labeled by Tn5-mob-sacB was transferred from 7653RT14 into HN308SR by tri-parental mating method, two stable indigenous plasmids of pMhHN308c and pMhHN308b of HN308SR was co-eliminated. Results of plant pot 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 failed to replace nitrogen fixation effect of pMhHN308b and pMhHN308c. Plasmid cured mutant HN308SRN14D harboring only pMhHN308a could also form nodules that demonstrated pMhHN308a was relevant to nodulation ability.When symbiotic plasmid pMh7653Rb::Tn5-mob-sacB was also transferred into HN3015SR by tri-parental mating method, its second largest pMhHN3015b was eliminated. The mutant HN3015SRN14D harboring pMhHN3015a and pMhHN3015c was obtained by plasmid curing tests from transconjugant HN3015SRN14. Results of plant pot nodulation tests showed that HN3015SRN14 formed only null nodule and HN3015SRN14D lost the nodulation ability.Primers of RC1 and RC3 were used to amplify the repC-like sequences from the above six trans-conjugants and their plasmid cured mutants, repC-like sequences were obtained from strains tested except of 7653R-1SRN18D and 7653R-1SRN29D. The sizes of the PCR products were about 750 bp. The repC sequences of ten strains tested showed 99% sequence similarity, but were obviously different from that of other rhizobia strains.更多还原