【作者】 缪礼鸿；

【导师】 周俊初；

【作者基本信息】 华中农业大学 ， 微生物学， 2001， 博士

【摘要】 利用带有枯草芽胞杆菌蔗糖敏感基因sacB的Tn5-Mob转座子标记了费氏中华根瘤菌(Sinorhizobium fredii)HN01、YC4B和WWG18不同的内源质粒；在含有10％蔗糖的TY培养基上，除YC4的第二大质粒外，均获得了消除标记的质粒的突变株。深入的比较研究结果证明： 1．在辅助质粒pRK2073的协助下，实现了费氏中华根瘤菌HN01、YC4B和WWG18中的6个标记内源质粒在种内不同菌株之间和费氏中华根瘤菌与农杆菌之间诱动转移。通过植物盆栽结瘤试验，从功能上证明HN01的第二大质粒pSymHN01b为共生质粒。将标记共生质粒pSymHN01转入大豆品种专一性结瘤菌株AB359后，可使后者成为能在黑农33等大豆品种上结瘤的广品种范围结瘤的菌株，从而证明费氏中华根瘤菌品种专一性结廇性状受共生质粒控制。 2．将HN01共生质粒pSymHN01b分别转入WWG18SR和C361SR后的质粒快速检测、质粒消除和植物盆栽结瘤试验结果证明：HN01的共生质粒与WWG18SR的共生质粒具有不相容性，但能与其非共生质粒相容。相反，HN01的共生质粒可与C361SR的共生质粒相容，但与其中一个非共生质粒具有不相容性。利用费氏中华根瘤菌不同菌株质粒间的不相容性，本研究成功地消除了WWG18SR的共生质粒和C361SR的一个非共生质粒。 3．将YC4B的标记共生质粒pSymYC4b导入HN01的研究结果证明YC4B的共生质粒pSymYC4b与HN01的非共生质粒pHN01c具有不相容性，并同时与其共生质粒pSymHN01b出具有不相容性。将YC4B的pSymYC4b导入HN01后，通过质粒间的不相容性在含蔗糖的平板上获得了同时消除HN01菌株中的两个稳定的内源大质粒(pSymHN01b和pHN01c)的突变株HND100。 4．从在大豆上正常结瘤和固氮的野生型菌株YC4中分离到两个结瘤性状改变的自发突变株YSC3和YC4B，其中YSC3在所有供试的大豆品种上只形成无效的根瘤，而YC4B则完全失去了在大豆上结瘤的能力。进一步研究证明，突变株YSC3的共生质粒发生了扩增，并引起结瘤因子(LCOs)组分的改变。突变株YC4B的质粒带未发生任何可见的变化。用TLC技术检测的LCOs结果还表明：YC4及其突变株YSC3产生的LCOs结构明显不同于其它4个野生型S.fredii菌株。培养温度对YC4及其突变株产生的LCOs量的影响不同。野生型菌株YC4在较低培养温度(23℃)和正常培养温度(28℃)下产生的LCOs的量变化不大，但突变株YSC3产生的LCOs的量在28℃下却大大增加。 5．发现HN01的共生质粒pSymHN01b在转入WWG18SR的过程中发生缺失，并导致转移接合子WWG18SRN1在大豆上结瘤的品种范围缩小的现象。通过盆栽结瘤试验证明：pSymHN01b的缺失导致转移接合子WWG18SRN1由广品种结瘤菌株转变为品种专一性结瘤菌株，并失去了在所有供试大豆品种上固氮的能力。 6．通过两亲本接合转侈，将豌豆根瘤菌(Rhizobium leguminosarum bv.viciae)1229的共生质粒pSym1229分别转入费氏中华根瘤菌HN01SR及其消除了共生质费氏中华根瘤菌内源质粒问的tI]互作用及J〔对共生固氮的影响 粒的突变株!INI)l()()‘1，。实验结果表I梦」:转移接合子1 IND x 00(psyml229)厂白二’!二的l矛C()s 组分与豌豆根瘤菌1229相同。IIND100(psyml229)能够在豌豆，{石种Nigra上形成 白色球状的无效根瘤;但豌豆根瘤菌1229则形成正常的长圆柱状的有效根瘤。 将psyml229转入}INolSR后，部分接合子表现出与}以D100(psyml229)珊·}11司的结 瘤性状，而另一部分接合子的结瘤性状与11丫olSR相同，显示了IIN0lSR与1229 r内异源共乙卜质粒之I司结瘤功能fl勺寺fl互扣J制作川。7.本研究采用发光酶丛因(ju川功标记技术，在无菌的砂培条件下测定了费氏【!，华 根瘤菌内源质粒和大豆品种对菌株竞争结瘤能力的影响。结果表明:供试菌株的 竞争结瘤能力主要取决于染色体携带的从因，内源质粒(包括共生质粒和非共生 质粒)对竞争结瘤能力没有明显的影响。结瘤试验还证明，消除】IN01和wwG18的 非共生质粒对它们的I司氮效率没有显著影响。将psymllNol卜转入AI3359后，转移 接合子在黑农33等大豆品种上表现出卜与!!功!相同的固氮效率。更多还原

【Abstract】 The indigenous plasmids of Sinorhizobium fredii HN01, YC4 and WWG18 were labelled with Tn5-Mob-sacB .Labelled plasmids were cured on TY medium containing 10% sucrose except the second larger plasmid of YC4 was identified the function of plasmids by pot plant experiment..The Labelled plasmids were transferred into Agrobacerium tumefaciens GMI9023 and S.fredii AB359 by tri-parental mating with the helper plasmid pRK2073 to identify the nodulation ability on soybean.The results indicated that the broad host characteristics of HN01 was determined by the symbiotic plasmid, pSymHNOl, since recepient strain AB359 recovered the ability to nodulate on Heinong33 by introduction of pSym HN01.The pSymHNOlb of HN01 was transferred into WWG18SR and C361SR. . The results of plasmid detection, curing and pot plant experiment revealed that the incompatibility was existed between pSymHNOlb and symbiotic plasmid of WWG18SR or the third plasmid of C361SR. But pSymHNOlb was compatible with the non-symbiotic plasmid of WWG18SR and symbiotic plasmid of C361SR. Using plasmid incompatibility, The symbiotic plasmid of WWG18SR and one nonsymbiotic plasmid of C361SR were eliminated successfully.A 270MD second larger plasmid named pSymYC4b of Sinorhizobium fredii YC4b, a spontaneous Nod~-_ mutant on soybean cultivar Heinong33, was mobilized into another S. fredii HN01SR which contained three indigenous large plasmids (pHN01a,pSymHN01b, and pHN01c).A new pattern of plasmid incompatibility was observed that two stable indigenous plasmids (pSymHNOlb and pHNOlc) of HN01SR were cured simultaneously by the introduction of pSymYC4b. Furthermore, an unstable plasmid named pHY4 whose molecule weight was larger than both pSymYC4b and pSymHN01b was detected together with pSymYC4b in one transconjuant.Two spontaneous nodulation mutants named YC4B and YSC3 were isolated from YC4.Mutant YSC3 formed ineffective nodules on all tested soybean cultivars and G.soja in the conditions of sand pot experiments and fewer bacteroid inside of nodules in comparison with the nodules formed by wild strain YC4. The results showed that the size of the second larger plasmid of YSC3 was larger than the pSymYC4b which indicated that pSymYC4b was amplified in YSC3. Mutant YC4B lost completely nodulation ability on all tested soybean cultivars and G.soja although its plasmid map was the same with YC4.YC4 producted the unique lipochitooligosaccharde Nod factors (LCOs) containing more hydrophobic substitution in comparison with LCOs from other four strains detected by Thin-layer chromatography (TLC). Mutant YSC3 producted more and different LCOs than that of its parental strain YC4. It was proved that incubation temperature has an important influence on the LCOs production of YSC3, LCOs production was greatly increased at 28℃ in comparison with at 23℃, but the LCOs production of YC4 and HN01 have no great change in two temperatures.WWG18SRN1 containing the deleted pSymHNOlb was obtained when pSymHN0lb was mobilized from HN01 into WWG18SR. It showed narrow host range on soybeancultivars comparing with WWG18SR, HN01 and another transconjugant WWG18SRN2 containing the complete pSymHN0lb. WWG18SRN1 formed only ineffective nodules on all G.soja and soybean cultivars tested.Reference strains were marked by luxAB genes and used for competitive nodulation tests in pot plant experiments. The results indicated that the competitive ability of strains tested was mainly determined by chormosomc genes. Both of symbiotic and non-symbiotic plasmids and soybean cultivars shown no significant influence on strain competitiveness.The effectiveness of mutants HN01 and WWG18 non-symbiotic plasmid cured showed no difference with their wild strains.The symbiotic plasmid of Rhizobium.leguminosarum bv.viciae 1229 was transferred into HN01SR and its pSym cured derivatives HND100. The transconjugant HND100(pSym1229) expressed the same LCOs as that of R.leguminosarum 1229 in free-living condition analysed by TLC method. The results of pot plant tests indicated that HND100(pSym1229更多还原