【作者】 李友国；

【导师】 周俊初；

【作者基本信息】 华中农业大学 ， 微生物学， 2000， 博士

【摘要】 本研究将来自苜蓿中华根瘤菌(Sinorhizobium meliloti)的四碳二羧酸转移酶基因(dctABD)经pIJ2925克隆到广宿主、稳定性质粒载体pTR102上，获得诱导型表达的重组质粒pHN202。在此基础上再引入来自pDB30所含的发光酶基因(luxAB)作分子标记，以pTR102为基础构建成带有dctABD和luxAB的重组质粒pHN205。再以pLAFR3为载体构建重组质粒pHN207，其上携带有dctABD基因、来自pTR102的parCBA／DE基因和发光酶标记基因luxAB。将来自肺炎克氏杆菌(Klebsiella pneumoniae)的共生固氮正调节基因nifA克隆到载体pTR102上构建成带有标记基因luxAB的重组质粒pHN306。进一步将dctABD基因和nifA基因同时克隆于一个稳定性质粒载体pTR102上，构建成带有发光酶基因标记的重组质粒pHN307。 再将所构建的重组质粒pHN205、pHN207、pHN306和pHN307经二亲本或三亲本接合转移分别导入费氏中华根瘤菌(Sinorhizobium fredii)HN01、GR3和YC4及大豆慢生根瘤菌(Bradyrhizobium japonicum)TA11和CB1809，获得一系列转基因重组大豆根瘤菌。在盆栽条件下比较研究了转基因重组根瘤菌与出发菌和参照菌的结瘤与共生固氮效率。盆栽试验结果表明：(1)重组根瘤菌HN01(pHN205)和GR3(pHN205)分别在大豆宁镇一号和川早一号上能显著提高植株地上部分干重、生物量和总氮量，YC4(pHN205)在大豆黑龙33上能同时显著提高植株地上部分干重、生物量、总氮量和根瘤鲜重。(2)重组质粒pHN207的导入可显著提高TA11和CB1809在大豆黑龙33、宁镇一号和渝豆一号上的共生固氮能力，使结瘤植物的地上部分干重、生物量和总氮量等指标较对照组有显著提高。(3)HN01(pHN306)和GR3(pHN306)分别在大豆渝豆一号和黑龙33上能显著提高瘤数、瘤重、植株地上部分干重、生物量和总氮量。YC4(pHN306)在大豆渝豆一号上能显著提高瘤数、瘤重和总氮量，对植株地上部分干重生物量也表现出一定的促进作用。TA11(pHN306)与大豆黑龙33共生时也能显著提高其结瘤植株地上部分干重和总氮量。(4)重组质粒pHN307导入受体大豆根瘤菌后，对于重组根瘤菌的结瘤和固氮效应的促进作用表现不一。(5)导入dctABD基因的重组根瘤菌在小区田间试验条件下，与宁镇一号或黑龙33共生时其小区产量显著高于出发菌和施氮肥对照组。 盆栽研究和小区试验结果表明：dctABD的导入可显著提高受体菌的固氮效率，nifA的导入主要影响重组根瘤菌的结瘤能力，导入额外拷贝dctABD、nifA或同时导入dctABD和nifA对结瘤和共生固氮效率的促进作用与受体根瘤菌和大豆品种等因素有关。 以luxAB为报告基因进行了转移接合子中外源重组质粒稳定性的探究，结果表明：pHN205、pHN207、pHN306和pHN307可在供试大豆根瘤菌中稳定遗传。同时还发现parCBA／DE基因的引入明显提高pLAFR3在TA11和CB1809中的稳定性。 两株代表性重组大豆根瘤菌HN01DL和TA113QD在灭菌土和非灭菌土缩影中的存活动态表现出相似的变化趋势。发现在灭菌土和非灭菌土缩影中供试根瘤菌的存活动态之间有显著差异，说明了在未灭菌土中存在来自生物因素的干扰并影响了引入根瘤菌的生长、繁殖和存活。华中农业大学20加年博士研究生毕业论文 供试菌株在根盒一土壤缩影中的定殖动态和结瘤研究结果表明:HN0lDL所形成的发光根瘤主要集中在种子下方A区段(O一4cm)范围内，并与在此根段部位定殖的根瘤菌群体密度一直很高密切相关，初步说明根瘤菌的根圈定殖动态和定殖水平对其结瘤和竞争能力具有重要影响。并且发现在此区段的主根瘤发光比例高达9叽以上，显著大于此区根段的侧根瘤发光比例，从而说明种子表面的接种根瘤菌主要是沿着主根的伸长而向下扩散定殖。 两个供试重组根瘤菌恻OIDL和TAll3QD在非灭菌土壤盆栽缩影中的根圈定殖动态显然不同于在灭菌土盆栽缩影中，表现为“先降后升”趋势，但二者之间在大豆根圈的定殖动态基本相同。初步观察到根瘤菌的生长速度与其在根圈的定殖动态和水平相关。 无论在灭菌土或在非灭菌土盆栽缩影中，两个供试重组根瘤菌HN01DL和TAI 13QD在四种供试大豆根圈的定殖动态存在一定的差异，且在宿主植物上的结瘤和共生效应也有一定程度的反映，尽管这种差异并未达到显著性水平，但本实验结果支持根瘤菌在植物根圈的群体定殖水平和动态特征与其结瘤数量和竞争能力直接正相关。 在灭菌土盆栽缩影中，三种不同的初始接种量对HNO IDL的结瘤数量没有产生显著影响。在非灭菌土盆栽缩影中，三种不同的初始接种量对刚OIDL在宿主植物根上的结瘤数量和占瘤率也没有产生显著影响。宿主植物似乎表现出对其根圈中根瘤菌定殖密度有较大的自主调节作用。 研究发现导入额外拷贝的n州能够显著提高受体根瘤菌HN01在根圈定殖竞争的适应性和占瘤率，HNO INL在根圈中的群体定殖水平高于出发菌和参比菌。并且观察到根圈定殖的动态与水平和其占瘤率呈正向关系，结果表明种子表面接种的重组根瘤菌能否成功地在根部定殖及其动态是竞争结瘤的重要基础。根瘤菌的根圈定殖密度与占瘤率表现出一种正向关系。 实验结果显示:消除了共生质粒的费氏中华根瘤菌更多还原

【Abstract】 5.75kb DNA fragment containing C4-dicarboxylic acid transport (dctABD) genes from Sinorhizobium meliloti was subcloned into the stable-broad-host plasmid pTR102 through pIJ2925, A inducible expression recombinant plasmid pHN202 was obtained. The reporter gene luxAB from plasmid pDB30 was also inserted into pHN202 and the recombinant plasmid pHN205 was constructed. And also a recombinant plasmid pHN207 containing dctABD, parCBA/DE genes from pTR102 and reporter genes luxAB was constructed by using pLAFR3 as the vector. A recombinant plasmid pHN306 containing nifA of Klebsiella pneumonias and luxAB was also constructed by using pTR102 as the vector. And finally a recombinant plasmid pHN307 containing dctABD, nifA and luxAB genes was constructed.The recombinant plasmid pHN205, pHN207, pHN306 and pHN307 were then introduced into Sinorhizobium fredH-HNOl, YC4 and GR3 and Bradyrhizobium japonicum TA11 and CB1809 by tri-or bi-parental mating. A series of recombinant rhizobium strains were screened. Plant pot experiments were carried out for the recipient strains and their transconjugants to compare their nodulation ability and symbiotic nitrogen fixation efficiency. The results showed: (1) Recombinant strains HN01(pHN205) and GR3(pHN205) could significantly increase the shoot dry weight(biomass) and total nitrogen content of the plant with soybean varieties of Ningzhen No.1 and Chuanzao No.1 respectively. Transconjugant YC4 (pHN205) could also significantly increase the shoot dry weight (biomass), total nitrogen content of the plant and nodule fresh weight with soybean variety Heinong 33. (2)The introduction of dctABD genes could significantly improve the symbiotic nitrogen fixation efficiency of TA11 and CB1809 with soybean varieties of Heinong 33, Ningzhen No.l and Yudou No.l. Compared with the control, the shoot dry weight (biomass) and total nitrogen content of the plants tested were significantly increased. (3)Recombinant strains HN01(pHN306) and GR3(pHN306) could significantly increase the nodule number, nodule fresh weight, shoot dry weight (biomass) and total nitrogen content of the plant with soybean varieties of Yudou NO. 1 and Heinong 33 respectively. Transconjugant YC4(pHN306) could also significantly increase the nodule number, nodule fresh weight and total nitrogen content of the plant with soybean variety Yudou No.l. (4) The introduction of pHN307 into the soybean rhizobia did not result in the expected further significant improvement effects on the symbiotic nitrogen fixation ability for all the recipient strains. (5) The soybean yield inoculated with recombinant strains of S. fredii with extra copy of dctABD gene was significantly greater than that of inoculated with recipient strains or nitrate fertilizer.The results of plant pot experiment and field experiment indicated that: the extra copy introduced dctABD gene or nifA gene could significantly increase the nitrogen fixation efficiency and the nodulation ability of the recipient strains respectively, and the effects were relevant to many factors such as soybean varieties, soil fertility and recipient rhizobia.By means of the luminescence detection, it was confirmed that all the recombinant plasmids constructed by pTR102 in this work were stable under free-living and symbiotic conditions, which proved that parCBA/DE genes could increase the stability of pLAFR3 in the transconjugants.The survival dynamics of two representative recombinant rhizobia strains HN01DL and TA113QD was similar to each other in the sterilized and non-sterilized pot-soil microcosms. It was proved that the survival dynamics of rhizobia tested in the sterilized pot-soil microcosms wassignificantly different from that of the non-sterilized pot-soil microcosms.The study on the relationship between rhizospheric colonization dynamics and nodulation in the non-sterilized rhizobox-soil microcosms suggested that: the luminescencent nodules formed by HN01DL were mainly located in the place of 0-4cm root segment from the coated seed, and it corresponded to the relativ更多还原