【作者】 谢波；

【导师】 周俊初；

【作者基本信息】 华中农业大学 ， 微生物学， 2005， 博士

【摘要】 根瘤菌的嘌呤合成途径在共生固氮过程中有重要作用，已知多数嘌呤缺陷型突变株都不能与宿主植物共生固氮。有报道认为嘌呤代谢途径中purL基因的转座子插入突变能导至腺嘌呤缺陷型和脂多糖LPS组分改变，该突变株与大豆只能形成微小无固氮活性的假瘤，虽在培养液中添加5-氨基咪唑-4-甲酰胺核苷酸或腺嘌呤和VB₁可以提高突变株的结瘤能力，但仍不能恢复固氮功能。为进一步探讨purL基因对根瘤菌共生固氮能力的影响，并通过改造purL基因表达方式实现根瘤菌应用中的生态防范，本研究对purL基因进行了不同时间和水平的表达改造，以探讨菌株共生能力、竞争结瘤与purL基因表达的关系，并获得了既能有效共生固氮、又可较快消亡的共生诱导表达重组菌。此外，本研究还考察了PurL突变对其它生理过程的影响。 本研究通过对费氏中华根瘤菌嘌呤合成酶purL的基因置换构建出腺嘌呤缺陷型突变株P825和P618，P825 purL的NotⅠ-ⅩhoⅠ片段被luxAB基因取代，P618的purL结构基因发生了1.98kb片段的缺失。两突变株只能在大豆上形成不固氮的假瘤，purL表达载体pBBR-PG在P825和P618中可恢复其在基本培养基上的生长情况和共生能力，同时对费氏中华根瘤菌SMH12、HN01、WH3和S11进行purL的基因置换，获得突变株SMH12-P1、HN01-P1、WH3-P1、S11-P1、SMH12-P2、HN01-P2、WH3-P2和S11-P2。 用fixK和fixR启动子启动purL表达的载体pHN801K和pHN801R，使P825仍需要添加腺嘌呤和VB₁才能生长，在1％琼脂的基本培养基穿刺培养中，P825（pHN801R）和P825（pHN801K）有弥散性的生长，表明两启动子能够在微氧条件下启动purL的表达。而两种purL诱导表达重组菌只形成假瘤，均没有恢复共生固氮的能力。以purL-gusA转录融合单元构建出purL基因渗漏表达载体pHN811（1／3野生型、组成型表达水平）、含nifH、nifQ、fixN启动子的共生诱导表达载体pTPG-PnifH、pTPG-PnifQ、pTPG-PfixN和超量表达载体pBBR-PG（10倍野生型水平）。结果表明pHN811和适宜的共生诱导表达的pTPG-PnifH与pTPG-PnifQ都可使重组菌与大豆在无外源嘌呤添加物时建立有效的共生固氮体系。添加腺嘌呤和VB₁，依然不能使突变株P618和purL不表达重组菌菌株P618（pTPG-PfixN）恢复共生固氮的功能，仅形成少数有侵染的无效根瘤。研究结果证明：只有表达了一定水平的purL基因才能进行有效侵染与共生固氮，而purL基因的表达在结瘤后期也是必需的。渗漏表达purL基因重组菌的现瘤时间晚于野生型2-3天，对植株鲜重和干重均、根瘤结构和含菌细胞中类菌体数量、固氮酶活的研究结果表明适宜的purL表达量对根瘤的正常发育和固氮效率发挥是必要的。 在植物砂培竞争结瘤实验中，purL基因渗漏表达、共生诱导表达和超量表达重组菌的竞争能力均显著下降，砂土盆载条件下重组菌的竞争能力均有所提高，添加更多还原

【Abstract】 The purine synthesis pathway plays an important role in the Rhizobium-plant interaction since most purine auxotrophs of rhizobia species are unable to form effective nodules on their host plants. Previous reports showed that a transposon induced PurL mutant of Sinorhizobium fredii induced pseudonodules on Glycine max, addition of 5-aminoimidazoie-4-carboxamide (AICA)-riboside or adenine and VBl to the plant could enabled the mutant to form infected nodules but no nitrogen fixation. To gain a better understanding of the impact of purL gene on the symbiosis formation, we studied the relationship between the purL gene and effective symbiosis on soybean by different transcriptional modification of S.fredii purL gene. The engineered genetic circuit of symbiotic-expressing purL was also applied in the biological containment study of rhizobia inoculum. In addition, other interested pleiotropic effects were studied with these PurL" mutants..Two different PurL" mutant, P825 and P618, were constructed from the wild type S. fredii strain HH103 by gene replacement. The strain P825 was the replacement mutant whose Xhol-Notl fragment in purL gene was replaced by luxAB, and the strain P618 was a 1.98kb deletion mutant in purL open reading frame. Both mutant required the supplement of adenine and VB1 to grow in minimal medium, and induced defective pseudonodules on soybean. Expressing vector pBBR-PG could restore the mutants to grow in minimal medium and the symbiotic abilities, which confirmed that they were the result of mutated purL gene. By the same method, four other PurL" mutants were constructed with S. fredii strain SMH12, HN01, WH3 and S11, to create the mutants SMH12-P1, HN01-P1, WH3-P1, S11-P1, SMH12-P2, HN01-P2, WH3-P2 and S11-P2, respectively. All these PurL" mutants were defective in symbiotic nitrogen fixation.Symbiotic expression vectors of purL gene using the promoter of fixK and fixR., pHN801K and pHN801R, could not restore the growth of P825 in minimal medium. In microaerobic stab inoculation in minimal medium, pHN801K and pHN801R could improve P825 a diffused growth like that of HH103, suggesting that purL could be expressed under the microaerobic conditions. However, neither two plasmids could restore the mutant to form infected nodules.New modification of purL expression containing a reduced expression plasmid(pHN811, 1/3 of the wild type level, constitutive) , symbiotic expression plasmids withpromoter of nifH, nifQ and fixN (pTPG-PnifH, pTPG-PnifQ and pTPG-PfixN), and aover-expression plasmid (pBBR-PG, 10 times of the wild type level, constitutive) wereconstructed. Plant results showed that the reduced expression of pHN811 and symbiotic更多还原