【作者】 季天委；

【导师】 方萍； 石伟勇；

【作者基本信息】 浙江大学 ， 植物营养学， 2003， 硕士

【摘要】 化肥的施用对农业生产的突破性发展功不可没，但长期大量、单一地施用化肥特别是化学氮肥所带来的肥料利用率降低、农产品品质下降和环境污染问题正日趋突出，已对农业的可持续发展产生严重的负面影响。与施用化学氮肥相比，生物固氮毋需消耗非再生能源，固氮产物可直接为植物吸收利用，故不易通过挥发、反硝化、淋溶损失而引起农业环境的污染，因此充分发掘和利用农业生态系统中的生物固氮潜能对农业的可持续发展意义尤为重大。大量国内和国外的水稻接种试验结果证明，用联合固氮菌接种水稻对提高植株干重、含氮量及产量效果显著，但接种效果在不同水稻基因型间的表现有较大差异。可见，要建立水稻根际高效联合固氮体系，不但需要分离、筛选并接种高效联合固氮菌株，而且，还要求具备能有效促进根际联合固氮作用的水稻品种。为此，有必要探明控制水稻根际联合固氮能力的遗传背景。 本文利用华中农业大学作物遗传改良国家重点实验室以珍汕97×明恢63杂交组合，经单粒传方法构建的重组自交系RIL(F₁₁)群体共241个株系，和本实验室拥有的分离自埃及棉花根际的高效固氮菌株W12和中国农业科学院提供的联合固氮菌株（菌种中心菌号为ACCC10084）FY为研究材料，在严格限菌条件下，采用改进的Spermosphere Model技术，测定供试群体的不同株系与各固氮菌株的联合固氮酶活性的表型值。利用华中农业大学作物遗传改良国家重点实验室构建的RIL群体的分子标记遗传连锁图谱，采用MAPMAKER/QTL软件对联合固氮能力及有关的表型性状进行QTL定位分析，得结果如下： 一、水稻根际联合固氮体系中固氮酶活性测定方法—Spermosphere Model技术改进 在固氮菌株与RIL群体共241个株系的联合固氮酶活性的测定中，若每个菌株与各株系重复测定10次，则整个群体与需测定2410次，对于如此庞大的测定规模，难以采用常规的乙炔还原法程序即在特定的反应时点测定固氮酶活性。因此，需要对现有的Spermosphere Model技术进行改进，研究确定适合大规模水稻根际联合固氮酶活性测定的条件，以保证测定数据的可比性。 1．水稻根际联合固氮体系中固氮酶活性的时间变化曲线测定表明，在Spermosphere Model系统中，接种W12固氮菌液和FY固氮菌液24h后，注入乙炔气再培养60小时，体系内的乙烯积累量趋于相对稳定，此后12小时中体系内的乙烯总量增加不显著，因此，在大批量的联合固氮酶活性测定中，以注入乙炔气再培养60小时作为开始测定时间，并保证同一批样品在此后10小时内完成测定。 2．为进一步提高 RIL群体各株系与 WIZ和 FY联合固氮体系中固氮酶活性测定之间的可比性，保证各联合固氮体系具有相同固氮时间，从3种化学灭菌剂各取2个浓度对2个供试固氮菌株的抑菌效果试验中，筛选出每种抑菌剂具有较强抑菌效果的浓度分别为：甲醛3．5％，氯化汞0二％，次氯酸钠2石％，其中以甲醛3．5％的抑菌效果最佳。 3．根据上述选定的3个抑菌剂的浓度，对山2个水稻品种与2个固氮菌株组成的4联合固氮体系进行酶促反应阻断试验。筛选出3．5％甲醛水溶液是一种理想的联合固氮作用阻断剂。 二、水稻根际联合固氮能力及相关性状的QTL定位分析结果 1．利用改进的 Spermosphere Model技术，分别测得 RIL群体及其亲本与固氮菌株 W 12和固氮菌株FY的联合固氮能力（nmol历0h）并记录各供试株系的糙米千粒重地)表型值。通过以’N标记的半固体琼脂培养基为生长介质，进行W12接种试验，测得ML群体及其亲本的叶绿素含量mPAD值L植株总干物重吨)和根干重吨)等性状表型值。t检验表明，与W12联合固氮能力、与FY联合固氮能力和糙米千粒重等3个性状在亲本间呈显著性差异；叶绿素含量、总干物重和根干重等3个性状间亲本差异未5％显著性水平。RIL群体的6个性状表型值均呈正态分布，满足QTLS作图的基本要求。 2水稻根际联合固氮能力QTL定位结果 门)检测到控制与固氮菌株 WIZ的联合固氮能力 QTL个，位于第 2染色体上的侧连标记 R712～RM29区域内，最大 LOD值为 2．sl，表现加性效应。 Q)检测到控制与固氮菌株 FY的联合固氮能力的 QTL个，位于第 2染色体上的侧连标记RM208～RM48区域内，最大LOD值为3．37，也表现加性效应。 两基因位点在第二染色体上的位置不同，说明不同控制两个固氮菌株联合固氮能力的遗传背景并不相同。由于本研究利用Spermosphere Model技术测定水稻根际联合固氮能力是苗期表型值，所定位的QTL也只能反映苗期表达的基因效应，控制不同生育期水稻根际联合固氮能力QTL有待进一步研究鉴定。 3，检出控制糙米千粒重的 QTL共 12个，分别位于第 lx 3、5、8、9和口染色体上，第 1染色体上有2个 QTLS，位于标记区附 R753～RG532和 RG532～RM259：第 3染 色体上有5个QTLS，位于标记区间C3～C63、RM232～C1087、C1087～R19、R19～ C746和 C746～C944，共解释性状变异的 69．5％；第 5染色体上有 2个 QTLS，标记区 间为 R830～R3166和 R3166～RG360，LOD值分别为 6．19和 10．28，第 8、9?更多还原

【Abstract】 Application of chemical fertilizer has promoted a great development in agricultural production. However, injudicious use of chemical fertilizer, especially chemical nitrogen fertilizer has induced a crucial negative impact to sustainable agriculture development, such as decrease of fertilizer utilization efficiency, reduction of agricultural product quality, and environmental pollution. Compared with the use of chemical nitrogen fertilizer, biological nitrogen fixation dispense with non-renewable resource. The products of N2-fixation could be directly assimilated and utilized by plants, so it is hard to be lost by volatilization, denitriflcation and eluviation from agro-ecosystem. Therefore, exhumation and utilization of biological nitrogen fixation in agro-ecosystem is particularly important to sustainable agriculture development. According to the results of field and pot experiments reported, inoculation with associative nitrogen-fixing bacteria can increase nitrogen concentrations in tissues, and biomass and grain yield of rice plants. But the response of inoculation for different rice genotypes showed great difference. The establishment of high efficient associative nitrogen-fixing system in rice rhizosphere, depends not only on inoculation with high nitrogen-fixing efficient bacteria strain, but on rice genotype that could effectively promote associative nitrogen fixation at rhizosphere as well. Hence, it is essential to find out the genetics background that controls the capacity of associative nitrogen-fixing in rice rhizosphere.A recombinant inbred lines (RIL) population and two strains of nitrogen-fixing bacteria denoted as W12 and FY were used in this study to detect the QTLs for associated nitrogen fixation capability by means of Spermosphere Model technique on condition of gnotobiotic culture. The RIL population (F11)consisting of 241 lines were developed by single seed descent procedure from a cross between an indica variety Minghui 63 (MH63) and an indica variety Zhenshan 97 (ZS97). The strain W12 with higher nitrogenase activity is isolated from cotton rhizosphere in Egypt. The strain FY, ACCC10084 is provided by Chinese Academy of Agricultural Sciences. Based on the molecular marker linkage map of RIL population generated by National Key Laboratory of Crop Genetic Improvement of Huazhong Agricultural University, QTLs for associated nitrogen fixation capability in rice rhizosphere and some related traits are detected using the program MAPMAKER/QTL. The resulted obtained are as follows:I. Modification of assay method for nitrogenase activity of associative nitrogen-fixing system in rice rhizosphere1. It is necessary to select a proper associative nitrogen-fixing period in the Spermosphere Model system after C2H2 injected for testing the nitrogenase activity of a batch of specimens. After inoculated with nitrogen fixing bacteria culture for 24h, 10% of the air in system was replaced by C2H2, then the amount of C2H4 reduced from C2H2 by nitrogenase in the system was detected by GC-14B of SHIMADZU CORPORATION at 24h, 48h, 60h, 72h, 90h and 120h after C2H2 injected. It was found that the amount of C2H4 was significantly increased from 24h to 48h, and the increment of C2H4 was not evident from 60h to 72h. So it is reasonable to finish nitrogenase activity detecting of a batch of specimens within 60 to 70h after C2H2 injected.2. For further ensuring the veracity of Acetylene reduction assay (ARA) in detecting numerous specimens, it is judicious to find a reagent to interdict the activity of nitrogenase catalyzing the acetylene reduction reaction in an associative nitrogen-fixing system at certain period. The effect of 3 bactericides with 2 concentrations each to restrain the growth of W12 and FY were test by means of the inhibition zone method. The results indicated that 3.5% formaldehyde, 2.6% sodium Hypochlorite and 0.1% mercuric chloride showed higher restricting effects and the best one is 3.5% formaldehyde.3. The bactericides selected previously were use更多还原