【作者】 李晓琳；

【导师】 孙杰； 王彦章；

【作者基本信息】 石河子大学 ， 作物遗传育种， 2010， 硕士

【摘要】 豆科植物能与土壤中的根瘤菌建立共生关系,在其根部形成固氮根瘤。共生固氮提供宿主植物生长所需要的氮,但产生过多的根瘤会影响植物的发育。因此,根瘤形成的数目受到宿主结瘤自我调节及环境因子的调控。本文对百脉根结瘤数减少的共生突变体rel3进行了表型鉴定和分子机理研究。在共生和非共生条件下,rel3的主根比野生型短,侧根数减少,结瘤数目约为野生型一半,结瘤区比率显著低于野生型。然而,突变体与野生型的固氮酶活力没有差异。这些表型分析表明rel3根瘤数目减少,可能是突变体结瘤区域变窄所致。用LacZ标记的根瘤菌接种突变体,发现rel3的侵染事件显著减少,这暗示rel3侵染频率的降低,使rel3的结瘤区域变窄,最终导致rel3的结瘤数目降低。为了进一步阐明REL3突变导致根瘤数目减少的分子机理,首先分析了rel3对结瘤负调控因子氮和乙烯的敏感性。结果表明,用硝酸盐和乙烯处理后,rel3和野生型产生相似的结瘤反应,这表明rel3中氮和乙烯对结瘤的调节不受影响。另外,通过野生型与突变体间的相互嫁接,结果显示rel3根瘤减少的表型主要受茎基因型控制。通过发根转化获得REL3启动子-GUS转化根系,分析表明REL3在根系的维管束组织特异表达,在根瘤器官起始处的维管组织中强烈表达,随着根瘤的发育,REL3继续在发育的维管束组织中表达。这表明REL3对于根瘤的起始起着重要的作用。REL3突变使植物体内TAS3 -siRNA的合成受阻,导致TAS3 -siRNA调控的靶基因即生长素应答因子ARF3和ARF4的表达上调。这些提示我们rel3结瘤减少可能与生长素有关。用生长素抑制剂NPA和TIBA处理后,rel3显示出更强的向地性丧失,表明rel3对生长素抑制剂的处理比野生型敏感,这暗示rel3根系中生长素的极性运输发生了改变。这种生长素极性运输的改变是否参与了根瘤起始的研究正在进行中。根据现有的研究结果,我们推测REL3的突变改变了根系中的生长素运输,使得根皮层细胞中的生长素浓度改变,导致根系中结瘤敏感区域根瘤起始所需要的生长素浓度范围变窄,因而产生较窄结瘤区域,从而形成较少的根瘤。本研究提供了ta-siRNA途径调控的生长素信号途径,直接参与了共生根瘤起始的证据。更多还原

【Abstract】 Rhizobia are capable of infecting the roots of leguminous plants, leading to the formation nitrogen-fixing nodules. While nitrogen-fixing nodule can provide nitrogen for host plants, too many nodules become undesirable. The number of nodules is tightly regulated by environmental factors and autoregulation of nodulation.In this study, we analyzed phenotypes and molecular mechanisms of Lotus japonicus rel3 symbiotic mutant with reduced nodules. In symbiotic and non-symbiotic conditions, the length of the main root is shorter and the number of lateral roots is less in rel3 than in wild type. Moreover, the rel3 mutant developed approximately half number of nodules found in the wild type. And the ratio of nodulation zone length to the main root length in the rel3 plants was less than that in the wild type. However, there is no difference in nitrogenase activity between rel3 and wild type. The phenotypic analysis indicated that rel3 showed fewer nodules due to narrow nodulation zone. We determined the infection frequency using-NZP2235/HemA::LacZ that constitutively expresses the (3-galactosidase reporter gene. After inoculation, infection thread formation was repressed in rel3 compared to that in the wild type. This suggest that the decreased infection frequency in rel3 produce narrow nodulation zone in rel3, leading to the reduced number of nodules formed in rel3.In order to investigate molecular mechanisms of rel3 with reduced nodules, we first analyzed the sensitivity of rel3 to negative regulators of the nodulation including nitrogen and ethylene. The results show that the mutation does not affect the negative regulation of nodule development by nitrate and ethylene. In addition, the grafting experiments of rel3 and wild type indicated that the phenotype of reduced nodule number in rel3 mutant is in control of shoot-derived signals.By Agrobacterium rhizogenes-mediated root transformation, we analyzed REL3 promoter-GUS activity in inoculated roots and nodules. The GUS activity showed that REL3 gene specifically expressed in root vascular bundle and nodule vascular strands, particularly in cells that were derived from the pericycle. And REL3 also expressed in vascular bundle of developing nodules. This indicated that REL3 plays an important role for nodule initiation.REL3 mutations blocked the synthesis of TAS3-siRNA in plants, resulting in the upregulated of its target genes, ARF3 and ARF4, auxin response factors. This prompt us to detect whether the reduced nodulation number of rel3 is correspond with Auxin. Treated with auxin inhibitors NPA and TIBA, rel3 showed nor agravitropic than wild type, suggesting that the rel3 mutant is sensitive to auxin inhibitors. This implies the polar auxin transport is altered in rel3 root. The study on alternation of polar auxin transport in rel3 is ongoing.Based on current data, we speculate that the alteration of polar auxin transport in rel3 roots make change of auxin level in root cortical cells, leading to narrow the range of auxin level that required for nodule initiation in root cortical cells, and thus fewer nodules are formed in rel3 roots. This study provides the evidence that ta-siRNA pathway regulated the auxin signaling pathway, directly involved in nodule development.更多还原