【作者】 孙超；

【导师】 郭泽坤； 刘华伟；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 我国52.5%的陆地面积为干旱半干旱地区，倘若干旱发生在小麦苗期，会阻碍小麦正常的生长发育。有研究发现，根瘤菌除自主生活和与豆科共生固氮外，还存在其作为内生菌与非豆科草本植物的联合作用。茎瘤固氮根瘤菌Azorhizobium caulinodans ORS571（A.caulinodans）作为一种植物内生固氮菌，它不但具有一定的固氮能力，能够促进寄主或非寄主植物的生长，而且具有增强植物抵御逆境胁迫和病原菌侵染的能力。1.本研究比较了A.caulinodans对小麦五种不同侵染方法的侵染效率，优化了侵染方式，建立限菌条件下A.caulinodans对小麦的侵染体系；同时，通过温室叶面接种A.caulinodans实验，探索A.caulinodans作为叶面肥对小麦生长的促进作用。2.利用gfp-A.caulinodans分别侵染小偃22种子、根系和叶片，通过徒手切片、制片，利用激光共聚焦扫描显微镜检测。结果表明，A.caulinodans可通过根毛和侧根裂隙处侵入小麦根部和根维管束组织，并在维管束组织和细胞间隙中定殖。同时，定殖于小麦根中的gfp-A.caulinodans可向上迁移至小麦叶内，定殖于小麦叶中的gfp-A.caulinodans又通过气孔从小麦叶内迁移到叶表。以上结果为在分子水平研究菌植互作提供了依据。3.本研究用A.caulinodans侵染小麦种子，提取接菌24h处理（TsR24）和对照（CsR24）总RNA，基于第二代高通量测序技术进行small RNA测序。测序结果表明，参与逆境胁迫的miRNA占全部的12%，其中抗旱相关的microRNA有14个表达量上调，7个表达量下调。在所有上调的miRNA中，miR1867、miR854上升差异达到极显著；在所有下调的miRNA中，miR1088、miR1126下降差异达到极显著。以上结果为根瘤菌与干旱小麦互作的研究提供新思路。4.本研究筛选出对A.caulinodans敏感的小偃22为干旱敏感型品种，测定干旱胁迫下A.caulinodans的生长曲线，表明A.caulinodans在干旱胁迫下能够繁殖。在开放条件下，A.caulinodans侵染干旱胁迫的小偃22种子，种子发芽势提高、发芽指数增大。A.caulinodans侵染干旱小麦幼苗叶片，叶片可溶性蛋白、相对含水量和脯氨酸含量均明显提高。表明A.caulinodans对小偃22种子的萌发有缓冲、调节和促进适应的能力，有助于小麦幼苗抗旱。综合上述研究表明，A.caulinodans能在小麦幼苗体内全身分布，对幼苗有明显的促生作用。种子侵染A.caulinodans24h后发现了表达差异显著的干旱相关miRNA。并在生理生化水平上表明A.caulinodans能提高小麦幼苗的抗旱能力。为小麦抗旱研究提供了一条新思路。更多还原

【Abstract】 In china,52.5%of the land is the arid and semi-arid area. If the drought occurredin wheat seedling stage, it will hinder the normal growth of wheat. Studies showedthat, rhizobium can live independently and has symbiotic nitrogen-fixing withleguminous, it as endogenous bacterium interacts with leguminous plant.Azorhizobium caulinodans ORS571(A.caulinodans) is a kind of plant endogenousnitrogen-fixing bacteria, it not only has a certain nitrogen-fixing ability and canpromote the growth of the host or non-host plant, but it has the enhancement theability against adversity stress and pathogen infect in the plants.1. This study established infection system of the A.caulinodans on wheatseedling. First of all, in the gnotobiotic conditions, we compared infection efficiencyof five different infection methods, to established infection system of theA.caulinodans on wheat seedling in the gnotobiotic conditions; at the same time, wedid foliar spray with A. caulinodans in the greenhouse. We explored A.caulinodans oneffect of the wheat growth as the promoting function of foliar fertilization. Finally,this study established infection system of the A.caulinodans on wheat seedling.2. In the gnotobiotic conditions, using the gfp-A.caulinodans infect respectivelyXiaoyan22seeds, roots and leaves, we got detection using fluorescence microscopeand laser confocal microscope after free-hand section and mounting technique. Theresults showed that A.caulinodans can through the root hair and lateral root fractureinto the roots and the vascular tissue, then colonized in vascular tissue and cell gapshall. At the same time, the gfp-A.caulinodans colonized in wheat root can move up tothe wheat leaf, the gfp-A.caulinodans colonized in wheat leaf can move from thestomata to leaf insid. These results will provide the theory basis for the molecule level research of bacteria-plant interaction.3. This research use A.caulinodans infect wheat seeds, extracting total RNA ofthe24h Treatment (TsR24) and control (CsR24), then small RNA sequencing basedon the second generation high-throughput sequencing technology. Sequencing resultsshowed that miRNA related to the adversity stress accounted for12%of all miRNA.Expression of14miRNA related to drought were up-regulated,7miRNA weresignificantly down-regulated. In the up-regulated miRNA, miR1867, miR854reacheda significant difference. In the down-regulated miRNA, miR1088, miR1126reached asignificant difference. The results provide new ideas for research of rhizobiuminteracting with drought wheat.4. This study screened wheat xiaoyan22as drought sensitive variety toA.caulinodans. We determinated the A.caulinodans growth curve in drought stress,results showed that A.caulinodans can reproduce in drought stress. In the openingcondition, we infected drought stress xiaoyan22seeds with A.caulinodans, the resultsshowed that seed germination potential was improved, germination index wasincreased. We infected drought stress xiaoyan22laminas with A.caulinodans, theresults showed that leaf soluble protein, relative water content and proline contentwere increased significantly. A.caulinodans has buffered, regulated and promotedfunction of the seeds germination on xiaoyan22and contribute to drought-relief ofwheat seeding.Based on these studies showed that A.caulinodans can systemic distribute in thewheat seedlings and promote seedling growth. We found expression differencessignificant miRNA related to drought after24h turough seed infection withA.caulinodans. A.caulinodans can improve the wheat seedling drought resistance onlevel of physiological and biochemical. This Study provides a new idea for wheatdrought resistance research.更多还原