【作者】 王明霞；

【导师】 邢邯；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2012， 硕士

【摘要】 作物在生长发育的过程中难免会受到各种胁迫的影响,比如干旱、高盐。这些胁迫能够影响作物生长和降低产量。然而作物已经形成复杂的机制来应对这种胁迫,但这是远远不够的。研究这种复杂的机制,挖掘发掘抗逆基因资源,培育抗逆高产新品种来应对逆境胁迫是刻不容缓的。本研究从耐盐大豆品种苏协1号克隆两个耐盐基因GmHAL3a和GmHAL3b,对其进行了序列比对分析,组织表达分析和非生物胁迫分析。同时构建了这两个基因的表达载体,并成功转入拟南芥,用高盐、山梨醇和锂离子处理纯系后代,初步分析两个基因在非生物胁迫反应中的功能。此外对这两个耐盐基因GmHAL3a和GmHAL3b,分别克隆了两个RNA干扰片段,构建四个RNA干扰载体,并成功转入根癌农杆菌EHA105中。主要研究结果如下：1.氨基酸序列比对发现这两个基因的蛋白序列含有和其它物种相似的保守位点,进化树分析结果说明其蛋白序列与拟南芥HAL3基因亲缘关系是最高的。2.理化性质分析结果为,两个蛋白都呈酸性；发现这两个基因都可能定位于叶绿体中；且它们都具有两个潜在的跨膜螺旋区域。组织表达分析发现,GmHAL3a和GmHAL3b基因在苗期到开花后30天都是在根中表达量最高,开花期在叶中表达量最低,在茎中的表达量随着植株的生长是呈上升趋势的；开花后55天之前,两个基因在荚中的表达量都高于籽粒中,从开花后55天开始,籽粒中两个基因的表达水平就高于荚中,开花后65天籽粒中两个基因的表达量都达到最高值。3. GmHAL3a和GmHAL3b基因都受盐、山梨醇和LiCl的诱导表达,受LiCl的诱导表达最强烈。4.将连接有GmHAL3a和GmHAL3b基因的过表达载体成功转入拟南芥,发现转基因株系的开花时间提前,叶片中的AtHAL3a、AtHAL3b和AtHIP1基因的表达量在胁迫前后都高于对照。推测过表达GmHAL3a和GmHAL3b基因能够激活拟南芥叶片中AtHAL3a、AtHAL3b和AtHIP1基因的表达。5.用NaCl、山梨醇、LiCl处理转基因拟南芥纯合株系,转基因拟南芥的萌发速度比对照植株快,根长也显著高于对照植株,转基因拟南芥叶片中的脯氨酸含量在处理前后都比野生型多,但活性氧含量比野生型少。这些结果表明过表达GmHAL3a和GmHAL3b基因能够提高植株的耐盐性和耐锂性,增强拟南芥的抗渗透能力。更多还原

【Abstract】 Crops will inevitably subject to a different environmental stress, such as drought and high salinity impact on plants growth and productivity. Plants have evolved complex mechanisms to control the expression of various genes to adapt to such environmental stresses. But this is not enough. We need to continue to explore the use of resilience genetic resources, cultivating adverse resistant and high yielding variety to cope with stress. n this study, we cloned GmHAL3a and GmHAL3b gene from soybean. Cloned Two RNAi fragment each of the two genes to construct four RNAi vectors, and successfully transferred to Agrobacterium tumefaciens strain EHA105; Analyzed expression pattern of the two genes with real-time quantitative PCR tissue expression analysis and abiotic stress analysis; studied on functional analysis of transgenic GmHAL3a and GmHAL3b gene Arabidopsis which were transformed via Agrobacterium mediation after treated with various stress. Our results are mainly as follows:1. The protein sequences of these two genes contain similar conserved sites with other species. The phylogenetic relationship appeared that GmHAL3a and GmHAL3b genes were more similar to Arabidopsis HAL3gene than others.2. Tissue expression analysis found, the expression of GmHAL3a and GmHAL3b genes were highest at the root from seedling to30days after flowering and lowest in leaves during the flowering stage. The expression level of the two genes in the stem were progressively upward with the growth of plants. Before55days after flowering, they expression higher in pods than seeds; Since then, the opposite, when65days after flowering, the expression of the two genes reached the highest value in seeds.3. Abiotic stress analysis of the GmHAL3a and GmHAL3b genes showed that they were induced by salt, sorbitol and LiCl, and most intense by LiCl. 4, After transformed GmHAL3a and GmHAL3b genes into Arabidopsis via Agrobacterium mediation, transgenic Arabidopsis.After Arabidopsis were treated with various stress. We found that AtHAL3a,AtHAL3b and AtHIPl genes expression levels were higher than that of the control Arabidopsis.These results indicate that overexpression of GmHAL3a and GmHAL3b genes can activates AtHAL3a, AtHAL3b and AtHIPl genes in Arabidopsis.5. After stress induced on transgenic pure lines by salt, sorbitol and LiCl, we found that the germination rate of seeds and root elongation of transgenic plants were increased. Also proline content in transgenic Arabidopsis was significantly higher than wild Arabidopsis, but active oxygen content in was lower. The results suggested that GmHAL3a and GmHAL3b genes enhanced the tolerance to salt and osmotic stress in Arabidopsis.更多还原