【作者】 安宝燕；

【导师】 张宪省；

【作者基本信息】 山东农业大学 ， 植物学， 2005， 硕士

【摘要】 土壤中的盐胁迫对植物的生长发育造成不利影响,渗透胁迫和离子毒害是盐对植物危害的两个主要的过程,导致植物细胞受到伤害。在盐胁迫中,Na~+是造成离子毒害的主要原因。为耐受高浓度的盐,植物通过Na~+/H~+ 逆向转运蛋白将Na~+运到液泡中或将Na~+从胞质排到细胞外从而避免了Na~+在胞质内的毒害,维持了渗透平衡。将Na~+转运到液泡中是通过定位于液泡膜上的Na~+/H~+ 逆向转运蛋白完成的。Na~+/H~+ 逆向转运蛋白在植物的耐盐性方面有重要作用,而紫花苜蓿是主要的豆科牧草,所以本研究以紫花苜蓿(Medicago sativa CV: WL323)为材料,利用RT-PCR 方法分离出与Na~+/H~+ 逆向转运蛋白基因具有较高同源性的基因,命名为MsNHX1,并在基因银行登记,序列登记号为AY513732。序列分析表明,该基因编码区长度为1623 碱基,编码含541个氨基酸的蛋白质。经过同源序列比较以及进化关系分析,初步推断MsNHX1 的编码产物为液泡型Na~+/H~+ 逆向转运蛋白。软件分析表明,该基因编码的多肽链有12 个跨膜结构域,可能与该蛋白的活性有关。Southern 杂交结果显示,该基因在四倍体苜蓿中存在一个小的基因家族。Northern 杂交表明该基因在紫花苜蓿的叶和根中皆有表达,并且用200 mM NaCl 分别处理苜蓿水培苗3、12、24 和48 小时,其表达水平明显上调,表明该基因受NaCl 诱导表达。由35S 启动子驱动的MsNHX1 基因已转入模式植物拟南芥,PCR 和RT-PCR 表明MsNHX1 整合到拟南芥基因组中,并且在转录水平上已表达。盐胁迫实验证明,在200 mM NaCl 处理条件下,野生型拟南芥生长状况较差,而转基因T2 代株系在该条件下基本能正常生长。并且在盐处理下,转基因植株比野生型对照的生物膜受破坏程度低,光合能力也较野生型更强。为获得耐盐能力提高的苜蓿新材料,我们将35S::MsNHX1 转化紫花苜蓿品种WL323,选用子叶、下胚轴和真叶作为外植体,通过农杆菌浸染的方法进行转化实验。目前从抗性愈伤组织上已经分化出绿色芽点,结果有待进一步观察。更多还原

【Abstract】 Salt stress in the soil has been existing for a long time, and the problem is more serious in recent years. High concentrations of salt can cause osmotic stress and ion toxicity, resulting in the damage for plant cells. Among the salt stress, Na~+ is the major cause of ion-specific damage in many plants. In order to tolerate high level of salt, plants compartment Na~+ into vacuoles or remove Na~+ out of cells from the cytoplasm through the operation of Na~+/H~+ antiporter to avoid the toxic effects of Na~+ in the cytosol and maintains osmotic balance. The compartmentation of Na~+ into vacuolar is achieved by the role of Na~+/H~+ antiporters on the vacuolar membranes. Vacuolar Na~+/H~+ antiporter is important in salt tolerance, and alfalfa is a kind of important leguminous plants. Therefore, an alfalfa (Medicago sativa cv: WL323) gene with high homology to Na~+/H~+ antiporter genes (named MsNHX1) was isolated by RT-PCR methods with homologous gene region and the Genbank accession number was AY513732. Sequence analysis showed the cDNA was 1623 bp in length and it encoded a protein of 541 amino acids. The deduced amino acid sequence showed high identities with other plant vacuolar type Na~+/H~+ antiporters, but had low identities with plasma membrane Na~+/H~+ antiporters. This indicated that the MsNHX1 encoded a putative vacuolar type Na~+/H~+ antiporter. Amino acid analysis with TMpred software indicated that the polypeptide chain contained 12 transmembrane motifs, which were related to the activity of the putative protein. Southern blot analysis revealed MsNHX1 exisited as a small gene family in the genome of tetraploid alfalfa. Transcripts of MsNHX1 were detected in alfalfa leaves and roots. In addition, the transcriptional level in root and leaf was upregulated when treated with 200更多还原