【作者】 郭善利；

【导师】 赵彦修； 张慧；

【作者基本信息】 山东师范大学 ， 植物学， 2005， 博士

【摘要】 土壤盐渍化是影响农林业生产,导致农林业生物量减少的主要非生物胁迫因子之一。盐分胁迫主要包括渗透胁迫、离子胁迫及其造成的一系列次级胁迫如氧化胁迫等,严重干扰植物体内业已存在的细胞及整株水平上的水分及离子稳态,造成植物细胞分子损伤,生长延滞甚至死亡。植物耐盐基因工程被认为是培育耐盐植物、开发利用盐碱荒地最有效、最经济的手段,但有效目的基因的缺乏,特别是来自盐生植物的耐盐基因,是限制植物耐盐基因工程发展的一大障碍。本论文的主要目的是利用真盐生植物补血草建立EST 数据库,结合盐生植物盐地碱蓬的EST 数据库,发掘来自盐生植物的耐盐基因,并且在林木模式植物杨树遗传转化方面做一些有益的尝试。1. 补血草(Limonium sinense)EST数据库的构建构建了一个补血草叶片的cDNA 文库,并从中随机挑选克隆单向测序,获得了1162 个EST序列,其中405 个EST序列与以前鉴定的基因序列具有很高的同源性。通过序列分析,鉴定了739 个独立的EST 序列,其中194 个序列与以前鉴定的基因具有同源性。所有的EST数据都将公布在GeneBank 的dbEST中。耐胁迫调控基因的EST划分为7 个主要的类别,占EST总数的3.4 %。2. 盐地碱蓬(Suaeda salsa)液泡膜焦磷酸酶基因的克隆与鉴定从400mmol/L NaCl处理的盐地碱蓬地上部分构建的λZap-cDNA文库中克隆了编码液泡膜H~+-PPase 的部分cDNA(SsVP),据已知序列设计引物利用PCR方法获得编码盐地碱蓬液泡膜H~+-PPase 的全长cDNA。对该基因的序列特征、基因组结构和在盐胁迫下的表达特性做了详细的分析。结果表明:SsVP 与已报道的红叶藜(Chenopodium rubrum)的液泡膜H~+-PPase 基因(CVP)同源性最高,Clustalx 软件对不同物种中液泡膜H~+-PPase 基因所作的系统进化分析表明SsVP与CVP 聚为一簇,均属于类型Ⅰ类液泡膜H~+-PPase;Southern杂交表明该基因在盐地碱蓬基因组中可能是多拷贝的;Northern杂交结果表明在盐胁迫和干旱胁迫下SsVP 的转录都是上行调节的,400mmol NaCl处理和干旱处理下,盐地碱蓬地上部分中SsVP 的mRNA增加。将SsVP 的全长cDNA克隆入植物表达载体pCAMBIA1300 中,导入根瘤农杆菌GV3101后,由花浸泡法进行拟南芥遗传转化,转化SsVP 的拟南芥在含潮更多还原

【Abstract】 Soil salinity is one of the major stress factors limiting the growth and productivity of crop and forest plants in agricultural and forestry fields. Salt stress consisting of osmotic and ionic stress,and then the secondary stress such as oxidative stress arising by them, can disrupt homeostasis in water potential and ion distribution. This disorder of homeostasis occurs at both the cellular and the whole plant levels. Drastic changes in ion and water homeostasis lead to molecular damage, growth inhibition and even death. Plant salt-tolerant genetic engineering was considered as the most effective and economical means to bring up salt-tolerant plants and to exploit salt wasteland. The lack of efficient genes, salt-tolerant genes specially coming from halophytes has been the obstacle to limit its development. The main objectives of the dissertation were to set up a cDNA library of halophyte Limonium sinense and to exploit new efficient genes, combining the cDNA library of Suaeda salsa, for some benefit attempts in forest model plant poplar salt-tolerant genetic engineering. 1. Construction of EST Database from Limonium sinense We constructed a cDNA library of Limonium sinense leaves and sequenced randomly selected clones. 1162 ESTs were generated. Among them, 405 ESTs had significantly homology to previously identified genes. By sequence analysis, 739 unique clones were identified and 194 of them showed homology to previous identified genes. All our EST Data will be submitted to dbEST in GenBank and will be available on the internet. At least 7 classes of genes were related to the stress tolerance, which accounted for about 3.4 % of total sequenced ESTs. 2. Molecular Cloning and Function Characterization of SsVP gene in Suaeda salsa We isolated one cDNA that may encode a vacuolar H~+-PPase (SsVP) from a λZap-cDNA library constructed from a 400 mmol/L NaCl-treated Suaeda salsa aerial tissue. We analyzed its sequence characterization, genomic structure and transcription level under salinity stress. The results indicated that SsVP showed the highest homology to the vacuolar H+-PPase (CVP) gene from Chenopodium rubrum and phylogenetic analysis indicated that SsVP and CVP share a cluster, which showed that they might be more similar in evolution. Southern blot analysis showed that there was more than one copy of SsVP in the Suaeda salsa genome. Northern blot analysis indicated that the transcription levels of SsVP in S. salsa aerial tissue were significantly increased after salt and drought stress. The expression levels of SsVP in S. salsa aerial tissue were significantly increased after 48h being treated with 400mmol/L NaCl and after 9 days being treated with drought. Then the SsVP ORF was integrated into the plant expression vector pCAMBIA1300. The integrated vector was introduced into Arabidopsis thaliana by in planta transformation method mediated by Agrobacterium tumefaciens GV3101. SsVP transformations were continuously screened on media with hygromycin (25mg/L). We obtained 14 homozygous T3 transgenic SsVP gene transformants. We selected two homozygous transgenic SsVP gene lines, named SsVP-1 and SsVP-2, which were used for molecular and physiological analysis. The activities of both the V-ATPase and the V-PPase in the SsVP transgenic line, SsVP-2, increased significantly compared with those in wild-type plant under NaCl and drought treatments. The results showed the increase of the H+ electrochemical gradient across the vacuolar membrane, which permit the secondary active transport of Na+ and other solute molecules, by up-regulation of the V-ATPase and the V-PPase activities. The net increase in the concentration of solutes in the cell must lead to an increase in the uptake of water and the retention of water under water deprivation condition, which make transgenic plants maintain turgor. The Arabidopsis plants overexpressing SsVP showed a tendency to accumulate more Na+ under saline condition than wild type plants. The increased accumulation of sodium is likely to be a consequence of the activity of the vacuolar secondary transports. The relative water content was higher in the transgenic plants in comparison with that of controls. The transgenic plants increase salt tolerance and drought tolerance. 3. Studies on transforming poplar by SsNHX1 gene SsNHX1 was heterogenously expressed in poplar and the transformants in different concentrations of salt was testified by molecular approaches. The results of the RT-PCR and the Northern blotting analysis showed the extraneous gene had been integrated into poplar genome. No visible differences were observed in growth and development between the poplar transformants and the wild-type plants in the absence of NaCl stress conditions, which indicated that the insertion and overexpression ofSsNHX1 had no effect on the normal growth of Populus plants. However, transgenic poplar plants exhibited improved salt adaptability in comparison with their non-transformed counterparts in the presence of salt stress. The overexpression of extraneous gene SsNHX1 can increase relative water content, accumulate less Na+ and more K+, maintain a high cytosolic K+/Na+ ratio and photosynthetic rate (Pn), increase the content of proline and then reduce the osmotic potential and MDA in the transgenic poplar compared with wild-type poplar under different concentrations of NaCl treatment. These results indicated that transgenic Populus plants overexpressing SsNHX1 had higher salt tolerance than wild type plants, which further indicated the importance of Na+ compartmentation in plant salt tolerance. This lay a fundation for breeding new varieties in salt tolerance of forest plants by plant genetic engineering. The innovations of this thesis can be summarized as follows: 1. We constructed a cDNA library of the halophyte Limonium sinense and analysed different expression of genes. It plays a key role in exploiting salt-tolerant genes of the halophytes. 2. It was the first time to clone and characterize the full cDNA coding vacuolar membrane H+-pyrophosphatase from the cDNA library of Suaeda salsa. We have constructed a plant expression vector with SsVP gene and integrated it into Arabidopsis by Agrobacterium tumefaciens. We also studied particularly the expression of Suaeda salsa SsVP under salt and drought stresses. 3. For the first time, the Suaeda salsa vacuolar Na+/H+ antiporter gene-SsNHX1 was heterogenously expressed in poplar, and the salt tolerance of the transgenic poplars was analyzed,which lay a fundation for breeding new varieties in salt tolerance of forest plants.更多还原