Exploitation And Application Of Salt-tolerant Genes Of Halophyte Limonium Sinense And Suaeda Salsa

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.