Physiological Responses To Salt Stress And Cloning Of An Na～+/H～+ Antiporter Gene From Betula Halophila And Its Overexpression Improves Salt Tolerance Of Tobacco

In plants, by the energy provided by transporting proton with its concentration gradient,vacuolar-type Na⁺/H⁺ antiporters delivered Na⁺ into the vacuoles against the electrochemical gradient generated by vacuolar H⁺-ATPase and H⁺-PPase. This compartmentation of Na⁺ provided an effective mechanism to avert the deleterious effects of Na⁺ in cytosol and maintain an osmotic potential. The enhanced salt tolerance of transgenic Arabidopsis, tomato, rice, corn and wheat by overexpressing tonoplast Na⁺/H⁺ antiporter genes indicated their important roles in plant salt tolerance.B.halophila was one of special halophytes belonging to Betulaceae family with high salt tolerance, so it was an important tree in Xinjiang for virescence and forestation. It was a levelâ…¡protected wild culture by country in severe danger located only in A Le Tai in Xinjiang. Discovering the salt tolerance mechnism had significant interest in improving crop and tree's salt tolerance. In this thesis, a series of studies have been carried on the isolation, sequence and expression analysis, salt tolerant function identification of B.halophila vacuolar-type Na⁺/H⁺ antiporter, and some physiological indexes of salinity tolerance were also determined basis on the materials from tissue culture. Then main results were as follows:Primers according to the homologous sequences from other plants were designed to amplify NHX1 specific DNA fragment using cDNA prepared from the leaves of B.halophila. The core fragment of interesting cDNA was obtained by RT-PCR. The full length cDNA was further isolated by 5'–RACE and 3'-RACE. The cDNA contained 2165 nucleotides with a 5'-untranslated region(5'UTR) of 205 bp, an open reading frame (ORF) of 1623 bp comprising 540 amino acid residues , and a 3'-untranslated region(3'UTR) of 337 bp. The deduced amino acid sequence showed high identities with other plant vacuolar-type Na⁺/H⁺antiporters from Penhx(DQ414512.1), Ptnhx(AY660749.1), Vvnhx(AY634283.1), which were 72%, 72%, 75% respectively. But had low identities with Na⁺/H⁺ antiporters from Arabidopsis and rice, which were 68% and 65% respectively.The formers were woody plants, but the latters were herbs. In addition to having high identities in nucleotides and amino acid residues, the grape had similar appearance with B.halophila, which showed that they might have closer relationship. It indicated that the full length cDNA encoding a putative vacuolar-type Na⁺/H⁺ antiporter.By the TMpred software, the deduced protein sequence contained the highly conserved 11 transmembrane (TM) segments at the N terminal and a C-terminal hydrophilic region. A putative amiloride binding domain (⁸⁵LFFIYLLPPI⁹⁴) was in TM3 in BhNHX1, it was the competitive binding site between amiloride and Na⁺. TM5, TM6 and TM3 seemed not to span the tonoplast in AtNHX1, They can polymerized to form a ion chunnel, TM3 was on the side of cytoplasm and TM5 and TM6 are on the side of vacuole. They could bind to Na⁺ and directed the influx of Na⁺ into the vacuole, BhNHX1 might have the same ion chunnel like AtNHX1.RT-PCR analysis indicated that the mRNA accumulation of BhNHX1 was strongly induced by salt stress. The mRNA expression of CaM was also high induced by all intimidates, indicating that CaM was one of universal signal transduction factor in plant. The transcription level of BhNHX1 was also up-regulated by drought, ABA (abscisic acid), cold,suggesting a non-specific salt stress response. It was also indicated that the promoter of BhNHX1 comprised the ABRE and DRE/ CRT element which could response to many kinds of adversity.The decrease of relative water content(RWC) and the K+/Na⁺ ratio was marked in plant after NaCl treatment. MDA did not change much from 0 to 200mM NaCl treatment, but it ascended sharply at 300 mM NaCl treatment, It could be explained by the CAT. The CAT increased from 0 to 200mM NaCl treatment, but it decreased sharply at 300mM NaCl treatment. This indicated that the plant was hurt at 300mM NaCl treatment. Crystals of calcium oxalate were widespread among plant's stem and leaf. But we didn't know how to form the crystal and what functions it played in this plant.Transgenic tobacco was analysed by genome PCR and RT-PCR with wild type control.The results indicated that the BhNHX1 might insert the tobacco genome. Under 400mM NaCl treatment, the transgenic tobacco had significantly less content MDA than the wild type.