Cloning Of IlVP Gene And Its Salt Tolerance Analysis In Iris Lactea H~+-PPase

Iris lacteal is one kind of perennial herb plant of Iris of Iridaceae, its place of growth is northeast, northwest, north and other places of China. Because its great resistance, make it become the indicative plant of degraded lowland and saline meadow, and widely used in the garden, its leaf color is dark green, long green period, color and elegant beauty, extensive management, make it become a good plant of city garden landscape configuration and green construction of the country park. Our research group sequencing the marker site ISSR841-320 of Iris lacteal, find that VP homology reached more than 60% with other plant vacuole membrane H+-PPase gene, among them, the homology of H+-PPase gene with HvVP gene of barley was 80%. H+-PPase gene is localized in the vacuole membrane, to assume the role of proton pump with H+-ATPase, composition of H+ transmembrane electrochemical gradient, regions of excess inorganic ions in the cytoplasm into vacuoles, reduce the damage of inorganic ions on plant. In this study, Iris lacteal as experimental materials, use Rapid amplification of cDNA end (RACE) technology, get the full length cDNA sequence of IIVP of Iris lacteal; By using atomic absorption spectrophotometer and RT-PCR real-time technology, to different concentration of NaCl to the lower and upper in the quantitative analysis of the expression level of IIVP of Iris lacteal; use Leaf disk transformation technology of Agrobacterium mediated by Agrobacterium, transferred the IIVP gene of Iris lacteal into tobacco W38, altogether obtain 25 transgenic tobacco plants, and through the experimental verification of Northern and Southern hybridization; analysis on physiological characteristics of leaves osmotic potential, relative plasma membrane permeability and relative water content of transgenic tobacco and wild type tobacco plants, aims to reveal the role of IlVP in salt tolerance of tobacco. The results obtained are as follows:(1) Successful cloning of IlVP gene from Iris lacteal, the total length of cDNA sequence is 2738 bp, contains an open reading frame (ORF) 2316 bp, the 5' untranslated region (UTR) is 103 bp and 3'-UTR is 319 bp; encoding 771 amino acids, the predicted molecular weight is 80.7 kDa, isoelectric point is 5.16, similarity comparisons showedthat they sharedover70% similarity in nucleotide sequence and over 79% similarity in amino acid sequence with those of other plants H+-PPase. Amino acid sequences contained three highly conserved fragments, KAADVGADLVGKVE, DVGADLVGK, and GGG, is PPI binding site of the vacuole membrane H+-PPase.(2) With different concentrations of NaCl (mM 0-200) treatment, IlVP is mainly expressed in the upper part of the earth, the transcript abundance increased with the increasing of concentration.(3) Through the traditional carrier construction method, pBI121-35S-IlVP-Nos recombinant plasmid was successfully constructed. And then is transformed into the tobacco W38 by Agrobacterium tumefaciens-mediated leaf disc,25 positive plants were identified from 28 transgenic tobacco plants by using 50 mg/L kanamycin and PCR.(4) After 200 NaCl mmol/L treatment 7 d, the salt tolerance of transgenic tobacco plants was stronger, no significant changes in growth status, but normal growth of wild type plants was inhibited, the leaves showed wilting dry state, its growth becomes slow. By Northern and Southern hybridization, further validation experiments have been successfully irisquinone IIVP gene was integrated into tobaccos, the highest expression abundance of T18 plant lines and the lowest expression abundance*of T3 plant lines were obtained.(5) After 200 NaCl mmol/L treatment 7 d, the fresh weight of transgenic tobaccos showed decreasing trend with the increase of NaCl concentration, the decline trend of transgenic plants was slow. With 200 NaCl mM treatment, the dry weight of root stem and leaf of wild type tobaccos decreased by 83.1%,69.4% and 73.5% respectively, fresh weight decreased by 89.6,74% and 67.1%, the dry weight and fresh weight of the transgenic tobacco T18 only decreased by 50.3%,63.7%,9.2% and 61%,42.2%,39.9%. The leaf growth inhibited of transgenic tobaccos was not obvious by NaCl salt stress, but the leaf area of the wild type tobaccos was significantly decreased, the leaf area of transgenic tobacco T18 was 68.9% larger than wild type tobaccos. Compared with wild type tobaccos, with NaCl salt stress, the relative water content of transgenic tobaccos T18 and T3 decreased by 5.3% and 4.8%, respectively, WT tobaccos decreased by 11.9%.(6) After 200 NaCl mmol/L treatment 7 days, the relative membrane permeability of leaves of transgenic tobaccos and wild type tobaccos increased with NaCl concentration increased, but the wild type tobaccos increased faster. The relative membrane permeability of transgenic tobaccos T18 and T3 was 25.5% and 10.6% lower than wild type tobaccos. With 200 NaCl mM treatment, Na+ and K+ in leaves of T18 plants were 20% and 123% higher than wild type tobaccos, Na+and K+ in stem were 29.5% and 115% higher than wild type tobaccos, Na+ and K+ in roots were 187% and 392% higher. And Ca2+ showed a decreasing trend with the increase of NaCl salt concentration, Ca2+ in root, stem and leaf of T18 were decreased by 45.6%,25.8% and 58.3% respectively compared with wild type tobaccos. The experimental results also showed that the overexpression of IlVP gene can protect the water utilization rate irisquinone of transgenic tobaccos. All the above research results show that, salt tolerance of tobacco plants was significantly increased by transferred the IIVP gene of Iris lacteal into tobaccos, it has laid an important scientific theoretical basis and technical support for the genetic improvement of salt tolerance of pasture and turf grass varieties, and played an important role and technical guarantee for the urban landscape and green space construction.