Cloning And Salt Tolerance Analysis Of Vacuolar H~+-pyrophosphatase Gene Of Malcolmia Scorpioides

Proton-pumping pyrophosphatase(H+-PPase) is a kind of pyrophosphatases existing in various organi-sms, such as land plants, alga, protozoa and bacteria. Plant vacuolar H+-PPase can catalyze the hydrolysis of PPi into two orthophosphate(Pi) molecules, providing free energy for H+ transmembrane transportation and pumping protons from the cytoplasm into the vacuole. Previous studies showed that enhanced H+-PPase activity could improve salt-stress in several plants.Malcolmia scorpioides is one of the spring ephemeral plants of Cruciferae, malcolmia genera, which is a close relative of Arabidopsis thaliana. M.scorpioides is located on the edges of deserts of Xinjiang and has very strong adaptability to the arid desert environment. It was characterized by high reproduction rate and rather short life-cycle which germinated and growed rapidly using melted snow in late March to Early April, with flowering in May and fruiting in June.In this research, the resistance of M.scorpioides was analysised using A. thaliana as a model plant. A vacuolar H+-PPase c DNA, named Ms VP1, was cloned from M.scorpioides and the sequence of bioinformatics was analysed. Ms VP1 expression was detected in salt and heat tolerant. The expression vector p BI121::Ms VP1 was constructed, and had been introduced into tobacco and the salt resistance of transgenic plant-s was analysed. The reaearch provided a reference for the salt-tolerant mechanism of M.scorpioides and laid the foundation for further use of Ms VP1.(1) Drought and salt resistance of M.scorpioides was studied and compared with A. thaliana. The results showed that M.scorpioides has greater ability to tolerate drought and salt stress than A. thaliana. For instance, seeds of M.scorpioides can germinate and grow at low tempreture of 4℃, seedings can withstand Na Cl stress at the 200mmol/L and 20% PEG6000 in 1/2MS culture.(2) A H+-PPase c DNA, named Ms VP1, was cloned from from M.scorpioides by RT-PCR and RACE technique. The sequence analysis has revealed that the full length c DNA of Ms VP1 was 2824 bp, containing a open reading frame of 2310 bp for 769 amino acids which has the characteristics of H+-PPase gene families. The deduced amino-acid sequence of Ms VP1 showed more than 85% homology with other plant VP1 proteins. Phylogenetic tree analysis indicated that M.scorpioides had close relationship with A. thaliana.(3) Expression analysis showed that MsVP1 mRNA could be induced by NaCl, PEG6000 and heat shock, and decreased first then increased over time. The amount of Ms VP1 m RNA was more than 7.1 times higher than control after NaCl stress for 24 h.(4) Physiological analysis of transgenic tobacco plants in response to salt stress: The expression vector p BI121::Ms VP1 was constructed. The Ms VP1 gene was transformed into tobacco by Agrobacterium through leaf disc transformation and the transgenic tobacco was obtained by Kanamycin selection. The transgenic plants were treated with different concentrations of Na Cl,then the leaf chlorophyll contents, relative water content and the MDA content was detected after treatment. The results showed that the leaf chlorophyll contents, the decrease of relative water content rate and the increase of MDAcontent rate in transgenic tobacco were significantly less than the wild type. The results of our study showed that overexpression of Ms VP1 gene could improve the water holding capacity and the tolerance to salt stress in transgenic tobacco plants.