CDNA Cloning Of The Vacuolar H～+-pyrophosphatase From Hordeum Brevisubulatum And Establishment Of Regeneration System Of Medicago Sativa L.

Soil salinity is a major constraint to food production because it limits crop yield and use of land previously uncultivated. Thus, it is important to solve the question of soil salinity with the increasing of the population of the world.With a homologous gene region we successfully isolated a Vacuolar H+-pyrophosphatase gene by RACE method together with RT-PCR from Hordeum brevisubulatum which is selected by salt for several years by our laboratory and named it HbVPl (accession number AY255181). The isolated cDNA is 2773 bp in length and encodes a deduced protein of 773 amino acid residues with a predicted molecular mass of 80.4 kDa and pl 4.90. The deduced amino acid sequence was similar to gene family that of previously identified in plant. The results showed that this gene was conserved in evolution. It shared high similarity with the sequences within predicted transmembrane segments and an catalytic site. Transmembrane domain analysis of the HbVPl predicated that this protein has 14 transmembrane helices, which indicated that this protein is a typical transmembrane protein.At the same time, we have established a good regeneration systerm of Medicago sativa for gene transformation through somatic embryogenesis way. During the process of tissue culture, the orthogonal design was used to plan the component of the culture media and multivariate statistical analysis was applied to analyse the experimental results. The results showed that there are lots of differences among the explants sources, genotypes and the base culture mediums. The dow-hypocotyl was prone to induce the embryoid compared with the cotyledon. The best media for inducing differentiation was UM + KT 0.5mg/L + 2,4-D 10.0mg/L + proline 20mM and 1/2MS + NAA 0.5mg/L + IBA 1 .0mg/L was the best one for rhizogenesis.Explants sources have severe effects on somatic embryogenesis in alfalfa. Induction on mature cotyledon embryos was effective, and the frequency of somatic embryo production was 100%.In general, the effective genetic transformation system of alfalfa was important to gene transformation in further studying.