The Cloning And Functional Analysis Of Vacuole H~+-ATPase Subunit Gene SiVHA-E From Foxtail Millet

The V-H+-ATPase plays important roles in processes of plant growth, development and stress adaptation. soil salinity is one of the most abiotic stresses, which affects plant growth and development and causes major reductions in crop productivity and quality. In recent years, some subunits of V-H+-ATPase had been studied, but most studies mainly focus on the genes functions, whereas the mechanism is still poorly understood. Thus, it is very meaningful that the V-H+-ATPase salt tolerance mechanism is researched.The prominent drought resistance features of foxtail millet, as well as its small sequenced diploid genome(about 490 Mb), make foxtail millet an excellent candidate model system for investigating abiotic challenges. This experiment uses millet material H214 as templates, to V-H+-ATPase subunit gene E, SiVHA-E, and around the gene to do the characteristic analysis and resistance identification, to obtain the following results:1. In this research, a V-H+-ATPase E subunit gene, SiVHA-E was identified from millet crop by sequencing Blast with the wheat known sequence. The gene is 696 bp, encoding 231 amino acids. Analysis results of SiVHA- E genetic characteristics show that the SiVHA-E was conserved in evolution and the SiVHA-E was the closer relatives with a V-H+-ATPase E subunit gene, ZmVHA-EL in maize(Zea mays). SiVHA-E gene promoter sequence analysis and the quantitative real-time PCR(qRT-PCR) analysis revealed that the SiVHA-E were response to all kinds of plant hormones and light.2. 8 transgenic Arabidopsis thaliana seedling was obtained by agrobacterium infection. The results of salt tolerant assay showed that the germination rate of SiVHA-E transgenic lines were significantly higher than that of wild type plant under salt stress. During seedlings period, the root lengths of SiVHA-E transgenic lines were significantly longer than that of wild type plant and fresh weight and survival rate of transgenic lines were significantly higher than that of wild type plant under salt treatment. Comparing with wild type plant, the content of Na+ reduced and the relative water content increased inside transgenic plant cells, respectively. In addition, the results of germination experiment used ABA showed that SiVHA-E transgenic Arabidopsis were more sensitive to ABA than wild type plant during post-germination. In short, overexpressing SiVHA-E in transgenic Arabidopsis lines enhanced salt tolerance, which might be results of positive regulation of ABA signaling pathway or reduction of Na+ accumulation and water lost in transgenic plants comparing with wild type plant.3. However, phenotype of transgenic plants were no obvious different with wild type Arabidopsis in other stress conditions(including PEG, low nitrogen, phosphorus, potassium).In conclusion: overexpressing SiVHA–E in transgenic Arabidopsis plants enhanced salt tolerance, which might be results of positive regulation of ABA signaling pathway or reduction of Na+ accumulation and water lost in transgenic plants comparing wild type plants.This study provides a new salt-tolerant crop genetic improvement candidate genes, and to understand the V- H+-ATPase salt-tolerant mechanism provides a new clue.