Saccharomyces Cerevisiae Three Salt-tolerant Genes Cloning And Its Transformation In Cotton

Salinity is the important factor limiting plant growth and productivity. Cotton?G. hirsutum L.?, as the pioneer of saline-alkali land crops, is one of ideal crops to development and utilization of salinization land. Through genetic engineering and transgenic technology to improve cotton breeding of salt resistance has become the current urgent demand and important direction.Related studies have shown that more than 200 genes are related to salt tolerance in Saccharomyces cerevisiae genome. ENA1?exitu natru 1? encodes a P-type Na⁺-ATPase that efflux of Na⁺ and Li⁺, and overexpression of ENA1 indeed enhances salt tolerance in Arabidopsis under both acidic and alkalin conditions. As a effector for ENA1, HAL1?halotolerance 1? promote the absorption of K⁺ by improving its absorption capacity in the feedback regulation. ScHAL1 transform into plants not only can improve the salt tolerance, and improve its resistance to alkali and crop yield. YCF1?yeast cadmium factor 1? protein is a S-GSH transporter that sequesters Cd⁺ and Hg²⁺ in vacuole membrane, and expression the Mg ATP-dependent gene in Arabidopsis may confer strong salt tolerance under the higher salt stress than the wild type.In this study, we obtained Saccharomyces cerevisiae salt-tolerance genes mRNA sequence by NCBI databases, cloned ENA1, HAL1 and YCF1 by using RT-PCR technology from Saccharomyces cerevisiae As2.375. The ENA1?GenBank: NM₀01180348. 1? gene was 3267 bp, coding 1091 amino acids; the HAL1?GenBank: EF015596.1? gene was 885 bp, coding 294 amino acids; the YCF1?GenBank: NM₀01180442.3? gene was 4548 bp, coding 1515 amino acids. Bioinformatics analysis with three proteins, isoelectric point of 5.53, 9.01 and 8.64 respectively. The ENA1 protein is acidity and negative charged while the others are alkaline and positively charged. With the hydrophilic-hydrophobic property, ENA1 and HAL1 belong to hydrophilic protein, and YCF1 belongs to hydrophobic. Three protein included alpha helix angle, beta-sheet, beta-turn and no rules volume.Expression the pBI121-ENA1::GFP, pBI121-HAL1::GFP and pBI121-YCF1::GFP fluorescence vectors for onion epidermal subcellular localization showed that, the ENA1 and YCF1 protein were expressed and located at the cytomembrane, and the HAL1 protein was located at the cytoplasm. The green fluorescence of the cotton pollen were obviously enhanced, indicating that three genes could be expressed in cotton pollen, which lay the foundation for the next step to obtain transgenic salt-tolerant materials with using the gene gun technology.Expression vectors were constructed and transformated into the cotton materials Zhong 12 by gene gun. Salt-resistant screening of T1 generation of the seeds was conducted with 0.6% NaCl, and shown that the transgenic seed germination rate and root growth were better than control Zhong 12 selfing species, which prove the three genetic transformation of cotton seeds can obviously increase the salt resistance. Molecular detection for transgenic seedlings, vector plasmid as positive control, the DNA of Zhong 12 selfing species as the negative control, with the detection primer amplification, the PCR products were sequenced directly. Comparative analysis of sequencing results showed that 18 transgenic plants had specific bands, for three are ENA1, nine are HAL1, and six are YCF1.In this study, we cloned three salt-tolerance genes ENA1, HAL1 and YCF1 from Saccharomyces cerevisiae and transformed into cotton by gene gun in vivo transformation technology, and we identified they can improve cotton salt resistance initially. Our study provide the basis to further explore the exact function of the yeast salt tolerance related genes in higher plants, and provide the foundation for breeding new varieties of cotton salt tolerance materials.