| As one of main abiotic stresses in nature, salt stress with high concentration of saline ions in soil does great harm to plants, and seriously affect the growth and development of plants. Simultaneously, the crops cultivated in the saline land undergo a wide range of underproduction. Cotton, a relative salt-tolerant crop, can largely adapt to environmental stress through its own regulatory mechanisms. At present, there is almost none of the salt-tolerant variety which can be used effectively in production. Therefore, it is particularly important to excavate salt-tolerant genes of cotton and innovate cotton salt-tolerance germplasm in order to lay the foundation for salt-tolerant cotton breeding. In this study, the salt-tolerant material, Zhong9835, and the salt-sensitive material, ZhongS9612, were treated by the method following 0.4% NaCl stress identification. Two salt-tolerance related genes, H~+-pyrophosphata- se gene and S-adenosylmethionine synthetase gene, were cloned from the salt-tolerance material on Gossypium hirsutum, which are named GhVP and GhSAMS, respectively. The bioinformatics analysis was conducted. At the same time, Real-time PCR analysis, prokaryote expression vectors, expression of SAMS in E.coli were performed and constructed. The main progresses were as follows:1. Gene cloning and sequence characteristics analysisThe full length sequences of GhVP and GhSAMS were obtained using RACE technology combined with RT-PCR. The full length of GhVP is 2678bp, including 5'-UTR 105bp, complete open reading frame 2301bp and 3'-UTR 272bp, which encodes a polypeptide of 766 amino acids. The full length of GhSAMS is 1576bp, including 5'-UTR 117bp, complete open reading frame 1182bp, 3'-UTR 250bp, and PolyA 27bp, which encodes a polypeptide of 393 amino acids.2. Bioinformatics analysisBioinformatics analysis shows that GhVP protein has the similarity of 90% with Arabidopsis and of 93% with Nicotiana. It forecastes that GhVP protein contains 14 transmembrane domain via transmembrane analysis, which infers it is a transmembrane protein. Hydrophobic protein annlysis shows that GhVP has strong hydrophobicity. Phylogenetic analysis shows that GhVP protein has the closest relationship with Kalidium and Chenopodium glaucum, which are halophyte. The homology of the GhSAMS protein with Arabidopsis thaliana, S. salsa, O.Sativa are 91%, 93%, 93%, respectively. Phylogenetic analysis shows that GhSAMS is closest to S. Salsa, while Secondary structure prediction shows that the protein is composed ofα-helix,β-sheet, turn and random coil. Meanwhile, GhSAMS protein contained three conserved domains.3 Real-time PCR analysesRoots are selected from salt-tolerance material and salt-sensitive material under different salt treatment stages, and RNA is extracted, then Real-time PCR analysis is conducted. The results show that, under the salt stress, GhVP and GhSAMS gene are induced, however, the induction were put off in salt-sensitive material. Therefore, we speculate that both genes are likely to play an important role in the process of adapting salt stress in cotton.4 Prokaryotic expression analysis Pokaryotic expression vectors, pET28a-GhVP and pET28a-GhSAMS, are constructed by the In-Fusion technology, which contain complete reading frame of GhVP and GhSAMS, then are transformed to E.coli BL21. It is nullity to GhVP in E.coli, though different temperature and concentration, we view GhVP might be toxicity to E.coli preliminarily, while GhSAMS is expressed successfully in E.coli via a 46.6KD fusion protein.Under salt stress, H~+-PPase provides proton driving force for Na~+/H~+ antiproton and promotes distribution of Na~+ in cell, and then eliminates Na~+ infection. SAM enhances the cell-to-cell pathway for water transport and reduces ions up-taking, and improves the adaptations of whole cotton under salt stress. With the development of stress cell biology, the function of GhVP and GhSAMS will be stated in further, which are hopeful to be important resource of salt-tolerance germplasm on cotton. |
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