Expression And Function Of The Preliminary Analysis Of Cucumber Phloem Protein CsCML1 Gene In Response To Salt Stress

Cucumber is an important vegetable crop in the world, and it is also one of the main vegetable crops in China. Nevertheless, cucumber is sensitive to soil salinization because of the shallow distribution and the weak regeneration ability. In recent years, the secondary soil salinization of facilities has seriously affected the growth, yield and quality of cucumber, and how to improve the salt tolerance of cucumber has attracted wide attention. CML is a family of plant-specifc Ca2+-responsive proteins that are involved in many important stress responses to plant stress. CsCML1 (Calmodulin-like protein 1-like) is a kind of calcium-binding protein of cucumber phloem in response to salt stress. It is important to understand the characteristics and function of CsCML1 in cucumber salt-tolerant cultivation and breeding.On the basis of previous studies, the open reading frame of CsCML1 gene was cloned and analyzed by bioinformatics firstly. The expression pattern of CsCMLl gene in roots steam and leaves was tested under salt stress. The CsCML1 gene was used as the target gene to construct the plant expression vector and transformed into Arabidopsis. Transgenic Arabidopsis plants were obtained. The salt tolerance of Arabidopsis thaliana was identified and the function of CsCMLl was preliminarily analyzed. The results are as follows:1. An open reading frame of CsCML1 gene about 573 bp was cloned from cucumber by PCR technique, The analysis of physical and chemical properties showed that the gene encodes 190 amino acids The encoded protein is a stable protein,and the hydrophilic part is larger than the hydrophobic part. It contains four EF-hand structures. The secondary structure of CsCMLl is predominantly alpha-helix, followed by random curl. Amino acid homology analysis showed that cucumber CsCML1 and CmCML32 were homologous and homologous to 95%.2. The conclusion had got from qRT-PCR that CsCML1 gene have expression in roots, stems and leaves of ’Xintai Mici’ Cucumis sativus, expressed in root in the biggest, followed by the stem and leaf. With the treatment of 75 mmol·L-1 NaCl, the expression of CsCMLl gene in roots,stems and leaves was up-regulated to some extent, and the expression level of root and leaf was the highest at 48 hours, which was 0.84 and 2.62 times of that of CK, respectively, The highest expression of stem in 24 hours, 2.15 times the control. Therefore, salt stress can induce the expression of CsCML1 gene in cucumber, and the induction of CsCMLl gene by salt stress.3. The target gene and the plant expression vector pCambia2301 were digested and ligated into E.coli competent cell-DH5a by heat shock method. The expression vector of pCambia2301-CsCMLl was obtained.4. Transferred the expression vector into agrobacterium (GV3101) by freeze-thawing, and the CsCML1 gene was transformed into Arabidopsis thaliana by agrobacterium tumefaciens-mediated transformation and the transgenic plants were obtained by PCR.5. The expression level of CsCCMLl gene in transgenic Arabidopsis was detected by qRT-PCR,and the overexpression of CsCML1 -3 was identified.6. The salt tolerance analysis of overexpressed lines showed that:The germination rate of transgenic Arabidopsis thaliana seeds under salt treatment was lower than that of wild type, and the differences were significant with the salt concentration increasing. With the increase of salt concentration, the root length of transgenic Arabidopsis plants was also inhibited, which was lower than wild type. Arabidopsis thaliana of four weeks seedling age was treated with salt stress for tow weeks, and the contents of fresh weight and dry weight ,chlorophyll, malondialdehyde and proline were determined. The results showed that fresh weight, dry weight and chlorophyll content of transgenic plants was lower than that of wild type plants under salt stress, but the content of soluble protein was not significantly different. The MDA content of transgenic plants increased with increasing concentration of salt treatment while the change of MDA content in wild type plants was not significant with salt concentration. The content of proline in transgenic plants was higher than that in wild type under normal growth condition and salt stress.Therefore, we believe that the transfer of CsCMLl gene to Arabidopsis plants is more sensitive to salt stress, reducing the salt tolerance of plants.