Cloning And Functional Research Of A SlMADS23-like Gene In Solanum Lycopersicum

As the Solanaceae annual herbaceous plants, tomato is an important crop in fresh fruits,vegetables and processing industries. Due to its short growth cycle, easy to observe the phenotype and relatively small genome conserved genomic structure, tomato is considered as a representative species of solanaceae fruits.MADS-box is a kind of important transcription factor, and was found expressed in different parts of organism and in different developmental stages. The myocardial development of animal; the pheromone response in fungi; regulate development of floral organs, flowering time, mature and fall off, fruit root, stem, leaf development in plants, and also involved in the resistance of plants. By NCBI database in the study of gene screening, we isolated and cloned tomato gene:SlMADS23-like. The development of biological information and tissue specificity expression of SlMADS23-like were analyzed. The silence of transgenic tomato cultivation, the main research results obtained and content are as follows:1, bioinformatics analysis showed that the gene ORF was 720 bp, encoded 239 amino acids, the molecular weight is 27.596 kD, the average value of the isoelectric point of the protein hydrophobic 9.84. SlMADS23-like(GRAVY):-0.804(aliphatic index) lipid solubility index was 86.86, showed that the SlMADS23-like protein is a hydrophilic lipid soluble protein. It was designated as unstable protein due to the instability coefficient forecast of 55.51. Two protein secondary structure prediction of 52.30% alpha helix, 6.28% beta folding, random coil and 25.10% extension chain 16.32%. Subcellular localization prediction does belong to the SlMADS23-like transmembrane protein.2, We set housekeeping genes SlCAC(accession number: SGN-U314153) as the reference, the expression pattern of SlMADS23-like was detected by real-time fluorescent quantitative PCR method. The results showed that the gene expressed in all tissues of tomato, and the expression was high in young leaves, mature leaves and fruit period(IMG, MG). The expression of fruit ripening is relatively low, and showed a decreased trend.3, in order to reveal the biological function of the gene, we constructed the silenced vector of SlMADS23-like gene by using Agrobacterium mediated method to its success into the wild type tomato, and then used the detection of NPT II positive strains and real-time quantitative RT-PCR technology, obtained 3 silenced-lines whose silence rate reached more than 90%.4, the phenotype of transgenic lines: mature leaves(ML) and the green fruit(IMG?MG) of transgenic lines were much lighter compared to the wild type. In order to confirm the observed phenotype, chlorophyll of mature leaves(ML) and green fruit(IMG?MG) were detected, and the results show that the chlorophyll accumulation of transgenic lines were much lower than the wild type. The detection of chlorophyll related genes(DCL, GLK1, GLK2) in the molecular level show that the expression of these three genes were down regulated significantly.5, Abiotic stress(salt treatment, dehydration treatment, high and low temperature treatment): the salt treatment, the expression of SlMADS23-like was low in root, but higher in the leaves; in high temperature treatment, the expression of Sl MADS23-like reached the maximum at 12 h, while in low temperature treatment, the expression of SlMADS23-like reached the maximum at 24h; in the process of dehydration, the expression of SlMADS23-like in reached the maximum at 4h and has a small decline in 8h, then up regulated from 8 to 24 h. That is to say, in addition to the expression of SlMADS23-like in leaves of salt treatment, was all changed in other abiotic stress, and the changes in leaves of salt and cold treatment were particularly obvious. On the seedling germination sensitivity experiments, transgenic gene lines were more sensitive to ABA than wild type and the result was opposite in salt treatment. The results showed that SlMADS23-like transgenic lines may exist resistance.6,The salt stress and cold treatment were carried out in two transgenic lines with the efficiency of 90%. We observed RNAi3-3, RNAi4-1 showed more resistance to salt, cold in transgenic lines, To further validate the phenotype, salt stress related genes(PR1, PR5, Cat1, Cat2, ER5, TAS14) and cold treatment related gene(Cat2, CBF1 and Cor) were detected. The results show that, the expression of salt stress related genes(PR1, PR5, Cat1, Cat2, ER5, TAS14) showed varying degrees of increase, while expression of cold treatment of genes(Cat2, CBF1 and Cor) showed different degrees of reduction.In summary, this study explored the function and molecular mechanism of tomato SlMADS23-like gene in tomato, SlMADS23-like gene may be regulate the Chlorophyll biosynthesis in tomato, and analysis function and mechanism of SlMADS23-like in abiotic stress, these all have laid a foundation for the comprehensive elucidation of the function and mechanism of SlMADS23-like gene in plants and the possible application in gene engineering in the future.