| Heat shock protein (HSP) is one of stress protein, which induced in plant to adapt itself to variety of environmental stresses including elevated temperatures, chilling temperatures, salinity, aridity, deprived of nutrition and heavy metal ions, etc. Under adverse conditions, heat shock proteins play a role as a molecular chaperone promote the stabling, assembling, refolding of other proteins, intracellular transport and degradation, promote repairing of damaged proteins and cell survival, and thus plays a very important role in improving abiotic stress resistance of plants. Base on the size of the molecular weight, heat shock protein family inplants are divided into five categories:HSP100, HSP90, HSP70, HSP60, sHSP (small HSP). Heat shock protein 70 (HSP70) is an important member of heat shock protein family, is widely distributed in various parts of the cell: cytoplasm, endoplasmic reticulum, mitochondria, chloroplasts,etc, playsimportant physiological functions under all normal circumstances and stress conditions.Wintersweet [Chimonanthus praecox (L.) Link] is a unique and precious flower in China, blossoming in winter, with strongly fragrance, and it is the best of cut flowers in the winter. Wintersweet widely used in ornamental aspects, its medicinal value and food value has also been developed and utilized. In recent years, the researchesof molecular biology inwintersweet attracted widespread attention. But the study of plant protein HSP70 gene family is still limited, we obtained a newheat shock protein gene from wintersweet and studied its function. This work will help to clarify the function of plant protein HSP70 family, and also help to explore the stress resistance mechanism in wintersweet under stress conditions.In this thesis, based on transcriptomic database of wintersweet in our lab, we screened two EST sequences of HSP70 gene family, named E-CpHSP70-l and E-CpHSP70-2, through the expression analysis of two genes, we selected E-CpHSP70-1gene for further study. RACE technology is used for cloning E-CpHSP70-1 sequence, and obtained the gene full-length sequence, and then carry out analyze its bioinformatics, subcellular localization and its ectopic expression both in tobacco and Arabidopsis. The main results are as follows:1. Characteristic gene expression analysis of E-CpHSP70-1 and E-CpHSP70-2Real-time quantitative fluorescence PCR (qf-PCR) were applied to analyze the expression patterns of E-CpHSP70-1 and E-CpHSP70-2 in different tissues, different stages in the growth cycle of a flower and under different temperature stress, we found that there are different spatio-temporal gene expressions of E-CpHSP70-1 and E-CpHSP70-2. The expression of E-CpHSP70-2 geneis significant difference in each tissue, and the gene expression in floral tissues was significantly higher than that in vegetative organs, and the highest expression level is detected in the outer petals blooming period. While E-CpHSP70-1 gene expressed in all tissues and organs, especially in mature leaveswith the highest gene expression, and high abundance expression was found in sprouting period and petal baring period of flower, so we speculated that this gene may be involved in the regulation of developmental processes of plant leafs and flowers. Compared with the control, E-CpHSP70-1 gene at stress of 4℃ and 1h after treatment have a higher expression level, at stress of 42℃ and 0.25h after treatment have rapid up-regulation expression, and the highest expression level was found at 1h after treatment. So it is illustrated that E-CpHSP70-1 gene may be involved in the high and low temperature resistance regulation process of Chimonanthus praecox.E-CpHSP70-2 gene has also significantly different expression in various tissues, the expression in floral organs was significantly higher than that in vegetative organs, the highest expression deteceted in the outer petals blooming stage. The expression of E-CpHSP70-2 gene showed no significant difference both in low temperature and high temperature stress condition.2. Cloning and bioinformatical analysis of CpHSP70-1 geneFrom the transcriptome database of Chimonanthus praecoxwe use the RACE technology cloned an HSP70 gene, named CpHSP70-1 (GenBank accesstion KR071130).CpHSP70-1 gene cDNA was 2520bp in full-length, encoding a protein of 653 amino acids, containing TATA Box, CAAAT Box and CAAT Box components, these components combined with heat shock factor HSF express the regulation of heat shock protein gene, in response to different environmental stimuli. The protein encoding of CpHSP70-1 gene is homologous with HSP70 protein in other species, the homologous as high as 96% with the musa acuminata, grapes, lotus, rice, lotus, and cocoa. The amino acid sequence of encoding protein CpHSP70-1 contains three typical HSP70 family signature motifs, two cross helical amino acid sequence, a fingerprint sequence, a cytoplasmic localization characteristic sequence. Clustering results showed that, encoding protein of CpHSP70-1 gene and the nuclear or cytoplasmic localization HSP70 protein are clustered into one branch, speculated that this protein might be an organelle protein. And the protein does not contain a signal peptide, only containing a small transmembrane region, subcellular localization prediction showed that this protein was mainly localized in the nucleus, and the CpHSP70-1 protein may play a role in the nucleus. The two level structure of the CpHSP70-1 protein consists of 40.43% beta sheet,7.81% alpha helix, 31.85% random coil and 19.91% extension chain. This protein has 14 serine phosphorylation sites, 13 threonine phosphorylation sites and 5 tyrosine phosphorylation sites, is a hydrophilic protein, suggesting that this protein after translation may be a variety of modification, and the effect of modification plays an important role in abiotic stress response of plants.3. Subcellular localization analysis of CpHSP70-1pMD19-T/CpHSP70-1 plasmid and pCAMBIA1300 plasmid were cut by the same of two BamHI and SalI restriction endonuclease enzymes, then CpHSP70-1 gene was inserted into pCAMBIA1300 plasmid. We constructed a pCAMBIA1300/CpHSP70-1 recombinant plasmid. pCAMBIA1300/CpHSP70-1 recombinant plasmid was reinserted into E. coli cell. The cutting of two restriction endonuclease enzymes and PCR validation results show that the subcellular localization of the pCAMBIA1300/CpHSP70-1 vector was successfully constructed. Following, pCAMBIA1300/CpHSP70-1 vector was transfered into onion epidermal cells by using gene gun bombardment method, the later expression results proved that CpHSP70-1protein was in the nucleus. These results of structural characteristics, cluster analysis, subcellular prediction of this gene results are all coincidence.4. Construction ofCpHSP70-1 expression vectorBy using two of BamH1 and XbaI restriction enzymes cut and link CpHSP70-1 gene from pMD19-T/CpHSP70-1 to pCAMBIA2301G we received pCAMBIA2301G/CpHSP70-1 recombinant plasmid.5. Transfer CpHSP70-1 gene into genetic transformation system of Arabidopsis thaliana and tobacco plantBy using electrophoresis method, Agrobacterium GV3101 with recombinant plasmid containing the target gene was inserted into tobacco and Arabidopsis cells by leaf disc method and floral-dip methods, after kanamycin (Kan) resistance screening, GUS staining, DNA and RNA identification, we obtained 9 positive transgenic tobacco plants and 9 T3 generation transgenic Arabidopsis.6. Analyze heat tolerance of transgenic plants containing CpHSP70-1 geneWe obtained different expression levels (high, medium, low) of positive transgenic tobacco and Arabidopsis plants,wild-type tobacco and Arabidopsis plants, and treated withhigh temperature stress conditions.The results of the heat resistance in tobacco plants show that, after heat treatment, leaf edges of the wild-type tobacco plants were curled, leaf emerged wilt and yellow phenomenon, while the phenotype of transgenic tobacco plant after treatment has no significant differences compared with its before treatment. And related physiological parameters were measured, results showed that after treatment with high temperature stress, MDA content in wild-type tobacco has an significantly increase than that in transgenic tobacco, and chlorophyll decomposition rate is also significantly faster than the transgenic tobacco plants, whereas proline content and SOD activity of the transgenic tobacco plants were higher than the wild-type tobacco. Results of MDA, proline, SOD and other physiological indicators of transgenic Arabidopsis is consistent with the trends of tobacco; and electrical conductivity rate of transgenic Arabidopsis after high temperature treatment change lower than the wild-type Arabidopsis; in addition, after high temperature treatment, seed germination rate of transgenic Arabidopsis was significantly higher than that of wild-type controls; hypocotyl length is more significant; after high temperature treatment of wild type Arabidopsis, its seedling rate in plate and soil are serious wilt, and the plant survival rate is far lower than that of transgenic Arabidopsis. After high temperature treatment, real time fluorescence quantitative PCR was used to detect the expression of CpHSP70-1 gene in different stems of transgenic plants, we found that with the increase of exposure time, the expression of the gene in the transgenic leafs were all increased. From the obtained results can describe that under high temperature conditions, CpHSP70-1 gene may accumulate in transgenic plants, affecting the synthesis of MDA and proline, and then improve the heat resistance ability in transgenic plants. |
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