| Rhomboid protein family is found in the recent years. We have found the homologue of rhomboid protein in archaea, bacteria, yeast, fruit flies and mammals, including humans, even plants and other organisms. Although the function of most rhomboids is not yet known, they have already been implicated in growth factor signaling, mitochondrial function, host cell invasion by apicomplexan parasites, and protein translocation across membranes in bacteria. By using the mechanism of cellular membrane trafficking, rhomboids have been evolved novel strategies to regulate the expression of proteolysis rhomboid family protein. The expression of rhomboid family genes have been regulated, and the catalytic substrate is specific, which may suggest that these proteins play a role in some important organizations, including signal transduction, development, apoptosis, it is able to cut a single transmembrane protein and transmembrane domain of serine proteases. Go through the hydrolysis of membrane proteins to activate many signaling proteins, or into the poor solubility of amyloid peptide fragments, so the study of this family of proteins is very important. Unfortunately, we still do not have a very clear understanding of its function. And the rhomboid family in plant is rarely known in the current.The Chimonanthus praecox (L.) Link is native to China, and it is tertiary relict plant and two rare endangered plants in China. Chimonanthus praecox deciduous shrub up to4-5meters high. The Chimonanthus praecox blooms in the cold winter, its flowers are yellow and like wax, the aroma of tangy, it is a typical plant which blooms in winter. It possesses many advantages, such as resistance of cold, drought, and secateurs. Among them, its main characteristic is chilling tolerance. As long as the temperature isn’t below-15℃, it can safely live through the winter. They are ornamental flowers in winter. After processing, the flowers of Chimonanthus praecox can be valuable medicinal herbs, they have Detoxification and fluid effect. This paper describes that the author Cloned the gene of rhomboid family from the organs of the flowers of Chimonanthus praecox, and named CpRBL.then the author build a plant carrier and it is expressed in tobacco. Next, the author discussed the functional analysis of transgenic tobacco, and achieved major results are the following:1. Cloning and Bio-informatics Analysis of CpRBLWe got EST sequence of1347bp by randomly selecting from Chimonanthus praecox cDNA library, which contains the largest open reading frame of993bp and encodes330amino acids. The BLASTX results show that it has a high homology with other rhomboid proteins, and the highest homology of,96%with Arabidopsis rhomboid-like protein10. Then, we initially conclude that the obtained cDNA encodes rhomboid protein and named CpRBL. Bio-informatics analysis showed that the protein secondary structure of the gene has no signal peptide sequence. At the same time, we found that it contains the transmembrane structure domain by the hydrophobicity analysis and the transmembrane structure prediction.2. Expression analysis of CpRBL geneExpression of CpRBL in the leaves, outer flap, flap, inner flap and stamens is very high and almost flat. This gene is not expressed in the root, the expression of CpRBL in the stem and pistil is rare. The expression levels of CpRBL in flower bud period and bud stage is the least. The expression has increased substantially in flap period, then the expression level was gradually reduced in the early opening period and the bloom period, and finally expression levels are highest in decline period. These results showed that the CpRBL gene may regulate the development of leaves and flowers. After PEG, high-salt treatment in different time periods (0.25h,1h,6h,12h), the Chimonanthus praecox seedlings compared with the untreated plants, it showed that the gene down regulated. It is reasoned that the gene’s Function greatly associated with plant resistance in the Chimonanthus praecox.3. Construction of Plant Expression VectorThe largest ORF fragment of CpRBL gene was connected to the pMD-19T vector, then Xba Ⅰ and SadⅠ restriction enzyme digested the pMD-19T-CpRBLvector and plant expression vector of pCAMBIA2301g, and we retrieved target fragments and pCAMBIA2301g vector. After the double digestion,the single digestion and PCR validation, we successfully constructed plant expression vector containing the gene.4. Resistance analysis of transgenic tobaccosWith agrobacterium-mediated transformation, we got transgenic tobacco plants by resistance screening, GUS staining, the PCR amplification detection of target gene and fluorescence quantitative analysis at the transcriptional level. According to CpRBL expression analysis in the Chimonanthus praecox, we chose PEG, high-salt treatment both on transgenic tobacco and wild tobacco in different time periods. Then we detected plant physiological indicators. The results showed that the drought tolerance in transgenic tobacco significantly weaker than the wild tobaccos. We speculated that the expression of the gene may be down regulated, and involved in plant drought-tolerant adjustment. At the same time, the analysis of the MAPKK gene expression in the wild and transgenic tobacco plants showed that CpRBL may regulate MAPKK gene expression and thus involved in drought resistance adjustment. |
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