Cloning And Functional Analysis Of The Peroxisome Generate Protein Gene CpPEX22from Chimonanthus Praecox

Peroxisome generate protein is a sort of proteide which involves in peroxisome’s biogenesis. Thise kind of protein, taken together, is called peroxin, which encoding gene is written as PEX. Until now, more than30peroxins have been identified. Peroxins involve in peroxidase body split, multiplication, transportion mechanism of peroxidase membrane protein and matrix proteins. Researches showed that PEX22p (A kind of protein, encoded by PEX22gene) is acted as anchor point of PEX4p on the peroxisomal membrane. PEX5p is a receptor protein of positioning signal PTS1in transformational process of peroxisome matrix protein. The synergistic effect of PEX22p and PEX4p can reduce destroying and modifying of PEX5p, and protect PEX5p from hydrolysising by Some Hydrolysis of protein. For the reason given above, PEX5p could participate in the transport of the peroxisomal matrix protein cyclically. Peroxysome, containing a mass of enzymes which are primarily oxidase, catalase and peroxidase, exists extensively in all types of cells in Eucaryon. Hydrogen peroxide is a potent cytotoxic agent which produced in the oxidation-reduction reactions catalyzesd by oxidase. Peroxidase and catalase both exist in peroxysome, thus cells are protected from being hurt. Herefore, PEX22gene contributes to peroxisome biogenesis, antioxidation and anti-aging.The change of peroxisome oxidative metabolism is a manifestation of plant fading. This study is departure to explore the features of peroxisome generate protein gene PEX22of Chimonanthus praecox, to cDNA library of Chimonanthus praecox builded by Sui Shunzhao of our laboratory for the material, cloning this gene randomly, named CpPEX22. And then analyzes the transcriptional expression in different tissues and under stress response. A plant expression vector is constructed and then transformed into tobacco, which provides a chance to analyze the function of the CpPEX22gene. The main results of the research are as follows:1. The bioinformatics analysis of CpPEX22gene and the protein encoded by CpPEX22The full length of CpPEX22cDNA sequence is1207bp, including an804bp open reading frame, which GC-content percentage is44.65%. CpPEX22encodes a peroxin being comprised of268amino acid residue with no intron. The structure characteristics of protein encoded by CpPEX22are analyzed in bioinformatics methods. The secondary structure of the protein encoded by CpPEX22is consist of random coil, alpha helix and extend strand, each accounting for46.82%,41.95%and11.24%respectively, which contains no signal peptide and17phosphorylation sites, including15Serine phosphorylation sites, one thremnine phosphorylation site and one tyrosine phosphorylation site. The protein encoded by CpPEX22gene contains transmembrane structure which is generally positioned in mitochondrial membrane, mitochondrial matrix, nucleus, and chloroplast thylakoid membrane.2. Analyze CpPEX22gene at the transcriptional levelQuantitative real-time PCR is used to compare the mRNA expression. The results reveal that CpPEX22gene expresses to varying degrees in different tissues while leaf tissues show the maximum expression quantity and three rounds of flower organs also have a good performance, especially mesopetalum. The highest peak of CpPEX22expression emerges in the withering period of flower. Under abiotic stress treatment, like high and low temperature, H2O2, ABA and so on, CpPEX22gene expression pattern changes. H2O2and ABA stress treatment induces the expression of CpPEX22. The results of transcriptional analysis reveal that CpPEX22, in all probability, plays a significant role in leaves, withering flower, and the process of abiotic stress response.3. The overexpression of CpPEX22gene in tobaccoThrough Agrobacterium-mediated method, the peroxin gene CpPEX22attaching to plant expression vector, is transformed into tobacco. After resistance selection of kanamycin, GUS assay, PCR analysis among these regenerated tobaccoes and luorescent quatititive PCR,18transgenic plants are obtained. Treated with H2O2stress, transgenic tobaccoes have better antioxidant and anti-aging ability than wild types. So the preliminary judgement suggests that CpPEX22has relations with peroxisome’s biogenesis, antioxidation and anti-aging.