Study On Cloning And Expression Of Copper/zinc Superoxide Dismutase Gene In Upland Cotton

The problem of earliness with decrepitude has worse effect on the yield in the short-season cotton in our country. Up to now ,about the problem of earliness with decrepitude in short-season cotton .careful research has been done on the physiological and biochemical level, the results of the formers: comparing the varieties of earliness with not-decrepitude with the varieties of earliness with decrepitude .their antioxidant enzyme activity are higher and steadier in the growth retrial period, the key enzymes are SOD. if we want to understand the molecular mechanism about earliness with decrepitude, it is very important of cloning the correlative genes. These genes can help us to improve the yield and quality.(1) the material CRI36 is choused because that is earliness but not decrepitude and has very widest planting acreage. First the leaves were treated with hurting and high temperature, then total RNA was extracted and obtained cDNA by RT-PCR, two Cu/Zn SOD genes were cloned by using RACE technology from cDNA of cotton leaves which are possessed of the biggest proportion in all the SOD(86%). these genes have two kinds of types: chloroplast Cu/Zn SOD gene and cytoplast Cu/Zn SOD gene, the accession number of chloroplast Cu/Zn SOD gene is DQ120514, the length of gene sequence is 1043bp,includes a ORF 645bp, which start from 98 to 745, coding 215 amino acids, there are two copies in the cotton genome, comparing cotton chloroplast Cu/Zn SOD protein with others, there are about homologous 65.7%-81% in the amino acid ingredients. Molecular weight of cotton chloroplast Cu/Zn SOD protein is 25.0KD, Theoretical pI 6.11 there are two active centers of enzyme, the protein maybe locate in the chloroplast cavity, maybe like a goblin.the accession number of cytoplast Cu/Zn SOD gene is DQ445093, the length of gene sequence is 682bp,includes a ORF 456bp, which start from 120 to 576, coding 152 amino acids, there are one copy in the cotton genome, comparing cotton cytoplast Cu/Zn SOD protein with others, there are about homologous 81%~87% in the amino acid ingredients. Molecular weight of cotton cytoplast Cu/Zn SOD protein is 15.03KD, Theoretical pI 6.09.there are two active centers of enzyme, the protein maybe locate in the cell liquid, should be a goblin.(2)The expression of chloroplast Cu/Zn SOD gene was determined using northern blotting analysis. The material include different tissue organ (root, flower, leaf stem and hypocotyls) and different development stages(seeding/beginning flower/open flower/fruiting period and bell opening period ).the chloroplast Cu/Zn SOD gene mRNA levels were high in the young leaves but below in the limit of detection in young stem and flower;the expression was different in each stage, the mRNA were extracted from different stage materials;the chloroplast Cu/Zn SOD gene mRNA levels were lower in the seeding period, then piece by piece hoisted, the highest level at beginning open flower then went down and the prophase levels are higher than the anaphase.For the cytosol Cu/Zn SOD gene mRNA levels , there are the highest levels at open flower , and the the anaphase levels are higher than the prophase . in the different tissue organs , there are the highestin the root.(3)The expression and detection of chloroplast Cu/Zn SOD gene was done in the Escherichia coli: a pair of premiers was designed in the light of the start codon and stop codon sites, the full-length gene was amplified by using cDNA template. , then , to construct middle-recombine plasmid:pUC-l-SOD;next to construct expression vector pET—Cu/Zn-SOD, the Escherichia coli was induced by IPTG, results showed enzyme activity of the translated bacteria had increased 17% than the comparison ,the expression protein had enzyme activity.(4) the plant translation vector of the chloroplast Cu/Zn SOD gene was built, a pair of premiers was designed in the light of the start codon and stop codon sites, the full-length gene was amplified by using cDNA template., then, to construct middle-recombine plasmid:pUC-2-SOD, next to construct plant expression vector pBIY-SOD, the results was testified by two enzyme cutting and PCR amplification.