Cloning And Functional Analysis Of Copper/Zinc Superoxide Dismutase Gene From Sugarcane

The relationships of copper/zinc superoxide dismutase(Cu/Zn-SOD) expression and plant resistance to adverse environment stresses have been confirmed, however, no report on cloning and expression analysis about sugarcane Cu/Zn-SOD has been found yet. In the present study, Cu/Zn-SOD gene was cloned from sugarcane. And its sequence features, tissue expression differences and expression patterns under different abiotic stresses were analyzed as well. The prokaryotic expression vector was constructed and the gene expressed in E. coli successfully. The sense and antisense plant expression vectors were built, and transformated into tobacco by Agrobacterium mediated leaf disk and transgenic tobacco plants were obtained. This laid the foundation for further study on the relationship between biological function of the gene and stresses. The main results are as follows:1. According to the NCBI database,the nucleotide sequence of Gramineae which has high homology with sugarcane was selected to design the upstream degeneration primers of the gene. The cDNA gotten from reverse transcription of the leaf total RNA of sugarcane variety GT28was used as the template,3SIDE as downstream primer, the full length of the Cu/Zn-SOD gene was cloned through PCR amplification, which was710bp in size, contained the codons of promoter ATG and terminator GGC, and registered in GenBank with accession number JQ958328. Bioinformatics analysis showed that the relative molecular weight of Cu/Zn-SOD protein was15.1kDa, and the isoelectric point was5.71. The copper-zinc superoxide dismutase function region of the gene was highly conservative. The phylogenetic tree analysis revealed that amino acid sequence of this gene was located in the same evolutionary branch with Gramineae.2. Based on the sequence of the cloned Cu/Zn-SOD gene, the primers for real-time fluorescence quantitative PCR (qRT-PCR) analyses were designed, and the tissue differential expressions were analyzed by using the cDNAs from root, stem and leaf of sugarcane variety GT28as template, and the results showed that the gene expressed most in the leaves, followed by the stems, and rarely in the roots. To study the relationship between the gene and stress, the gene expressions also detected under four exogenous stresses including polyethylene glycol (PEG6000), NaCl, low temperature (4℃) and H2O2, and the results showed that the four exogenous stresses could induce the expression of the gene, but the expression patterns were different. It was speculated that the gene is related with sugarcane resistance to osmotic stress.3. The prokaryotic expression vector of Cu/Zn-SOD gene was constructed in this study. Under the induction of1.0mmol.L-1IPTG, a fusion protein strip with molecular weight of about22.0kDa was detected, and it is basically consistent with the expected size, which suggested that the prokaryotic expression vector had been constructed successfully, and had no wrong code or shift of amino acids. This laid the foundation for further studying the function of the gene and preparation of the antibodies.4. The antisense plant expression vector of Cu/Zn-SOD gene was constructed, which has been verified through digestion and sequencing analyses, transferred into tobacco by using Agrobacterium-mediated leaf disc, and transgenic plants were obtained and confirmed by PCR test. This laid the foundation for studying the relationship between the gene and adverse environments, and for resistant breeding of sugarcane.