| Reactive oxygen species (ROS), including hydrogen peroxide, singlet oxygen, superoxide anionand reactive hydroxyl radicals and et al., is a kind of oxygen-containing groups which possesoxidation activity or partially be reduced, there will be accumulated quantity of ROS in plant cellswith the invasion of pathogenic microorganism. If these ROS can not be scavenged efficiently, theplant cells should be injured and even programmed cell death was induced during its growth. In thepathogen-plant ecosystem, an enzymes promoting scavenge system, was formed, which caneffectively scavenge ROS including superoxide dismutase (SOD), ascorbate peroxidase (APX),catalase (CAT) and peroxidase (POD) in the process of incompatible interaction. Relying on theenzyme promoting scavenge system, plant can defend against the damage of ROS which wereinduced by pathogen infection.Sugarcane is the most important sugar crop in China. Sugarcane smut caused by Ustilagoscitaminea Syd. has become one of the most serious disease in the sugarcane planting areas whichreduces the output of sugarcane significantly. In sugarcane-smut interaction system, the research onthe key enzyme genes related to ROS metabolism has theoretical significances and practical values,which should be help in revealing the smut resistance mechanism of sugarcane, exploring theresistance genes, developing the technology of molecular marker-assisted selection and establishingthe new technology of disease resistance identification. At present, the research on the CAT and APXgenes, which involved in the ROS metabolic pathways during this interaction system, has notreported.Three genes, namely SoAPX, SoCAT1and SoCAT2, were obtained by in silico cloning andsplicing. Then these genes were analyzed and the corresponding nucleotide sequences, relativemolecular weights, pH, pro/hydrophobics, trans-membrane structures, sub-cellular localization,primary and secondary structures and3D-structures of moleculars of them were predicted bybioinformatics tools.Ya05-179is a smut resistance sugarcane genotype which possessed the kinship of Erianthusarundinaceum., RT-PCR amplification was conducted to isolate the above three genes by Using the buds of Ya05-179as plant materials. And then, buds of sugarcane genotypes of Ya05-179withhighly smut resistance and Liucheng03-182with highly smut susceptibility were occupied for testthe gene expressions by qRT-PCR and the enzyme activities of SoAPX and SoCAT1and SoCAT2.Besides, the expression and the ability of stress response of SoCAT1in prokaryotic expressionsystem were investigated. The main results and conclusions are as follows:1. Cloning and expression analysis of SoAPX gene in sugarcaneThe cDNA sequence of APX gene from sugarcane was cloned by in silico cloning. Thesequence is905bp in length, containing an ORF of498bp, which encodes a polypeptide of165amino acid residues. According to the prediction, SoAPX, a hydrophilic protein withouttrans-membrane structure which is located in the mitochondria,. The protein has a relatively highhomology of up to99%when aligned with the amino acid sequence of the corresponding genesfrom sorghum (gi|242075852|). Thus,it is indicated that SoAPX protein was highly conserved in therelative plant species.The target gene fragment of663bp was obtained by RT-PCR and it belonged to the cDNAsequence of SoAPX based on sequencing. Under the stress of U. scitaminea Syd., the APX enzymeactivity in buds in smut resistant genotype of ya05-179is much higher than that in smut susceptiblegenotype. After the pathogen infection, the enzyme activity of both genotypes was upregulated andtwo peaks were found at the time points of12h and48h, respectively. Specially, for both genotypes,the maximum APX enzyme activity appeared at the time point of48h. In the resistance genotype,two peaks of the enzyme activities were3.81times and9.15times than that of the control at thebeginning of infection. However, in the susceptible genotype, two peaks of the enzyme activitieswere only3.42times and6.08times that of the control at the beginning. Simultaneously, theexpressions of SoAPX gene in two different genotypes was analyzed by real-time quantitative PCRafter infection of U. scitaminea, In buds of sugarcane stressed by the pathogen infection, theexpression patterns of SoAPX gene in both sugarcane genotypes appeared to be similar ie."rising (0h-12h)-decreasing (12h-24h)-rising (24h-48h)–decreasing (48h-72h)-rising (72h-96h)".In the resistance genotype, the relative expression of SoAPX at the time point of48h were3.8timesthat of the control at the time point of0h. However, in the susceptible genotype, this value was only2.2times that of the control at the same compare. The expression of SoAPX in the resistant genotypeis higher than that in the susceptible genotye. It showed that the expression of SoAPX gene was positively correlated with the disease-resistance of sugarcane, indicating that the expression of thisgene was beneficial for sugarcane resistance to the pathogen infection.2. Cloning and expression analysis of SoCAT1in sugarcaneBased on in silico cloning, a novel sugarcane gene, termed as SoCAT1, was identified. ThecDNA sequence of this gene is2,046bp in length, contains a complete ORF of1,479bp and itencodes a492aa polypeptide. The transcript of the gene, which is located in the cytoplasm, is ahydrophilic protein without the transmembrane domains. SoCAT1protein has the highest similarity,98%, with that of the corresponding gene in Sorghum bicolor, which indicates that SoCAT1ishighly conserved in relative species.A1,658bp target gene was obtained by RT-PCR, which was found to be in according with thecDNA sequence by in silico cloning through sequencing and alignment. During the time period of0h-96h after the stress of the sugarcane smut fungus, the variation trend of SoCAT enzymaticactivity in the resistant sugarcane genotype presented "rising (0h-6h)-decreasing (6h-12h)-rising(12h-24h)-decreasing (24h-96h)". At the time point of6h, the first expression peak appeared,while at the time point of24h, the maximum expression appeared, which was4.3times that of thecontrol. The variation trend of SoCAT enzymatic activity in the susceptible sugarcane genotypepresented "decreasing (0h-6h)-rising (6h-12h)-decreasing (12h-24h)-rising (24h-48h)-decreasing (48h-96h)", and the maximum expression appeared at the time point of48h.Real-time quantitative PCR analysis showed that the gene expression of SoCAT1in theresistant and susceptible genotypes had the same variation trend with that of the SoCAT enzymaticactivity. The maximum expression appeared at the time point of24h in the resistant genotype,which was3.3times that of the control at the time point of0h, while the maximum expressionappeared at the time point of48h in the susceptible genotype. The maximum expression appeared atthe time point of24h in the resistant genotype, which was1.5times that of the control at the timepoint of0h. SoCAT1expressed a57kDa protein in E. coli BL21, of which the molecular weightwas similar to that of the predicted protein. However, the expression of the SoCAT1wasunfavorable for the growth of the recombinant bacteria, because the non-recombinant bacteria grewmuch faster in15%PEG stress.Thus, it suggested that the SoCAT1was not response to abioticdrought stress (PEG) though the expression of the SoCAT1was beneficial to smut resistance insugarcane. 3. Cloning and expression analysis of SoCAT2gene in sugarcaneThe cDNA sequence of another CAT gene from sugarcane was cloned by in silico cloning. Thesequence is2,009bp in length, containing a complete ORF of1,482bp, which encodes apolypeptide of493aa residues. According to the prediction, SoCAT2, which is located in thecytoplasm, is a hydrophilic protein without trans-membrane structure. The protein has a highlyhomology up to97%when aligns with the amino acid sequence of CAT from sorghum(gi|242063776|). The target gene fragment of1,547bp was obtained by RT-PCR amplification and itbelongs to the cDNA of SoCAT2gene based on the sequencing data. The expression analysis ofthe SoCAT2gene in buds in two sugarcane genotypes infected by U. scitaminea was conducted byreal-time quantitative PCR. The expression patterns of SoCAT2gene in disease-resistant genotypeappeared to be "rising (0h-6h)-decreasing (6h-12h)-rising (12h-24h)-decreasing (24h-72h)-rising (72h-96h)". The maximum expression value, which was3.9times that of the control atthe beginning of infection, appeared at the time point of12h. However, in susceptible genotype, theexpression patterns of SoCAT2appeared to be "decreasing (0h-6h)-rising (6h-12h)-decreasing(12h-24h)-rising (24h-48h)-decreasing (48h-96h). The maximum expression value, which was1.8times that of the control at the beginning of infection, appeared at the time point of48h. Inaddition, the expression level of SoCAT2gene in resistance genotype is higher than that insusceptible genotype at all the time points. It suggested that the expression of SoCAT2increased thesugarcane smut resistance. |
PDF Full Text Request