甘蔗Cu/Zn-SOD的克隆和表达分析

【目的】克隆甘蔗铜/锌超氧化物歧化酶（Cu/Zn-SOD）基因，并分析其序列特征、组织特异性表达及在不同逆境胁迫下的表达模式。【方法】以甘蔗桂糖28号为材料，利用RT-PCR技术克隆获得Cu/Zn-SOD，采用生物信息学方法分析所推测的氨基酸序列，通过实时荧光定量PCR研究Cu/Zn-SOD在不同组织及逆境条件下的表达情况。【结果】克隆获得甘蔗Cu/Zn-SOD，NCBI登录号为JQ958328，其开放阅读框为456 bp，编码151个氨基酸，与禾本科植物聚于同一进化分支。实时荧光定量PCR分析表明Cu/Zn-SOD在根、茎、叶中均有表达，为组成型表达，在叶中表达量最高；Cu/Zn-SOD在聚乙二醇（PEG）、NaCl、低温（4℃）和H2O2四种非生物胁迫下均诱导表达，表达模式因调控机制的不同而异。【结论】克隆获得Cu/Zn-SOD，其主要在甘蔗绿色组织中表达，其在铜/锌SOD超氧化物歧化酶的功能区域保守型很高，可能与甘蔗抵御渗透胁迫相关。

Molecular Cloning of Sugarcane Cu/Zn Superoxide Dismutase (Cu/Zn-SOD) and Its Expression Analysis

【Objective】The aim of this study was to clone the full-length cDNA of a key enzyme gene Cu/Zn-SOD related to superoxide anion removal in sugarcane (Saccharum spp.), investigate its sequence characteristics and analyze its expression in different organs and under different stresses. 【Method】 The Cu/Zn-SOD gene cDNA sequence was cloned from the leaves of sugarcane variety GT28 using RT-PCR techniques. The bioinformatics methods were used to analyze the putative amino acid sequence, and real-time fluorescence quantitative PCR (qRT-PCR) method was used to study the expression of Cu/Zn-SOD gene in different tissues and under different stresses. 【Result】 The full-length cDNA of Cu/Zn-SOD (GenBank accession number: JQ958328) in sugarcane was cloned. The sequence included an open reading frame of 456 bp, encoding a polypeptide of 151 amino acids, which was clustered in the same phylogenetic branch of the Cu/Zn-SODs of gramineae. Real-time fluorescence quantitative PCR results showed that the Cu/Zn-SOD expressed in root, stem and leaf of sugarcane plant, belonging to constitutive expression, but showing the highest in leaf. The Cu/Zn-SOD expression could be induced under the treatments of polyethylene glycol (PEG), H2O2, low temperature and NaCl stresses, but the expression models were different depending on the regulation mechanisms. 【Conclusion】 The gene Cu/Zn-SOD was cloned, and it mainly expressed in the green tissue in sugarcane plant. Its copper- zinc superoxide dismutase functional areas are highly conservative, and is possibly associated with osmotic stress resistance of sugarcane.