小麦光温敏雄性不育系BS366铜锌超氧化物歧化酶基因的克隆及表达分析

超氧化物歧化酶(SOD,superoxide dismutase)是植物中一种主要的抗氧化酶,在植物应对逆境胁迫及抗衰老中起重要作用。本研究从基因芯片数据中筛选获得小麦Cu/Zn-SOD基因的EST序列,通过序列比对后拼接得到小麦Cu/Zn-SOD的候选基因,利用PCR技术在小麦光温敏雄性不育材料BS366中克隆并获得该基因。通过对Cu/Zn-SOD基因序列进行生物信息学分析,结果表明,该基因拥有连续且完整的开放阅读框,长495bp,编码164个氨基酸。氨基酸序列分析发现,该蛋白具有保守的Cu/Zn-SOD功能结构域与典型的Cu/Zn-SOD三维结构,且定位于细胞质中。通过同源进化分析表明,该蛋白与二穗短柄草(Brachypodium distachyon(L.)Beauv.)和大麦(Hordeum vulgare L.)的Cu/Zn-SOD蛋白亲缘关系较近,相似度分别为89%和94%。利用实时荧光定量PCR技术对其在小麦不同组织的表达特异性及不同逆境胁迫下的表达模式进行分析,结果表明,该基因在根、茎、叶、雌蕊、雄蕊、颖壳中均有表达,属于组成型表达,且在小麦的地上部含叶绿体的组织中含量较高;同时受多种胁迫诱导,可能参与了多种胁迫诱导调控途径。通过对该基因在不同育性环境中BS366育性转换期花药中的表达模式分析,发现可育环境下,在小孢子母细胞时期和减数分裂期的表达量分别约为对照的8倍与16倍;而不育环境下,该基因表达水平无明显变化。因而推测,小麦Cu/Zn-SOD基因可能参与了光温敏雄性不育系BS366的育性调控。本研究为深入研究Cu/Zn-SOD基因在小麦中的作用机理奠定了重要基础。

Cloning and Expression Analysis of a Cupro-Zinc Superoxide Dismutase Gene from Wheat Photo-thermosensitive Genic Male Sterile Line BS366

Superoxide dismutase (SOD) is a major antioxidant enzymes, which is important for plants to cope with adversity stress and anti-aging effect. A cDNA of Cu/Zn-SOD gene defining by the EST sequence assembly from Microarray data, was cloned from the photo-thermosensitive male sterile wheat (PTGMS) line BS366. The length of ORF sequence is 495bp, which encoded a 164-amino-acid polypeptide. Protein sequence contained a typical Cu/Zn-SOD gene family functional domain and 3 dimensional structures. Besides, the protein was located in the cytoplasm by ubcellular localization analysis. Evolution and clustering analysis showed that the Cu/Zn-SOD protein of wheat shared 89% and 94% sequence similarities with the Cu/Zn-SOD protein of Brachypodium distachyon and Hordeum vulgare, respectively. The methods of quantitative real-time PCR (qRT-PCR) were utilized to analyze the expression patterns in different wheat tissues and abiotic stresses. It was showed that the Cu/Zn-SOD gene expressed in root, stem, leaf, pistil, stamen and glume, belonging to constitutive expression. The highest expression level was found in chlorenchyma such as leaf, stem and glume. In addition, the gene expression was induced by a variety of stressesrevealing that the Cu/Zn-SOD gene of wheat might involve in multiple stress regulation pathway. To illuminate the regulation of Cu/Zn-SOD in wheat fertility, expression profile of Cu/Zn-SOD in PTGMS line BS366 pollen during fertility transformation was performed. The results showed that the relative expression of Cu/Zn-SOD at microsporocyte stage and meiosis stage in fertile environment were 8 times and 16 times higher than control, respectively, while there was no significant changes in sterility environment, suggesting that Cu/Zn-SOD might be involved in the regulation of fertility transformation of wheat PTGMS line BS366. This information lays a foundation for further functional study of Cu/Zn-SOD in wheat.