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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。
Superoxide dismutase (SOD), a major antioxidant enzyme in plants, plays an important role in plant response to stress and anti-aging. In this study, EST sequences of wheat Cu / Zn-SOD gene were screened from gene chip data. The candidate genes of Cu / Zn-SOD wheat were spliced by sequence alignment. The PCR products were used to identify the wheat light-sensitive genic male sterile material BS366 And cloned and obtained the gene. The bioinformatics analysis of Cu / Zn-SOD gene sequence showed that the gene has a continuous and complete open reading frame, which is 495 bp in length and encodes 164 amino acids. Amino acid sequence analysis showed that the protein has a conserved Cu / Zn-SOD functional domain and a typical three-dimensional structure of Cu / Zn-SOD, and localized in the cytoplasm. Phylogenetic analysis showed that the protein was closely related to the Cu / Zn-SOD protein of Brachypodium distachyon (L.) Beauv. And Hordeum vulgare L., and the similarities were 89 % And 94%. Using real-time fluorescence quantitative PCR, we analyzed the expression patterns in different tissues of wheat and under different stress conditions. The results showed that the gene was expressed in roots, stems, leaves, pistils, stamens and glumes, Which belongs to the constitutive expression and has a high content in the chloroplast-containing tissues of the aerial part of the wheat; it is also induced by a variety of stresses at the same time and may be involved in various stress-inducing regulatory pathways. Through the analysis of the expression pattern of anther in BS366 fertility-transformable stage in different fertility environments, we found that the expression level of anther in the fertile environment at the microspore mother cell stage and meiosis stage were about 8 times of the control 16 times; while under sterile conditions, the gene expression level did not change significantly. Therefore, it is speculated that the Cu / Zn-SOD gene in wheat may be involved in the fertility regulation of the photo-thermo-sensitive male sterile line BS366. This study laid an important foundation for further study on the mechanism of action of Cu / Zn-SOD gene in wheat.