番茄白粉病菌钙调蛋白基因的克隆与分析 摘 要：钙调蛋白是细胞第二信使系统的重要成分，在Ca2+信号系统传导中起着关键作用，调控生理代谢及基因表达，控制细胞正常的生长和发育。本研究拟克隆番茄白粉病菌钙调蛋白基因（Calmodulin, CaM）的序列以及对其进行系统发育分析。首先参考GenBank上已发表真菌的钙调蛋白氨基酸序列的保守区域设计并合成简并引物，然后从番茄白粉病菌中提取gDNA，以此为模板，PCR克隆了番茄白粉病菌钙调蛋白的部分基因并构建至克隆载体，鉴定后测序；最后利用MEGA 3.1软件对所得序列进行系统发育分析。结果表明：不同宿主间的白粉病菌CaM基因同源性较高，真菌的CaM基因具有一定的保守性，但随着物种间的进化其保守性逐渐降低。
关键词：番茄白粉病菌；钙调蛋白；PCR克隆；序列分析
Cloning and Sequence Analysis of Calmodulin Gene from Tomato Powdery Mildew
Abstract: Calmodulin was an important component of the cellular second messenger system, it played a key role in the Ca signal transduction system, regulating physiological metabolism and gene expression, controlling normal growth and development of cells. In this study, cloning of calmodulin (CaM) from tomato powdery mildew and phylogenetic tree analysis were conducted. Firstly, the tomato powdery mildew calmodulin was cloned with degenerate primer, which was designed by referring to the conserved amino acid sequences of fungal calmodulin, which have been published on the GenBank. The calmodulin gene was cloned by the gDNA, which was extracted from the tomato powdery mildew and calmodulin gene was constructed to the cloning vector. After PCR identification of the recombinant vector, the positive clones were sequenced by the Biotechnology company. Finally, phylogenetic tree analysis of the sequence obtained were conducted by MEGA 3.1 software. The results indicated that the homology of CaM gene of powdery mildew between different hosts was high. The CaM of Fungi was conservative, but with the evolution of species, the conservation was gradually reduced.
Key words: Tomato powdery mildew; Calmodulin; PCR clone; Sequence analysis
目 录
摘 要 1
Abstract 1
引言 2
1. 材料与方法 2
1.1 实验材料及试剂 2
1.1.1 供试材料 2
1.1.2 主要试剂 3
1.1.3 主要仪器 3
1.2 试验方法 3
1.2.1 引物的设计与合成 3
1.2.2 番茄白粉病菌gDNA的提取 3
1.2.3 PCR扩增目的基因 4
1.2.4 pMD18-T载体与目的基因的连接反应 4
1.2.5 感受态细胞的制备和目的基因的转化 4
1.2.6 重组载体的PCR鉴定 4
1.2.7 重组载体的测序鉴定 4
1.2.8 CaM序列比对和系统发育分析 5
2. 结果与分析 5
2.1 番茄白粉病菌gDNA的检测 5
2.2 目的基因克隆的检测 5
2.3 重组载体的PCR检测结果 6
2.4 重组载体的测序结果 6
2.5 系统发育树的构建 7
3. 讨论 8
参考文献 9
致谢 11，3034