甘薯几丁质酶02314基因cDNA的克隆及序列分析
毕业论文关键词：甘薯 几丁质酶 cDNA 序列 表达量 序列分析甘薯几丁质酶 02314基因 cDNA的克隆及序列分析
The Cloning and Sequence Analysis of Chitinase cDNA 02314 in Ipomoea Batatas
Abstract Two different resistance varieties to black spot, which are Nanjing 92 (high resistance to black spot) and Yantai 252 (high susceptible to black spot), are considered as materials. 02314, which is one of the chitinase gene sequences, was cloned and sequenced. The expression was studied by quantitative real-time PCR. The gene sequence, structure and function of protein were analyzed by the methods of bioinformatics. The results pointed to the following. The gene sequences only had one copy in Nanjing 92 and Yantai 252, and the cDNA sequences were same, but the expression of chitinase was positively correlated with the resistance to black rot in various resistant varieties of Ipomoea Batatas. The analysis by a series of bioinformatics software showed the full-length of the sequence was 975bp and encoded 324 amino acids. The molecular weight was 34.512KDa. The isoelectric point was 4.27, so it belonged to acidic chitinase. It was non-transmembrane Family 18 chitinase. It belonged to Class Ⅲ chitinase, so it not only had the activity of chitinase, but also had the activity of lysozyme. In the secondary structure of the protein, the helix occupied 30.8% of the sequence, and the strand was 13%, and the coil was 56.2%. Homology analysis of phylogenetic tree showed the similarity of this sequence and Class Ⅲ chitinase in other advanced plants reached up to 73%.
Key Words: Ipomoea Batatas Chitinase cDNA Sequence Expression Sequence Analysis
目录
摘要.I
AbstractII
图清单..IV
表清单..IV
1引言1
1.1实验材料、试剂及仪器...1
2实验方法与步骤...3
2.1基因02314的克隆与测序...3
2.2荧光实时定量PCR...6
3结果与分析...7
3.1总RNA提取结果.7
3.2RNA反转录结果...8
3.3PCR产物检测结果8
3.4测序结果8
3.5实时荧光定量PCR结果...9
3.6cDNA序列及其蛋白质序列分析..10
3.7进化树...13
4讨论..14
参考文献..15
致谢..17
1 引言 甘薯种植区域较为广泛，是世界上最重要的农作物之一【1】，并且作为一种健康食品越来越受到欢迎。甘薯黑斑病是危害甘薯生产的主要病害之一，是由真菌侵染产生的植物病害【2】。甘薯黑斑病的危害巨大，我国因黑斑病每年甘薯产量损失在 10%以上【3】。目前，甘薯黑斑病较难根治，以农业防治为主的综合防治措施，其他措施为辅的防治措施才能对甘薯黑斑病奏效。目前，尚未发现黑斑病免疫的甘薯品种【4】，因此研究和利用抗病基因进行分子设计育种，是防治黑斑病的最经济有效的途径【5】。 几丁质酶广泛存在于生物体中，尤其是高等植物中，通过对病原真菌细胞壁的降解【6】，在植物中对病原真菌起重要抗性作用【7】。本实验组在前期研究中通过大量生理研究发现甘薯中品种几丁质酶活性与抗病性成正相关【8】，并且通过分子相关实验证明甘薯中部分品种几丁质酶活性与抗病性成正相关， 但未证明本实验所用甘薯材料南京92（高抗）与烟台252（高感）两个品种的几丁质酶活性与抗病性的关系。 本实验通过甘薯基因库筛选得到一批几丁质酶类似序列，比对分析其几丁质酶蛋白保守区域，得到 2 个几丁质酶基因。以对黑斑病抗性不同的两个品种南京92（高抗黑斑病）和烟台 252（高感黑斑病）的块根为材料，对其中的一个几丁质酶02314 基因进行了克隆和测序，研究不同抗性品种的几丁质酶 02314 基因序列是否存在差异，源`自*优尔?文.论~文`网[www.youerw.com又通过荧光定量PCR，分析南京92 与烟台252 两个品种中几丁质酶的表达量是否存在差异。并通过生物信息学对其结构和功能进行了分析，以期为下一步将该基因构建表达载体、转化至不抗病或抗病弱的品种，获得优质高抗病性的甘薯品种奠定基础。