摘要本文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基因——18136进行了克隆和测序，通过实时荧光定量PCR对其表达量进行了研究，通过生物信息学的方法对基因序列和蛋白质的结构及功能进行了分析。结果如下：几丁质酶18136基因在烟台252中有一个拷贝，南京92中有两个拷贝，并且其中一个与烟台252相同，另一个发生了变异；实时定量PCR显示，18136基因几丁质酶的表达量与甘薯品种抗病性呈正相关；生物信息学分析显示两序列全长均为804bp，均编码267个氨基酸，分子量均为28KDa，等电点均为6.56；均属Class Ⅰ几丁质酶，两基因编码的蛋白质都属于几丁质酶19家族。两蛋白质二级及三级结构有差异；进行相似性分析表明这两个几丁质酶基因与其他高等植物几丁质酶I类几丁质酶基因相似性达50%-90%。42688

毕业论文关键词：甘薯  几丁质酶  cDNA  实时荧光定量PCR  序列分析 

 甘薯几丁质酶18136基因cDNA的克隆及序列分析

 Gene Cloning and Sequence Analysis of Chitinase cDNA 18136 in Ipomoea Batatas

Abstract

This thesis used two disease resistant varieties of Ipomoea Batatas – Nanjing 92 (high resistance to black spot) and Yantai 252 (high susceptible to black spot) as experimental materials for gene cloning and sequencing of one chitinase gene – 18136. We used Quantitative Real-time PCR for the relative quantification of chitinase in Ipomoea Batatas with different resistant levels, and analyzed the sequences and structures of genes and proteins using bioinformatics software. It shows that this gene has one copy in Yantai 252 and two copies in Nanjing 92, and one of these copies is the same in Yantai 252, while the other one has s quite numbers of mutations; it also shows that the chitinase 18136 geng expression levels are positively correlated with the resistance of the Ipomoea Batatas. Bioinformatics analysis shows that the full-length of this two chitinase genes is 804bp, and both encode 267 amino acids; their PI is 6.59 and molecular weight is 28KDa; BLAST analysis also shows that these two genes belong to classⅠchitinase, which proteins belonging to family 19 chitinase. We construct their binary structures and 3D-structures respectively and find that these two proteins are different in structures. Similarity analysis shows that the two genes and other plants with chitinase similarities range from 50% to 90%. This is used for the phylogenetic tree.

Key Words: Ipomoea Batatas  chitinase  cDNA  Real Time PCR  sequence analyze  

目  录

摘  要 I

Abstract II

引言 1

1 材料与方法 1

1.1材料与仪器 1

1.2 实验试剂的配置 2

1.3 方法 3

2结果与分析 7

2.1几丁质酶基因克隆及测序 7

2.2 几丁质酶表达量分析 9

2.3 几丁质酶基因生物信息学分析 10

3 讨论 14

参考文献 16

致谢 17

引言

甘薯，薯蓣科薯蓣属。甘薯在世界粮食、饲料和工业生产产业中占极大比重。甘薯病害较多，其中由真菌侵染引起的甘薯黑斑病是危害甘薯生产的主要病害，且该病较难根治，目前多在综合防治的基础上辅以控制[1]，但始终缺乏治本之法，因此最直接有效的方法——选育抗病能力强的品种——得到许多人的重视。现如今，国内外不乏许多优秀的育种学家通过分子生物学技术对其抗病性进行改造，但还尚未出现黑斑病免疫品种。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。