目录
大豆疫霉PsSCP26的基因功能初探 I
摘要 I
关键词 I
ABSTRACT II
KEYWORDS II
引言
1 材料与方法 1
1. 1 供试试剂 1
1. 2 供试菌株及培养条件 2
1. 3 PsSCP26基因的克隆 2
1. 3. 1 设计引物 2
1. 3. 2 基因扩增PCR 2
1. 3. 3 DNA琼脂凝胶电泳 2
1. 3. 4 观察目的基因条带 2
1. 3. 5 基因回收 2
1. 3. 6 酶切 3
1. 3. 7 连接 3
1. 3. 8 转化 3
1. 3. 9 验证 3
1. 3. 10 提质粒 3
1. 4 敲除大豆疫霉PsSCP26基因 3
1. 4. 1 构建质粒sgRNA 4
1. 4. 2 构建HDR质粒 4
1. 4. 3 CRISPR/cas9 体系敲除PsSCP26 基因 4
1. 5 提取候选转化子的总DNA 6
1. 6 初步验证疫霉菌PsSCP26基因敲除转化子 6
1. 7 验证转化子 7
1. 8 单孢分离 7
1. 9 致病性观察 7
1. 10 定量分析 7
2 结果与分析 8
2. 1 PsSCP26 在大豆疫霉侵染初期的表达情况 8
2. 2 sgRNA 设计结果 8
2. 3 PsSCP26上下游各1000bp 碱基序列测序结果 8
2. 4 三对验证引物在基因上的位置 9
2. 5 大豆疫霉效应子PsSCP26敲除转化子的验证结果 10
2. 5. 1 利用PsSCP26的基因引物验证敲除转化子 10
2. 5. 2 利用引物 F1/R1、F2/R2验证敲除的转化子 10
2. 6 致病性观察 11
3. 1 实验结论 13 *51今日免费论文网|www.51jrft.com +Q: ￥351916072￥ 

3. 2 实验的注意事项 13
3. 2. 1 CRISPR/Cas9技术的使用 13
3. 2. 2 培养基的注意事项 13
3. 2. 3 原生质体转化实验过程的注意事项 14
致谢 15
参考文献 16
大豆疫霉PsSCP26的基因功能初探
摘要
大豆疫霉是大豆农业生产中极具毁灭性的一种病原菌。它属于植物卵菌门的病原菌，可以引起大豆根或茎的腐烂病以及产量的减少。为了抵抗疫霉菌的侵染，大豆进化出一套复杂的免疫系统。疫霉菌为了抑制植物的免疫，会分泌大量效应子进入大豆的质外体空间。在大豆疫霉与寄主植物的长期选择进化中，这些效应子能够调控植物的免疫并为病原菌的侵染提供有利条件。其中，本课题研究的大豆疫霉质外体效应子PsSCP26属于CAP超家族蛋白的一员，它在大豆疫霉侵染大豆的初期上调表达。CRISPR/Cas9系统是植物基因功能研究的一项重要技术，在多个领域的基因研究中发挥着重要的作用。其工作原理是通过DNA双链的断裂激活植物本身的内源修复途径，进而对目标位点定点突变或对目标基因随机插入与替换。为了深入研究在疫霉菌与植物互作过程中大豆疫霉质外体效应子PsSCP26的功能，本文利用CRISPR/Cas9系统将PsSCP26基因替换为mCherry基因进而敲除了PsSCP26。通过接种实验发现大豆疫霉菌在缺失PsSCP26基因后致病能力有所减弱，说明大豆疫霉质外体效应子PsSCP26对大豆疫霉的致病力有一定的贡献。
PRELIMINARY STUDY ON PSSCP26 GENE FUNCTION OF
PHYTOPHTHORA SOJAE
ABSTRACT
Phytophthora sojae is identified as one of the most catastrophic plant pathogens in soybean production around the world. P.sojae is a member of the genus Phytophthora of oomycetes, which can reduce soybean yield and cause root or stem rot. In order to resist the infection of Phytophthora, soybean has evolved a very complicated immune system. However, during P.sojae infection, it secretes a large amount of effector proteins into the soybean apoplast to escape the plant immunity. In the longterm selective evolution of P.sojae and host plants, these effector proteins can inhibit the plant immunity to promote Phytophthora infection. Among them, P.sojae apoplastic effector PsSCP26 is a member of the CAP superfamily protein, which is the main content in our subject. When P.sojae infecting soybean, PsSCP26 is upregulated in the initial stage. In the research about plant gene function, the CRISPR/Cas9 system is the most promising gene editing technology. CRISPR/Cas9 can activate the endogenous repair pathway of the plant by breaking the DNA doublestrand. In this way, it makes mutations on the target site or inserting and replacing the target gene at the specific sites. In order to study the function of the P.sojae apoplastic effector PsSCP26, we used the CRISPR/Cas9 system to replace the PsSCP26 gene with the mCherry gene. We found that the pathogenicity of P.sojae is compromised when delete the gene PsSCP26. Therefore, the P.sojae apoplastic effector PsSCP26 contributes to the pathogenicity of Phytophthora.

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