拟南芥LysM受体样激酶的克隆与筛库载体的构建
关键词：拟南芥；LysM基因；pGBKT7载体；功能验证
The Construction of the Recombinant Screening Vector of Yeast Two-hybrid of Arabidopsis thaliana LysM Gene
Abstract: A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis thaliana. This research focuses on constructing yeast two-hybrid library-screen vector pGBKT7 of Arabidopsis LysM. The primers were designed and synthesized based on the published LysM sequence in Genbank(ID: AB367524.1). The target gene LysM can be obtained by polymerase chain reaction (PCR) and then the PCR products and pGBKT7 were ligated after purification and double digestion by restriction endonuclease SalⅠ and NdeⅠ. After that the constructed vector was transformed into DH5α. Finally the transformed DH5α was identified by PCR and sequencing. The results illustrated that the recombinant vectors were constructed successfully. The research results lay foundation for the function identification of LysM-interrelated gene.
Key words: Arabidopsis thaliana; LysM gene; pGBKT7 vector; Function identification
目 录
摘 要 1
Abstract 1
引言 1
1. 材料与方法 2
1.1 实验材料及仪器 2
1.1.2 主要仪器 3
1.1.3 PCR引物的设计与合成 3
1.2 实验方法 3
1.2.1 筛库载体pGBKT7-LysM的构建流程 3
1.2.2 拟南芥叶片RNA的提取及目的基因的扩增 4
1.2.3 质粒pMD18-T与目的片段LysM的连接 4
1.2.4 pMD18-T-LysM重组质粒和pGBKT7质粒的双酶切与纯化回收 4
1.2.5 目的片段LysM与pGBKT7片段的连接 4
1.2.6 重组载体pGBKT7-LysM的转化与阳性克隆的挑取 4
1.2.7 重组载体pGBKT7-LysM的电泳分析 5
1.2.8 重组载体pGBKT7-LysM的PCR鉴定 5
1.2.9 双酶切处理重组质粒pGBKT7-LysM检测 5
1.2.10 重组载体pGBKT7-LysM的测序鉴定 5
2. 结果与分析 5
2.1 拟南芥叶片RNA的提取及目的基因的检测 5
2.2 pMD18-T-LysM重组质粒与pGBKT7载体的双酶切检测 6
2.3 重组质粒pGBKT7-LysM琼脂糖电泳比较大小检测结果 6
2.4 重组质粒pGBKT7-LysM的PCR检测结果 7
2.5 重组质粒pGBKT7-LysM双酶切电泳结果 7
2.6 重组质粒pGBKT7-LysM测序结果 8
3. 讨论 8
参考文献 10
致谢 12,4381
拟南芥LysM受体样激酶的克隆及筛库载体的构建
引言
在4亿多年前，地球上出现了高等植物[1]，从其诞生以来，它们就一直生活在一个充满多种病原微生物的环境中。植物与动物不同，大部分植物在其生长过程中无法移动，缺少对有害环境的机动性反应反应。但植物至今仍未走向衰退，说明了植物有一套防御病原菌入侵的免疫系统[2-3]。研究表明，植物的免疫防御系统至少包括两个层次，第一层为病原相关分子模式(pathogen associated molecular patterns, PAMPs)激发的免疫性(PAMP-triggered immunity, PTI)，这一层次的防御免疫主要靠植物细胞表面的模式识别受体(patters recognition receptors, PRRs)对病原菌的PAMPs进行分子识别，从而启动植物的防卫反应；第二层为病原菌效应子激发的免疫性(effectors-triggered immunity, ETI)，当毒性强的病原菌通过产生效应子(effectors)抑制PTI时植物的第一道防线被突破，植物又进化出新的分子受体以侦察病原菌效应子并启动第二道防卫反应[4]。植物防御机制需要在病原菌的PAMP作用下才能启动，自然界中存在的PAMP多种多样，几丁质就是其中的一种。
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