摘 要

由樟疫霉(Phytophthora cinnamomi)引起的病害是农林生产上的严重病害，对农田、森林、园艺等经济体系造成巨大影响，所以本研究对其编码基因进行了研究，以明确编码基因的生物学功能，为研发新型杀菌剂提供有效依据。CRISPR/Cas 系统是一种新型的基因组编辑技术，Cas9 核酸酶能够通过20 bp 的引导序列而导向特异的基因组位点而使DNA发生双链断裂。G蛋白偶联受体(G protein coupled receptor，GPCR)是真核生物细胞膜表面最大的一类受体家族，负责将体外的环境信号转变为细胞内部的信号。卵菌中已经鉴定出一类新型的GPCR-PIPK基因，同时具有GPCR的跨膜结构域和PIPK的催化活性，这种新型的GPCR目前只在卵菌和盘基网柄菌中发现。利用生物信息学筛选出GPCR-PIPK在樟疫霉中有10个编码基因，针对樟疫霉GPCR-PIPK基因位点，设计两条sgRNA（single guide RNA），随后将其构建入sgRNA 表达载体，在此基础上利用大豆疫霉稳定转化和CRISPR-CAS9系统对樟疫霉中的PcGPIPK2编码基因进行了初步的功能分析，明确PcGPIPK2编码基因的生物学功能，初步阐明樟疫霉的生长发育和致病等重要生理过程中的作用机制，为新型杀菌剂的研发提供可能的分子靶标，从而为有效防控樟疫霉引起的病害提供理论依据。

关键词：樟疫霉；G蛋白偶联受体；PIPK；转化

Phytophthora cinnamomi PcGPIPK2 clone and genetic transformation

ABSTRACT

Phytophthora cinnamomi cause of disease is one of the serious problems of agriculture and forestry production, the farmland, forest, garden and so on economic system cause huge impact. CRISPR/Cas system is a new type of genome editing techniques, Cas9 nuclease can  guide by 20 bp the boot sequence of the specific genomic loci and the DNA double  chain rupture. G protein coupled receptor ( GPCR) is one of the largest class of eukaryotic cell membrane surface receptor family, is responsible for the in vitro signals into the cell's internal environment. Oomycetes in have identified a new type of GPCR - PIPK gene, at the same time has a GPCR across membrane structure domain and PIPK catalytic activity, this new type of GPCR currently only the oomycetes and set the base handle bacteria found in the net. GPCR - PIPK in phytophthora cinnamomi has 10 coding genes,Phytophthora cinnamomi in GPCR - PIPK gene loci, design two sgRNA (single guide RNA), which is then built into sgRNA expression vector,on the basis of using soybean phytophthora and CRISPR - CAS9 system stability of camphor phytophthora PcGPIPK2 coding gene has carried on the preliminary function analysis, clear PcGPIPK2 the biological function of genes encoding,phytophthora cinnamomi growth and pathogenic preliminary clarify important role in the process of physiological mechanism, such as for the research and development of new fungicide possible molecular targets, disease caused by phytophthora cinnamomi for effective prevention and control to provide theory basis。

Key words:Phytophthora cinnamomi ; G protein coupled receptor ; PIPK;transformation

目 录

1 文献综述 1

1.1 研究目的 1

1.2 樟疫霉的研究进展 1

1.2.1樟疫霉的生活史 2

1.3 G蛋白偶联受体概况 2

1.3.1 G蛋白偶联受体的重要性.......................................................................................4

1.3.2 G蛋白偶联受体的结构...........................................................................................4

1.3.3 G蛋白偶联受体的分类...........................................................................................4

1.3.4 G蛋白偶联受体在卵菌中的研究概述...................................................................5

1.3.5 GPCR-PIPK概述.....................................................................................................5

1.4 CRISPR/Cas9技术...........................................................................................................6

2 樟疫霉PcGPIPK2基因的遗传转化研究 8

2.1 材料 8

2.1.1 实验菌株..................................................................................................................8

2.1.2 培养基的制备..........................................................................................................8

2.1.3 仪器与设备.................................................................................................. .........8

2.1.4 数据库信息和同源检索........................................................................................8

2.1.5 数据库信息和同源检索........................................................................................9

2.1.6 樟疫霉RNA-Sequencing测序样品的准备..........................................................9

2.1.7 RNA的提取........................................................................................................ 10

2.1.8 载体设计..............................................................................................................11

2.1.9 sgRNA靶点选择及其寡核苷酸链合成.............................................................12

2.2 转化载体构建 13