摘 要

樟疫霉（Phytophthora cinnamomi）是卵菌纲(Oo．mycetes)藻菌。可引起农作物和林木的腐烂枯死。北美洲、澳洲、大洋洲以及我国的台湾省等热带和亚热带地区均有分布，在国外十分常见。可侵染多种林木、果树和农作物，是植病中一种重要病原菌。CRN基因家族是卵菌中发现的一组基因，与RXLR家族类似，属于寄主胞质效应子。由于这类效应子在致病疫霉中被报道能引起烟草的细胞死亡，因此命名为CRN(Crinkleramp;nbsp;and necrosis protein)。本研究采用生物信息学的方法对樟疫霉家族进行了分析，表明该基因家族为卵菌所特有，且具有保守序列原件LQLFLAK和HVLVVP。本研究利用生物信息学，根据其余物种已报道的大量效应子的序列对所研究的效应子在樟疫霉的其他变种中进行同源性搜索，通过比对表明其属于樟疫霉家族。并通过农杆菌介导的PVX病毒表达，对樟疫霉中CRN5、CRN55的生物学功能进行了初步的功能分析。通过在本氏烟上瞬时表达这两个樟疫霉CRN家族效应子，验证了其能抑制老鼠细胞凋亡前体蛋白bax或者激发子INF1引起的植物细胞死亡，并且激发植物的免疫反应，从而对了解寄主与樟疫霉在分子水平上的互作机理产生重要意义。

关键词：CRN；樟疫霉；卵菌；效应子

BIOINFORMATICS ANALYSIS AND PRELIMINARY FUNCTIONAL STUDY OF CRN5 AND CRN55 OF PHYTOPHTHORA CINNAMOMI

ABSTRACT

Phytophthora cinnamomi is an algae belonging to the class Oo.mycetes. It can cause forest trees or crops to rot and die. Phytophthora cinnamomi is a common disease in foreign countries. It is distributed in tropical and subtropical regions such as North America, Australia, Oceania, and Taiwan Province of China. It can infect a variety of trees, fruit trees and crops and is an important pathogen in plant diseases. The CRN gene family is a group of genes found in oomycetes that are similar to the RXLR family and belong to the host cytoplasmic effector molecule. It was first reported to cause tobacco cell death in P. infestans, and was named accordingly CRN (Crinkler and necrosis protein). In this study, bioinformatics methods were used to analyze the family of P. capsici, indicating that the gene family is unique to oomycetes and has a conserved LQLFLAK and HVLVVP sequence element. In this study, bioinformatics was used to search homologously for the effectors studied among other variants of Phytophthora blight based on the sequence of a large number of effectors reported in other species. The comparison indicates that they belong to the family of Phytophthora. A eliminary functional analysis of the biological functions of CRN5 and CRN55 in P.panicola was performed using the Agrobacterium-mediated PVX virus expression system. By transiently expressing the two CRN effectors which belong to the family of Phytophthora on Nicotiana benthamiana, it was verified that it can inhibit plant cell death caused by apoptotic precursor protein bax or elicitor INF1 and stimulate the plant's immune response. It is of great significance to understand the interaction mechanism between the host and the pest at the molecular level.

Keywords：CRN; Phytophthora cinnamomi; Oomycetes; Effector molecule

目录

1引言 1

1.1 樟疫霉研究进展 1

1.1.1 樟疫霉分类地位及危害 1

1.1.2 樟疫霉生活史及生活特性 1

1.1.3 樟疫霉分离鉴定 2

1.2 植物病原卵菌效应子的研究进展 3

1.2.1 效应子与植物先天免疫系统 4

1.2.2 卵菌RxLR效应子结构 5

1.2.3 RxLR效应子的转运机制 6

1.2.4 RxLR效应蛋白的毒性功能 6

1.2.5 CRN效应蛋白 7

1.3 研究展望 7

2 研究材料与方法 8

2.1 实验试剂和设备 8

2.2 供试烟草和供试菌株的保存于培养 8

2.3 序列资源 8

2.4 樟疫霉RxLR效应子鉴定 9

2.5 序列进化树的构建 9

2.6 樟疫霉菌丝基因组DNA提取 9

2.7 樟疫霉RxLR效应子基因克隆 10

2.8 大肠杆菌DH5α的质粒提取 11

2.9 马铃薯X病毒载体pGR107 12

2.10 Infusion 技术构建载体 12

2.10.1重组反应 13

2.10.2大肠杆菌转化反应产物、涂板 13

2.10.3 验证转化子 14

2.11农杆菌GV3101感受态细胞的制备和转化方法 14

2.11.1农杆菌感受态细胞的制备 14

2.11.2农杆菌感受态细胞的转化 15

2.12效应子筛选 15

2.13樟疫霉CRN蛋白结构域的查找 16

3 结果与分析 17

3.1 CRN5、CRN55效应子蛋白二级结构预测 17

3.2 樟疫霉CRN蛋白结构域分析 17

3.3 效应子基因克隆 18

3.4 PVX载体连接和大肠杆菌转化 19

3.5 菌落PCR验证转化子 19

3.6 提取质粒测序结果 20

3.7 电转农杆菌 20

3.8 PVX::CRN55-SP注射烟草 21

4 结果与讨论 22

参考文献 23

附录：樟疫霉AVH53效应蛋白功能研究 25

1 材料和方法 25

1.1 供试菌株 25

1.2 樟疫霉RxLR效应子鉴定 25

1.3 序列进化树的构建 25

1.4 序列进化树的构建 25

1.5 樟疫霉菌丝基因组DNA提取 25

1.6 樟疫霉RxLR效应子基因克隆 25

1.7 大肠杆菌DH5α的质粒提取 25

1.8 马铃薯X病毒载体pGR107 25

1.9 效应子筛选 25

1.10 Infusion 技术构建载体 26

1.11 农杆菌GV3101感受态细胞的制备和转化方法 26

2 结果与分析 27

2.1 樟疫霉效应子信号肽预测 27

2.2 樟疫霉AVH53基因属于RXLR效应蛋白家族成员 27