摘 要

杨树（Populus）是林木中的模式树种，也是重要的速生用材树种，有着重要的经济价值。众多病害中，杨树黑斑病对杨树的威胁最为突出，它是由真菌——杨生褐盘二孢菌引发的。其发生范围相当广泛，是影响木材产量和质量的最严重的威胁之一。但目前为止，对杨盘二孢菌的致病机理不清楚，且关于该菌生长发育和致病相关基因克隆尚未见诸于报道。各种遗传转化技术中，根癌农杆菌介导遗传转化（Agrobacterium tumefaciens-mediated transformation，ATMT）技术在真菌的基因组研究中扮演着重要角色的，广泛应用于随机插入突变体库的构建和基因打靶中。本课题研究将选用该遗传转化技术，从而遗传转化杨盘二孢菌，最终获得大容量的ATMT突变体库，筛选可以致病的突变体，为在分子层面明确病菌的致病原理打下基础。本研究以杨生褐盘二孢菌强致病力菌株214-1作为出发菌株，利用以潮霉素和绿色荧光蛋白基因为标记基因的敲除载体PCAMBIA1300，采用ATMT技术进行遗传转化。在实验中探究最佳的农杆菌浓度；将农杆菌同分生孢子共同培养混合，探究最佳比例；共培养时长等遗传转化的条件，构建稳定且效率高的根癌农杆菌介导的杨盘二孢菌遗传转化体系。

关键词：杨盘二孢菌；根癌农杆菌；ATMT；转化效率

Construction and optimization of Agrobacterium mediated transformation of Marrssonina brunnea

ABSTRACT

Populus is a model tree species in forest, and also an important fast-growing forestry species which plays an important role in economy. Among all kinds of diseases, Poplar black spot disease is the most serious one, its pathogen is a kind of fungus -- Marrssonina brunnea which has a wide range of occurrence and is one of the most horrible threats to wood yield and quality. But until now, the pathogenic mechanism of Marrssonina brunnea remains unclear, and the growth and disease related to gene cloning has not been reported. Agrobacterium tumefaciens mediated- genetic transformation (ATMT) is a powerful tool for the study of fungal genomics, which is also widely used in the construction of fungal random insert mutant libraries and gene targeting. In this research, we will use the genetic transformation of Marrssonina brunnea, in order to get construction of large capacity ATMT mutant library, screening of pathogenicity related mutants, so that we can reveal the pathogenic mechanism of bacteria from molecular level to lay the foundation. In this study, Populus Marrssonina brunnea virulent strain 214-1 will be used as starting strain, hygromycin and green fluorescent protein gene will be uesd as marker gene knockout vector PCAMBIA1300, genetic transformation will be carried out by ATMT technology. We have tried to establish a stable and efficient Agrobacterium tumefaciens mediated by Agrobacterium tumefaciens, optimization of Agrobacterium tumefaciens concentration, mixing ratio of Agrobacterium and conidia and co-culture time.

Key words：Marrssonina brunnea；Agrobacterium tumefaciens；ATMT；transformation efficiency

目 录

1 文献综述………………………………………………………………………………………1

1.1 杨树的病害及其致病因子………………………………………………………………1

1.2 根癌农杆菌介导的遗传转化……………………………………………………………………1

1.3 根癌农杆菌介导的应用……………………………………………………………………2

2 研究内容………………………………………………………………………………5

2.1 材料……………………………………………………………………5

2.2 菌种培养………………………………………………………………………5

2.3 培养基及主要供试试剂制备…………………………………………………5

2.4 根癌农杆菌介导杨盘二孢菌转化体系条件的摸索…………………………6

2.5 转化与筛选……………………………………………………………………7

2.6 T-DNA 插入的分子检测及稳定性分析……………………………………8

2.7 转化子PCR验证……………………………………………………………10

3 结果与分析………………………………………………………………………………11

3.1 潮霉素B对杨盘二孢菌分生孢子萌发和菌落生长的作用…………………………………11

3.2 不同共培养方式对转化效率的影响……………………………………………………………12

3.3 T-DNA 插入的分子鉴定……………………………………………………………………13

3.3.1 转化子的 PCR 验证………………………………………………………………13

3.3.2 转化子荧光观察验证……………………………………………………………………14

3.3.3 转化子稳定性测试…………………………………………………………………15

结论…………………………………………………………………………………………………………16

致谢…………………………………………………………………………………………………………17

参考文献……………………………………………………………………………………………………18

1 文献综述

1.1 杨树的病害及其致病因子

1.1.1 杨树黑斑病

杨树（Poplar）是林木中的模式树种，也是重要的速生用材树种，有着重要的经济价值。在一个完整的生活周期中，可以威胁、危害杨树的病虫害非常多，如杨树黑斑病。

1.1.2 杨生褐盘二孢菌

引起该病害的为半知菌亚门腔孢纲黑盘孢目盘二孢属Marrssonina中的三种真菌，包含：杨盘单格孢菌（Marrssonina populi）；杨生褐盘二孢菌（Marrssonina brunnea）；白杨盘二孢菌（Marssonina castagnei），且病害灾区分布相当广泛，已严重影响木材产量和质量^[1]。在欧洲、美洲、大洋洲以及亚洲，调查之中都有发现该真菌的记录。Han等选取了一种特殊的分类方法，中国境内的杨生褐盘二孢菌分成：单芽管专化型（Marssonina brunnea f. sp. monogermtubi）和多芽管专化型（Marssonina brunnea f. sp. multigermtubi）两种^[2]。

1.1.3 目前研究中存在的问题