摘 要

栗树成长过程中会遭遇很多病虫害侵袭，这些病虫害中，以栗疫病菌（Cryphonectria parasitica）引起的栗疫病最为严重。欧美等地区用含有 dsRNA 的低毒力菌株取得了很好的治疗效果。无论是在碱基水平还是氨基酸水平，中国产的低毒病毒 CHV1-CN280 与来自欧洲的 CHV1-EP713 相比，差异都很大，包括 P29 编码区。我们使用外源基因导入法来对 CHV1-CN280 的 p29 基因进行了研究。实验中，我们构建了 p29 基因表达载体，将其导入栗疫菌中。结果发现，该基因的表达会引起寄主的产孢子量减少、黄色色素产量减少、生长变快以及漆酶（laccase）活性降低等低毒力性状。研究认为，这两种病毒各自的 p29 基因功能相似，但又并不完全一致，这主要是因为两种病毒的性质差异，它们各自 P29 的氨基酸序列对性状也有影响。

关键词：栗疫病菌；CHV1-CN280；低毒力病毒；p29基因

p29 which present in hypovirulent strain CHV1-CN280 in Cryphonectria parasitica

Abstract: The growth process of chestnut often encounters a lot of pests and diseases. And the chestnut blight fungus (Cryphonectria parasitica) is the most serious one. Over the past few years, Europe and the United States and some other regions have achieved good therapeutic effects in biological control by using the low toxicity strains which contain dsRNA. Whether at base level or amino acid level, the difference between the low virulence virus CHV1-CN280 from China and that of CHV1-EP713 from Europe is very great, and the variation of P29 coding region is also very great. Therefore, we use the method of foreign gene transformation to research the function of p29 gene in chestnut blight fungal. In the experiment, we cloned the p29 gene of CHV1-CN280, connected it to different expression vectors, and then introduced it into the genome of the chestnut fungus. The results showed that in the host, the expression of this gene in the chestnut blight fungus caused the decrease of the spores number, the decrease of pigment, the increase of the growth rate and the decrease of laccase activity which were similar to those of low virulence. Studies suggest that the function of the each p29 gene in CHV1-CN280 and CHV1-EP713 is similar, but not indentical. This is mainly due to the differences in properties between these two kinds of viruses, and to a certain extent, differences in amino acid sequences of the P29 also affected the traits of the host.

Key words: Cryphonectria parasitica; CHV1-CN280; hypovirus; p29

目录

1 文献综述 - 1 -

1.1 栗疫病的发生 - 1 -

1.1.1 栗疫病 - 1 -

1.1.2 栗疫病发生的历史 - 1 -

1.2 低毒病毒的发现 - 1 -

1.2.1 低毒病毒简介 - 2 -

1.2.2 低毒病毒在栗疫病防治中的应用 - 2 -

1.3 CHV1基因组功能研究 - 3 -

1.4 研究展望 - 4 -

2 研究内容 - 5 -

2.1材料与方法 - 5 -

2.1.1 材料 - 5 -

2.1.2 p29基因的克隆及序列分析 - 5 -

2.1.3 栗疫菌原生质体的制备和转化 - 5 -

2.1.4 转化子的筛选和验证 - 6 -

2.1.5 栗疫菌中突变体表型的分析测定 - 7 -

2.2结果与分析 - 8 -

2.2.1 p29基因的克隆转化子的筛选和序列分析 - 8 -

2.2.2 突变体菌落形态观察及生长速率分析 - 11 -

2.2.3 突变体分生孢子产量 - 12 -

2.2.4 突变体对漆酶的影响 - 13 -

2.2.5 突变体致病力测定 - 13 -

3 结论与讨论 - 15 -

3.1结论 - 15 -

3.2讨论 - 15 -

致 谢 - 16 -

参考文献 - 16 -

1 文献综述

1.1 栗疫病的发生

1.1.1 栗疫病

栗树（Castanea spp.）是一种具有良好经济效应的作物，栗树的果实板栗可以食用，栗树的心材结构粗、纹理直、坚硬，是一种优质的木材。但是板栗生产中也会遇到多种病虫害侵袭，其中最为严重的就是板栗疫病。板栗疫病主要发生在枝干上。栗疫病发病初期，栗树的枝干褪绿，逐渐变成褐黄的圆点，之后进一步发展，出现形状不规则的较大红褐色病斑，最后病斑围住整个树干，病斑继续纵向扩展。病部稍稍凹陷，病斑呈水肿状隆起的黄色小点，这就是板栗疫病菌的分生孢子器，病斑的内部潮湿、腐烂，有酒味，待病斑干燥后，树皮纵向裂开，可见到树皮内组织为枯黄色^[1]。此病轻则会导致板栗产量的减少，重则将会引起整片栗林绝收，造成极大的经济损失。

1.1.2 栗疫病发生的历史