希金斯炭疽病菌蛋白激酶ChSAT4的生物学功能研究毕业论文_林学毕业论文

希金斯炭疽病菌蛋白激酶ChSAT4的生物学功能研究毕业论文

2021-04-21更新

摘 要

希金斯炭疽菌是一种世界性病原真菌,能够侵染十字花科植物,分布广泛,造成巨大的经济损失和观赏价值的丢失。希金斯炭疽病菌是目前少数被全基因组测序的几个植物病原真菌之一,由于模式植物拟南芥是其的一个侵染寄主,因此利用拟南芥——希金斯炭疽病菌的互作平台开展丝状真菌的致病性分子机制也成为近年来分子植物病理学研究的热点之一。胞内磷酸激酶在真核生物中广泛存在,且进化上高度保守的一种蛋白。胞内蛋白激酶催化下游蛋白质的磷酸化实现胞内信号的传导。在模式真菌酵母菌中,SAT4作为胞内磷酸激酶,通过磷酸化蛋白,促进细胞对Na 、K 离子的吸收,以此来调节细胞内的质子平衡和电荷平衡,维持胞内环境的稳态。目前,在希金斯炭疽病菌也发现了SAT4的同源蛋白,但关于该基因的功能尚不清楚。

本研究利用同源重组成功将希金斯炭疽病菌的SAT4基因敲除,获得基因缺失突变体△ChSAT4,并通过与野生型菌株的对比分析,初步分析了其生物学功能。研究结果表明,ChSAT4参与调节希金斯炭疽病菌的营养生长与无性繁殖;ChSAT4对希金斯炭疽病菌的胞内Na 和K 的积累;ChSAT4还参与调控希金斯炭疽病菌的细胞壁完整性和胁迫物质的抗性;通过接种油菜进行致病性分析,发现ChSAT4基因缺失突变体的致病性显著下降。表达模式分析发现,ChSAT4在孢子、附着胞、菌丝和芽管菌丝中均有表达且定位于细胞质中。以上研究为进一步阐释ChSAT4的基因功能及其分子调控机制奠定重要基础。

关键词:炭疽病;磷酸化;激酶;致病性;Na /K 离子通道

Biological function of protein kinase ChSAT4 in Colletotrichum higginsianum

ABSTRACT

Colletotrichum higginsianum is a worldwide pathogenic fungus that infects cruciferous plants, creating enormous economic losses and ornamental value. Colletotrichum higginsianum is one of the few plant pathogenic fungi that is sequenced by the whole genome. Since the model plant Arabidopsis thaliana is one of its infected hosts, the interaction of Arabidopsis thaliana-Colletotrichum higginsianum. The pathogenic molecular mechanism of filamentous fungi has also become one of the hotspots in molecular pathology research in recent years. Protein kinase(PK) is widely present in the eukaryotic cell, which regulates intracellular signal transmission, gene expression, mitosis and a series of life processes. And is highly conserved in evolution. Intracellular protein kinase catalyzes the phosphorylation of downstream proteins to achieve intracellular signaling. In the model fungal yeast, SAT4 as an intracellular phosphokinase, promoting the absorption of Na , K through the phosphorylated protein, in order to regulate the intracellular proton balance and charge balance, maintain the intracellular environment steady state. At present, the homologous protein of SAT4 is also found in Colletotrichum higginsianum, but the function of the gene is unclear.

This study cloned gene named ChSAT4 from Colletotrichum higginsianum gene. We knocked this gene and obtain the △ChSAT4 , and compared with the wild-type strains, conducted a series of biological function analysis. Experimental results showed that ChSAT4 have played a regulation role in utilizing nutrients and asexual reproduction; ChSAT4 also have played a regulation role in the accumulation of Na , K in Colletotrichum higginsianum. Experimental results also showed that ChSAT4 participates in regulating the Colletotrichum higginsianum bacteria species of cell wall integrity and stress resistance. The pathogenicity of mutant was found to be significantly decreases by inoculation of rape. Expression pattern analysis showed that ChSAT4 was expressed in the spores, appressorium, germ tube and hyphae, and located in cytoplasm. The research laid an important foundation for further explaining the gene function and molecular regulation mechanism of ChSAT4.

Key words:anthrax;phosphorylation;kinase;pathogenicity;Na /K ion channel

目 录

1 前言 – 1 –

1.1 希金斯炭疽菌研究概况 – 1 –

1.2 磷酸激酶 – 1 –

1.3 研究目的与意义 – 1 –

2 实验材料与方法 – 4 –

2.1实验材料 – 4 –

2.2 实验方法 – 4 –

2.2.1敲除载体构建 – 4 –

2.2.2希金斯炭疽菌原生质体制备及转化 – 4 –

2.2.3粗提转化子gDNA – 5 –

2.2.4转化子PCR验证 – 5 –

2.2.5 精提菌丝gDNA – 6 –

2.2.6突变体Southern Blot验证 – 6 –

2.2.7突变体的基因回补及表达定位模式分析 – 8 –

2.2.8营养生长实验 – 9 –

2.2.9真菌细胞完整性分析 – 9 –

2.2.10 渗透胁迫测定 – 10 –

2.2.11过氧化氢胁迫测定 – 10 –

2.2.12菌丝中Na 、K 含量测定 – 10 –

2.2.13 ChSAT4的致病性测定及菌丝穿透实验 – 10 –

2.2.14 SAT4在希金斯炭疽病菌和赤霉菌中的功能保守性分析 – 11 –

3 结果与分析 – 12 –

3.1 ChSAT4 缺失突变体的获得 – 12 –

3.2 ChSAT4基因缺失对希金斯炭疽病菌营养生长的影响 – 12 –

3.3 ChSAT4基因缺失对菌丝Na 和K 含量的影响 – 13 –

3.4 ChSAT4缺失突变对细胞壁完整性的影响 – 14 –

3.5 ChSAT4缺失突变对希金斯炭疽病菌渗透胁迫抗性的影响 – 15 –

3.6 ChSAT4缺失突变对希金斯炭疽病菌过氧化物胁迫抗性的影响 – 16 –

3.7 ChSAT4的致病性测定及菌丝穿透实验 – 16 –

3.8 ChSAT4蛋白表达定位模式 – 18 –

3.9 SAT4在希金斯炭疽病菌和赤霉菌中的功能保守性分析 – 18 –

结 论 – 20 –

致 谢 – 22 –

参考文献 – 23 –

附录A – 24 –

1 前言

1.1 希金斯炭疽菌研究概况

希金斯炭疽菌(Colletotrichum higginsianum)属于真菌门,腔孢菌纲,黑盘孢目,黑盘孢科,刺盘孢属真菌。希金斯炭疽菌引起的植物炭疽病是一种世界性的真菌病害,主要分布于中国、日本、印度、美国以及东南亚地区,主要侵染十字花科植物,造成严重的经济损失[1]。该病害在我国各地也普遍发生。蔬菜大棚内的高温高湿环境以及我国南方地区温暖湿润的气候更加适宜该病发生,发病严重地区可导致30~40%的产量损失[2]。近年来,由于栽培面积和复种指数的增加以及连作障碍加剧,导致该病害在我国的发生与危害逐年加重。

希金斯炭疽病菌的农业栽培技术防治包括:十字花科植物与非十字花科作物交替轮作;加强作物田间管理及时清除病残体,积水增加空气湿度及传播,因此避免田间有积水;合理施肥,增施钾肥;避病栽培等。

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