飞蝗对绿僵菌IMI330189和MAD1蛋白的免疫应答

论文摘要

绿僵菌菌株（IMI330189）是一种能有效防治害虫的昆虫病原真菌。MAD1蛋白是由绿僵菌产生、协助绿僵菌附着在昆虫体表对昆虫产生毒力的一种蛋白。本实验通过饵剂饲喂的方法研究了绿僵菌菌株（IMI330189）和MAD1蛋白单独或混合取食对飞蝗的免疫相关酶和Toll通路基因的影响。研究表明当绿僵菌菌株（IMI330189）和MAD1蛋白混合处理飞蝗时,其累计死亡率最高为55%。MAD1+IMI330189混合处理使飞蝗PO的活性增加,单独使用MAD1蛋白处理时致使飞蝗体内的PO、POD和SOD活性降低。在处理72小时后,检测了飞蝗的中肠和体内Toll通路4个基因（MyD88、Cactus、Pelle和CaN）的表达量,发现当MAD1蛋白和MAD1+IMI330189混合处理后,MyD88在中肠和虫体的表达明显下降,但在中肠与虫体的下调程度不一致。IMI330189显著提高了Cactus在中肠和体内的表达。而MAD1+IMI330189混合处理显著降低了Cactus在中肠和体内的表达。单独使用MAD1蛋白和IMI330189处理可显著增加Pelle基因在飞蝗中肠中的表达,而混合处理MAD1+IMI330189可抑制Pelle基因的表达。仅IMI330189和MAD1+IMI330189处理中,Pelle基因在虫体内的表达水平明显升高。另一方面,MAD1显著增加了中肠内CaN的表达。三种处理均显著影响和抑制了蝗虫虫体CaN基因的表达。由此可见,绿僵菌和MAD1蛋白混合处理显著影响了Toll信号通路上的基因,最终增强了蝗虫的感病力。但是,这些影响在飞蝗的不同部位存在显著差异,对飞蝗中肠中Toll通路相关的基因的影响明显大于对虫体中相关基因的影响。本实验同时检测了饵剂饲喂后飞蝗体内解毒酶（MFO）、几丁质酶（CHI）、和抗菌肽（AMP）的活力。研究发现,当蝗虫取食绿僵菌IMI330189和MAD1蛋白72小时后,宿主体内的解毒酶多功能氧化酶（MFO）、几丁质酶（CHI）和AMPs的活性显著增加。MFO、CHI和AMPs活性的增加激活了蝗虫的免疫系统。通过用酶联免疫吸附法测定经喷雾法处理后的飞蝗在第3、5、7天时,内的6种抗氧化酶的酶活,包括几丁质酶（CHI）、酚氧化酶（PO）、超氧化物歧化酶（SOD）、过氧化物酶（POD）、活性氧（ROS）和抗菌肽（AMP）;并通过qPCR法检测了处理后的蝗虫在第3、5、7天时体内的Chitinase、Diptericin、GNBP3这3种基因的表达量。结果表明,在MAD1蛋白、MAD1+IMI330189和IMI33019处理后的第3、5、7天,抗氧化酶几丁质酶、酚氧化酶、超氧化物酶、过氧化物酶、活性氧和抗菌肽活性均显著升高。qPCR结果显示,IMI330189在处理后第3天、5天和第7天显著抑制Chitinase活性,而MAD1、MAD1+IMI330189处理组在第5天显著抑制Chitinase。MAD1+IMI330189处理后第3、5、7天对Diptericin有明显抑制作用。第7天,MAD1+IMI330189和IMI330189显著抑制Diptericin的表达。MAD1+IMI330189在处理第3、5、7天对GNBP3有明显的抑制作用。与对照组相比,MAD1蛋白在处理后第3天能显著抑制GNBP3的表达。结果表明,飞蝗体内的几丁质酶、酚氧化酶、超氧化物酶、过氧化物酶、活性氧、抗菌肽等在体内增高从而激活飞蝗对MAD1和绿僵菌IMI330189侵入的免疫应答。而Chitinase、Diptericin和GNBP3等基因的表达受到抑制,会导致飞蝗感病力增强,容易致使飞蝗死亡。从防治应用的角度来看,建议采用MAD1+IMI330189混合用药对飞蝗有较好的防治效果。结论:1、通过饲喂法发现MAD1蛋白能够增加PO、SOD、MFO、CHI和AMPs活性,并能抑制MyD88,Pelle,Cactus和CaN基因表达量,从而抑制蝗虫的免疫反应而致使飞蝗死亡。2、通过喷施法检测MAD1蛋白能增强东亚飞蝗的受体识别,并引起飞蝗的免疫反应,而AMP的表达量表现为先升高后降低的趋势,飞蝗的死亡率增加。

论文目录

摘要

Abstract

Abbreviations

CHAPTER 1 INTRODUCTION

1.1 Locust Background

1.2 Chemical control

1.3 Biological control

1.4 MAD1 Protein

1.5 Mode of infection of Metarhizium anisopliae

1.6 Host defense

1.7 The Phenoloxidase System

1.8 Antioxidant enzymes and immune related genes

1.9 Toll Pathway

1.10 Objectives

1.11 Technical route of study

Chapter 2 Influence of Metarhizium anisopliae（IMI330189）and MAD1 Protein on enzymatic activities and Toll-related genes of Locusta migratoria

2.1 Summary

2.2 Materials and Methods

2.2.1 Ethics Statement

2.2.2 Locusta migratoria rearing methods and experimental conditions

2.2.3 Fungal cultures

2.2.4 MAD1 protein culture

2.2.5 Bioassay

2.2.6 Preparation of enzyme solution

2.2.7 Enzyme Activity Assay

2.2.7.1 Phenoloxidase

2.2.7.2 Superoxide dismutase and Peroxidase

2.2.8.Real-Time qRT-PCR

2.2.8.1 Total RNA Isolation and cDNA synthesis

2.2.8.2 Quantitative real-time reverse transcription-polymerase reaction

2.3 Data analysis

2.4 Results

2.4.1 Virulence to Locusta migratoria

2.4.2 Enzymatic activities in Locusta migratoria

2.4.3 Relative quantitative expression of Myd88 m RNA in Locusta migratoria

2.4.4 Relative quantitative expression of Cactus m RNA in Locusta migratoria

2.4.5 Relative quantitative expression of Pelle m RNA in Locusta migratoria

2.4.6 Relative quantitative expression of Ca N m RNA in Locusta migratoria

2.5 Discussion

Chapter 3 Involvement of Multi-Function Oxidase,Chitinase and Attacin in the resistance of Locusta migratoria against Metarhizium anisopliae strain（IMI330189）and and MAD1 Protein

3.1 Summary

3.2 Material and Methods

3.2.1 Locusta migratoria rearing and Experimental conditions

3.2.2 Fungal Culture

3.2.3 MAD1 protein culture

3.2.4 Bioassay

3.2.5 Enzyme preparation

3.2.6 Enzyme activity assay

3.2.7 Data analysis

3.3 Results

3.3.1 Application of Metarhizium anisopliae and MAD1 causing virulence to Locusta migratoria

3.3.2 Influence of Metarhizium anisopliae and MAD1 protein on the activity of MFO in Locusta migratoria

3.3.3 Influence of Metarhizium anisopliae and MAD1 Protein on the activity of Attacin in the Locusta migratoria

3.3.4 Influence of Metarhizium anisopliae and MAD1 protein on the activity of Chitinase in Locusta migratoria

3.4 Discussion

Chapter 4 The immune response of Locusta migratoria to Metarhizium anisopliae and MAD1 Protein

4.1 Summary

4.2 Material and Methods

4.2.1 Locust rearing and Experimental conditions

4.2.2 Fungal culture

4.2.3 MAD1 protein culture

4.2.4 Bioassay

4.2.5 Enzyme assay and enzyme activity assay

4.3 Real-Time qRT PCR

4.3.1 Total RNA extraction and cDNA synthesis

4.3.2 Quantitative Real-Time reverse transcription-polymerase chain reaction

4.3.3 Data analysis

4.4 Results

4.4.1 Virulence to Locusta migratoria

4.4.2 Influence of Metarhizium.anisopliae strain（IMI330189）and MAD1 on the activity of Chitinase in Locusta migratoria

4.4.3 Influence of Metarhizium anisopliae strain IMI330189 and MAD1 on the activity of PO in the Locusta migratoria

4.4.4 Influence of Metarhizium anisopliae strain IMI330189 and MAD1 on the activity of SOD in the Locusta migratoria

4.4.5 Influence of Metarhizium anisopliae strain IMI330189 and MAD1 on the activity of POD in the Locusta migratoria

4.4.6 Influence of Metarhizium anisopliae strain IMI330189 and MAD1 on the activity of ROS in the Locusta migratoria

4.4.7 Influence of Metarhizium anisopliae strain IMI330189 and MAD1 on the activity of AMP in the Locusta migratoria

4.4.8 Relative quantitative expression of Chitinase m RNA in the Locusta migratoria

4.4.9 Relative quantitative expression of Diptericin m RNA in the Locusta migratoria

4.4.10 Relative quantitative expression of GNBP3 m RNA in the Locusta migratoria

4.5 Discussion

Chapter 5 Conclusion

References

Appendix

Acknowledgements

Author Biography

文章来源

类型: 博士论文

作者: Nazir Ahmed Abro

导师: 张泽华

关键词: 绿僵菌菌株,蛋白,飞蝗,通路,酶活

来源: 中国农业科学院

年度: 2019

分类: 基础科学,农业科技

专业: 生物学,农业基础科学,植物保护

单位: 中国农业科学院

分类号: S432.44

DOI: 10.27630/d.cnki.gznky.2019.000036

总页数: 80

文件大小: 4444k

下载量: 2

飞蝗对绿僵菌IMI330189和MAD1蛋白的免疫应答

论文摘要

论文目录

文章来源

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