腸道微生物促進(jìn)宿主桔小實(shí)蠅成蟲抵抗低溫脅迫的分子機(jī)制
發(fā)布時(shí)間：2021-03-12 22:20
桔小實(shí)蠅Bactrocera dorsalis（Hendel）是最具入侵性和多食性的害蟲之一,為害250多種水果和蔬菜作物,造成嚴(yán)重?fù)p失。桔小實(shí)蠅的高入侵性原因取決于多方面因素,如寄主范圍廣、繁殖力強(qiáng)、擴(kuò)散能力強(qiáng)、對(duì)環(huán)境脅迫壓力的快速適應(yīng)性等。對(duì)寒冷環(huán)境的適應(yīng)可能是其地理擴(kuò)張的一個(gè)關(guān)鍵因素之一。在環(huán)境脅迫條件下,昆蟲腸道共生菌被證明可顯著提高宿主的環(huán)境適應(yīng)性。然而,在長期的低溫脅迫下,腸道微生物對(duì)宿主適應(yīng)性的作用仍不清楚。本文旨在研究明確腸道微生物在協(xié)助宿主桔小實(shí)蠅抗10℃低溫脅迫中的功能,篩選鑒定其關(guān)鍵腸道共生細(xì)菌,并通過轉(zhuǎn)錄組學(xué)和代謝組學(xué)關(guān)聯(lián)分析和功能研究,闡明腸道微生物影響宿主桔小實(shí)蠅成蟲抵抗低溫脅迫的分子機(jī)制。1.腸道微生物增強(qiáng)宿主桔小實(shí)蠅成蟲對(duì)低溫脅迫的抵抗力首先研究了腸道共生菌在桔小實(shí)蠅成蟲抵抗低溫脅迫中的作用。結(jié)果表明,通過抗生素處理去除腸道菌群后,將桔小實(shí)蠅暴露于10℃的低溫脅迫中,中位生存時(shí)間顯著降低至正常種群組桔小實(shí)蠅的約68%。通過回補(bǔ)篩選,我們發(fā)現(xiàn)Klebsiella michiganensis BD1 77是影響桔小實(shí)蠅成蟲抗寒性的一種關(guān)鍵共生細(xì)菌,通過回補(bǔ)實(shí)...
【文章來源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校
【文章頁數(shù)】：226 頁
【學(xué)位級(jí)別】：博士
【文章目錄】：
摘要
Abstract
List of Abbreviations
Chapter 1: General introduction and literature review
1.1 Background of oriental fruit fly
1.2 Management strategies of oriental fruit fly
1.2.1 Chemical control
1.2.2 Cultural control
1.2.3 Attractant cue-lures
1.2.4 Male annihilation technique
1.2.5 Biological control
1.2.6 Sterile insect technique
1.2.7 Possible applications of gut bacteria for the management of insect pests
1.3 Fruit Fly Gut Microbiome Diversity
1.3.1 Tephritidae Fruit fly microbiota structure
1.3.2 Factors influencing gut microbial diversity
1.4 Gut microbe functions for the fitness improvement of host fruit flies
1.4.1 Gut bacteria provide nutrients to the host
1.4.2 Development and reproduction
1.4.3 Gut symbionts assist to overcome host plant defences
1.4.4 Resistance to pathogens
1.4.5 Symbiont-mediated insecticide resistance
1.5 Practical applications of gut microbiota in fruit flies
1.5.1 Probiotics used in mass-reared SIT
1.5.2 Attractant cue-lures
1.6 Knowledge gaps
1.6.1 Combination of incompatible insect technique （ⅡT） and SIT
1.6.2 Harnessing gut bacteria to promote the efficiency of mass-reared flies
1.6.3 Symbiont-mediated stress resistance
1.6.4 Possible applications of gut bacteria for the management of fruit flies
1.7 Identification and characterization of stress related genes stimulated by gut microbiota
1.7.1 Functions of molecular chaperones under extreme temperature stress
1.7.2 Importance of molecular cryoprotectants during extreme temperature stress
1.7.3 Symbiont-mediated stimulation of molecular cryoprotectant genes during low-temperature stress
1.8 Research objectives
Chapter 2: Gut microbiota enhances the host resistance to low-temperature stress in adultBactrocera dorsalis
2.1 Introduction
2.2 Materials and Methods
2.2.1 Fly culture
2.2.2 Isolation and identification of cultivable bacteria
2.2.3 Antibiotic treatment
2.2.4 Fitness parameter tests
2.2.5 Real-time quantitative PCR（RT-qPCR）
2.2.6 Statistical analysis
2.3 Results
2.3.1 Effect of the gut microbiota on the survival of B. dorsalis under low-temperature
2.3.2 A key symbiotic bacterium, K. michiganensis, enhances the resistance of B. dorsalis tolow-temperature stress
2.3.3 K. michiganensis maintenance in the gut improves host health throughout lifepostexposure to 10℃
2.3.4 K. michiganensis enhances the major nutrients level of B. dorsalis during low-temperature stress
2.3.5 Gut symbionts does not promote survival under UV stress treatment
2.4 Discussion
Chapter 3: Impact of gut microbiota on the metabolomic and transcriptomic responses of the hostto low-temperature stress in Bactrocera dorsalis
3.1 Introduction
3.2 Materials and Methods
3.2.1 Insect culture
3.2.2 UPLC/MS analysis
3.2.3 RNA sequencing
3.2.4 Primer designing
3.2.5 Real-time quantitative PCR（RT-qPCR）
3.3 Results
3.3.1 Influence of gut bacteria on the metabolomic response of the host to low-temperaturestress
3.3.2 Impact of gut bacteria on the transcriptomic response of the host to low-temperaturestress
3.4 Discussion
Chapter 4: Gut microbiota promotes host resistance to low-temperature stress by stimulating itsarginine and proline metabolism pathway in adult Bactrocera dorsalis
4.1 Introduction
4.2 Materials and Methods
4.2.1 Insect culture
4.2.2 Strains and plasmids
4.2.3 Laboratory reagents and kits
4.2.4 The Experimental equipment
4.2.5 Antibiotics, reagents, medium and buffer preparation
4.2.6 Microinjection
4.2.7 ATP assay
4.2.8 Real-time quantitative PCR（RT-qPCR）
4.2.9 dsRNA synthesis and delivery by injection
4.2.10 Transmission electron microscopy （TEM）
4.2.11 Quantification of distorted mitochondria percentage
4.2.12 Statistical analysis
4.3 RESULTS
4.3.1 Effect of gut microbiota on the regulation of the transcriptomic and metabolomicpathways of the host
4.3.2 Gut bacteria aids the host to stimulate gene expression levels of the arginine andproline metabolism pathway during low-temperature stress
4.3.3 RNAi mediated silencing of arginine and proline genes reduce the survival ofconventional flies under low-temperature stress
4.3.4 Combined effect of key genes silencing on the survival of conventional fliespostexposure to low-temperature stress
4.3.5 Functional validation of arginine and proline for the improvement in low-temperaturestress resistance
4.3.6 Gut bacteria maintains mitochondrial morphology and ATP levels during low-temperature stress
4.4 Discussion
Chapter 5: Characteristics and expression analysis of arginine and proline metabolism genes inBactrocera dorsalis
5.1 Introduction
5.2 Materials and Methods
5.2.1 Insect culture
5.2.2 Characteristics, sequence alignment and phylogenetic analysis
5.2.3 Developmental stages and tissues
5.2.4 Temperature and 20E treatment
5.2.5 Total RNA extraction and reverse transcription
5.2.6 Real-time quantitative PCR（RT-qPCR）
5.2.7 Statistical analysis
5.3 Results
5.3.1 Identification and characterization of five APMs
5.3.2 Expression of APMs at different developmental stages
5.3.3 Expression levels of APMs in different tissues
5.3.4 Expression of APMs in response to thermal stress
5.3.5 Expression regulation of APMs by 20E
5.4 Discussion
Chapter 6: Summary, innovations and future perspectives
6.1 Summary
6.2 Innovations
6.3 Future perspectives
References
Appendices
Appendix A. Significantly Enriched metabolites from metabolomics analysis
Appendix A1. Differentially expressed metabolites in the conventional/ antibiotic treated（ABX） fruit flies
Appendix A2. Differentially expressed metabolites in Klebsiella michiganensis/ antibiotictreated （ABX） fruit flies
Appendix B.Gene ontology （GO） enrichment analysis of DEGs from transcriptomics data
Appendix B1. Overrepresented GO terms in the conventional/ antibiotic treated （ABX） fruitflies
Appendix B2. Overrepresented GO terms in Klebsiella michiganensis/ antibiotic treated（ABX） fruit flies
Acknowledgements
【參考文獻(xiàn)】：
期刊論文
[1]The proline synthesis enzyme P5CS forms cytoophidia in Drosophila[J]. Bo Zhang,?mür Y.Tastan,Xian Zhou,Chen-Jun Guo,Xuyang Liu,Aaron Thind,Huan-Huan Hu,Suwen Zhao,Ji-Long Liu. Journal of Genetics and Genomics. 2020(03)
[2]橘小實(shí)蠅植物源引誘活性物質(zhì)的生物測(cè)定[J]. 王波,韓英,黃居昌,陳家驊. 應(yīng)用昆蟲學(xué)報(bào). 2012(06)
[3]橘園常用殺螨劑對(duì)巴氏鈍綏螨和柑橘全爪螨的選擇毒性[J]. 肖順根,余麗萍,舒暢,鐘玲,李愛華,夏斌. 植物保護(hù). 2010(03)
[4]前裂長管繭蜂對(duì)桔小實(shí)蠅的寄生效能研究[J]. 邵屯,劉春燕,陳科偉,曾玲. 華南農(nóng)業(yè)大學(xué)學(xué)報(bào). 2009(02)
[5]廣東橘小實(shí)蠅寄生蜂調(diào)查[J]. 姚婕敏,謝翠紅,何衍彪,邱波,陳華燕,許再福. 環(huán)境昆蟲學(xué)報(bào). 2008(04)
[6]布氏潛蠅繭蜂對(duì)橘小實(shí)蠅幼蟲寄生作用的研究[J]. 呂增印,黃居昌,季清娥,楊建全,陳家驊. 華東昆蟲學(xué)報(bào). 2007(03)
[7]桔小實(shí)蠅不同發(fā)育階段過冷卻點(diǎn)的測(cè)定[J]. 侯柏華,張潤杰. 昆蟲學(xué)報(bào). 2007(06)
[8]長尾潛蠅繭蜂對(duì)橘小實(shí)蠅幼蟲的寄生效能[J]. 林玲,黃居昌,陳家驊,季清娥,楊建全. 華東昆蟲學(xué)報(bào). 2006(04)
[9]阿里山潛蠅繭蜂對(duì)橘小實(shí)蠅卵的寄生效能[J]. 郭慶亮,黃居昌,季清娥,楊建全,陳家驊. 華東昆蟲學(xué)報(bào). 2006(04)
[10]球孢白僵菌對(duì)桔小實(shí)蠅致病力的測(cè)定[J]. 潘志萍,李敦松,黃少華. 華中農(nóng)業(yè)大學(xué)學(xué)報(bào). 2006(05)
本文編號(hào)：3079081
【文章來源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校
【文章頁數(shù)】：226 頁
【學(xué)位級(jí)別】：博士
【文章目錄】：
摘要
Abstract
List of Abbreviations
Chapter 1: General introduction and literature review
1.1 Background of oriental fruit fly
1.2 Management strategies of oriental fruit fly
1.2.1 Chemical control
1.2.2 Cultural control
1.2.3 Attractant cue-lures
1.2.4 Male annihilation technique
1.2.5 Biological control
1.2.6 Sterile insect technique
1.2.7 Possible applications of gut bacteria for the management of insect pests
1.3 Fruit Fly Gut Microbiome Diversity
1.3.1 Tephritidae Fruit fly microbiota structure
1.3.2 Factors influencing gut microbial diversity
1.4 Gut microbe functions for the fitness improvement of host fruit flies
1.4.1 Gut bacteria provide nutrients to the host
1.4.2 Development and reproduction
1.4.3 Gut symbionts assist to overcome host plant defences
1.4.4 Resistance to pathogens
1.4.5 Symbiont-mediated insecticide resistance
1.5 Practical applications of gut microbiota in fruit flies
1.5.1 Probiotics used in mass-reared SIT
1.5.2 Attractant cue-lures
1.6 Knowledge gaps
1.6.1 Combination of incompatible insect technique （ⅡT） and SIT
1.6.2 Harnessing gut bacteria to promote the efficiency of mass-reared flies
1.6.3 Symbiont-mediated stress resistance
1.6.4 Possible applications of gut bacteria for the management of fruit flies
1.7 Identification and characterization of stress related genes stimulated by gut microbiota
1.7.1 Functions of molecular chaperones under extreme temperature stress
1.7.2 Importance of molecular cryoprotectants during extreme temperature stress
1.7.3 Symbiont-mediated stimulation of molecular cryoprotectant genes during low-temperature stress
1.8 Research objectives
Chapter 2: Gut microbiota enhances the host resistance to low-temperature stress in adultBactrocera dorsalis
2.1 Introduction
2.2 Materials and Methods
2.2.1 Fly culture
2.2.2 Isolation and identification of cultivable bacteria
2.2.3 Antibiotic treatment
2.2.4 Fitness parameter tests
2.2.5 Real-time quantitative PCR（RT-qPCR）
2.2.6 Statistical analysis
2.3 Results
2.3.1 Effect of the gut microbiota on the survival of B. dorsalis under low-temperature
2.3.2 A key symbiotic bacterium, K. michiganensis, enhances the resistance of B. dorsalis tolow-temperature stress
2.3.3 K. michiganensis maintenance in the gut improves host health throughout lifepostexposure to 10℃
2.3.4 K. michiganensis enhances the major nutrients level of B. dorsalis during low-temperature stress
2.3.5 Gut symbionts does not promote survival under UV stress treatment
2.4 Discussion
Chapter 3: Impact of gut microbiota on the metabolomic and transcriptomic responses of the hostto low-temperature stress in Bactrocera dorsalis
3.1 Introduction
3.2 Materials and Methods
3.2.1 Insect culture
3.2.2 UPLC/MS analysis
3.2.3 RNA sequencing
3.2.4 Primer designing
3.2.5 Real-time quantitative PCR（RT-qPCR）
3.3 Results
3.3.1 Influence of gut bacteria on the metabolomic response of the host to low-temperaturestress
3.3.2 Impact of gut bacteria on the transcriptomic response of the host to low-temperaturestress
3.4 Discussion
Chapter 4: Gut microbiota promotes host resistance to low-temperature stress by stimulating itsarginine and proline metabolism pathway in adult Bactrocera dorsalis
4.1 Introduction
4.2 Materials and Methods
4.2.1 Insect culture
4.2.2 Strains and plasmids
4.2.3 Laboratory reagents and kits
4.2.4 The Experimental equipment
4.2.5 Antibiotics, reagents, medium and buffer preparation
4.2.6 Microinjection
4.2.7 ATP assay
4.2.8 Real-time quantitative PCR（RT-qPCR）
4.2.9 dsRNA synthesis and delivery by injection
4.2.10 Transmission electron microscopy （TEM）
4.2.11 Quantification of distorted mitochondria percentage
4.2.12 Statistical analysis
4.3 RESULTS
4.3.1 Effect of gut microbiota on the regulation of the transcriptomic and metabolomicpathways of the host
4.3.2 Gut bacteria aids the host to stimulate gene expression levels of the arginine andproline metabolism pathway during low-temperature stress
4.3.3 RNAi mediated silencing of arginine and proline genes reduce the survival ofconventional flies under low-temperature stress
4.3.4 Combined effect of key genes silencing on the survival of conventional fliespostexposure to low-temperature stress
4.3.5 Functional validation of arginine and proline for the improvement in low-temperaturestress resistance
4.3.6 Gut bacteria maintains mitochondrial morphology and ATP levels during low-temperature stress
4.4 Discussion
Chapter 5: Characteristics and expression analysis of arginine and proline metabolism genes inBactrocera dorsalis
5.1 Introduction
5.2 Materials and Methods
5.2.1 Insect culture
5.2.2 Characteristics, sequence alignment and phylogenetic analysis
5.2.3 Developmental stages and tissues
5.2.4 Temperature and 20E treatment
5.2.5 Total RNA extraction and reverse transcription
5.2.6 Real-time quantitative PCR（RT-qPCR）
5.2.7 Statistical analysis
5.3 Results
5.3.1 Identification and characterization of five APMs
5.3.2 Expression of APMs at different developmental stages
5.3.3 Expression levels of APMs in different tissues
5.3.4 Expression of APMs in response to thermal stress
5.3.5 Expression regulation of APMs by 20E
5.4 Discussion
Chapter 6: Summary, innovations and future perspectives
6.1 Summary
6.2 Innovations
6.3 Future perspectives
References
Appendices
Appendix A. Significantly Enriched metabolites from metabolomics analysis
Appendix A1. Differentially expressed metabolites in the conventional/ antibiotic treated（ABX） fruit flies
Appendix A2. Differentially expressed metabolites in Klebsiella michiganensis/ antibiotictreated （ABX） fruit flies
Appendix B.Gene ontology （GO） enrichment analysis of DEGs from transcriptomics data
Appendix B1. Overrepresented GO terms in the conventional/ antibiotic treated （ABX） fruitflies
Appendix B2. Overrepresented GO terms in Klebsiella michiganensis/ antibiotic treated（ABX） fruit flies
Acknowledgements
【參考文獻(xiàn)】：
期刊論文
[1]The proline synthesis enzyme P5CS forms cytoophidia in Drosophila[J]. Bo Zhang,?mür Y.Tastan,Xian Zhou,Chen-Jun Guo,Xuyang Liu,Aaron Thind,Huan-Huan Hu,Suwen Zhao,Ji-Long Liu. Journal of Genetics and Genomics. 2020(03)
[2]橘小實(shí)蠅植物源引誘活性物質(zhì)的生物測(cè)定[J]. 王波,韓英,黃居昌,陳家驊. 應(yīng)用昆蟲學(xué)報(bào). 2012(06)
[3]橘園常用殺螨劑對(duì)巴氏鈍綏螨和柑橘全爪螨的選擇毒性[J]. 肖順根,余麗萍,舒暢,鐘玲,李愛華,夏斌. 植物保護(hù). 2010(03)
[4]前裂長管繭蜂對(duì)桔小實(shí)蠅的寄生效能研究[J]. 邵屯,劉春燕,陳科偉,曾玲. 華南農(nóng)業(yè)大學(xué)學(xué)報(bào). 2009(02)
[5]廣東橘小實(shí)蠅寄生蜂調(diào)查[J]. 姚婕敏,謝翠紅,何衍彪,邱波,陳華燕,許再福. 環(huán)境昆蟲學(xué)報(bào). 2008(04)
[6]布氏潛蠅繭蜂對(duì)橘小實(shí)蠅幼蟲寄生作用的研究[J]. 呂增印,黃居昌,季清娥,楊建全,陳家驊. 華東昆蟲學(xué)報(bào). 2007(03)
[7]桔小實(shí)蠅不同發(fā)育階段過冷卻點(diǎn)的測(cè)定[J]. 侯柏華,張潤杰. 昆蟲學(xué)報(bào). 2007(06)
[8]長尾潛蠅繭蜂對(duì)橘小實(shí)蠅幼蟲的寄生效能[J]. 林玲,黃居昌,陳家驊,季清娥,楊建全. 華東昆蟲學(xué)報(bào). 2006(04)
[9]阿里山潛蠅繭蜂對(duì)橘小實(shí)蠅卵的寄生效能[J]. 郭慶亮,黃居昌,季清娥,楊建全,陳家驊. 華東昆蟲學(xué)報(bào). 2006(04)
[10]球孢白僵菌對(duì)桔小實(shí)蠅致病力的測(cè)定[J]. 潘志萍,李敦松,黃少華. 華中農(nóng)業(yè)大學(xué)學(xué)報(bào). 2006(05)
本文編號(hào)：3079081
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