拟南芥谷胱甘肽转移酶基因家族的抗病功能分析

摘要

谷胱甘肽S-转移酶(GSTs)是具有降解有毒物质和避免氧化损伤功能的酶的超家族。GSTs广泛参与各种生物学过程，包括生物和非生物胁迫以及调控过程。因此，包括农业生物技术在内的各个领域都对该酶非常关注。目前在植物中鉴定到了14类GSTs，其中Phi和Tau类是最大最重要的类别。Tau类的进化已经明确，但Phi类GSTs(GSTFs)的分布情况还不确切。在本研究中，分析了非植物物种中GSTFs的整体分布和进化关系，并进一步阐述了拟南芥GSTs对强破坏性坏死营养型真菌病原Sclerotinia sclerotiorum抗性的作用及其机制。获得的主要结果如下:
　　1.在非植物物种包括子囊菌、粘菌以及原生生物尾刺耐格里原虫（Naegleria gruberi）和Aureococcus anophagefferens中鉴定获得了GSTF序列。GSTFs在这些细菌和原生生物中的分布与其基因组大小及其栖息地相关，而在子囊菌中的分布和丰度则与生活方式关联。序列比较、基因结构合系统发育分析结果显示非植物GSTFs存在显著差异，说明进化过程的多元起源特点。
　　2.利用Atgst敲除突变体分析明确了AtGST基因在S.sclerotiorum抗性中的作用。结果表明，拟南芥GST家族中只有部分成员在S.sclerotiorum抗性中起作用。它们包括AtGSTL2以及Phi类GST大部分成员，但它们所起作用的重要性各不相同。这些AtGSTs涉及在病菌侵染后不同阶段起作用。
　　3.利用防卫信号传导基因突变体的分析结果显示，水杨酸途径充当了病原侵染后AtGSTs基因表达的放大扩增器。在诱导AtGSTF2中NPR1的作用比水杨酸含量更重要。乙烯途径不同因子对组成性以及病原侵染诱导的AtGSTs表达的调控作用各不相同。EIN2对这两类AtGSTs表达均起重要调控作用但调控作用方向相反。结果还表明，AtGSTF2和AtGSTF6的表达受多种因子的紧密而不同效应方向的调控。这些因子包括数个AtWRK Ys和AtCAMTA3。AtWRKY70负调控S.sclerotiorum抗性，是病原诱导激活AtGSTF2所必需，也是病原诱导AtGSTF6部分需要。AtWRKY70作用于NPR1以及大多数防卫反应通路调控因子的下游。此外，AtGSTF1强烈正向影响EIN2的组成性表达。
　　4.感S.sclerotiorum的AtGST突变体呈现改变的PAMP激发的活性氧积累。表明这些突变体早期提高的感病性可能与早期MAMP触发免疫信号传导的改变有关。
　　5.采用无胶无标记定量蛋白质组学方法，分析揭示了AtGSTL2抗病调控机制与多条防卫反应途径相关。该基因的敲除突变抑制了多条防卫途径体系，从而导致对S.sclerotiorum的高度敏感性。Atgstl2突变体与野生型对照差异表达蛋白涉及不同途径及许多生物学过程，绝大多数被下调表达。病原菌侵染后，Atgstl2突变体与野生型对照相比只有16个显著差异表达蛋白，这些蛋白涉及防卫反应、氧化还原调节和谷胱甘肽代谢。
　　6.鉴定分析结果显示，拟南芥潜在S-亚硝基化蛋白germin3(GER3)是应对S.sclerotiorum关键致病因子草酸的重要防卫因子候选。另外，蛋白BRI1suppressor1(BSU1)-like1可能在针对S.sclerotiorum的PAMP触发免疫信号传导中起重要作用。

目录

声明

Acknowledgments

Contents

Abstract

摘要

Chapter 1 General Introduction and Review of Literature

1．1 Glutathione S transferases(GSTs)and their evolutionary diversification in plant GSTs

1．2 Structural organization of plant cGST proteins

1．3 Functions of Plant GSTs

1．4 Applications of GSTs

Chapter 2 Phi class of Glutathione S-transferase gene superfamily widely exists in nonplant taxonomic groups

2．1 Abstract

2．2 Introduction

2．3 Materials and methods

2．3．1 Database searches

2．3．2 Sequence alignment and construction of phylogenetic tree

2．3．3 Sequence comparison and gene structure analysis

2．3．4 Prediction of functional partners of the identified GSTSTF sequences

2．4 Results

2．4．1 Putative Phi class GSTST sequences are widely distributed in nonplant kingdoms

2．4．2 Putative nonplant GSTSTFs show phylogenetic and gene structure divergence

2．4．3 Protein sequence analyses further demonstrate that the identified nonplant GSTs belong to Phi class

2．4．4 Protein partner prediction reveals functional divergence of nonplant GSTFs

2．5 Discussion

2．6 Conclusion

Chapter 3 Functional analysis of Arabidopsis GSTs and the mechanism underling the resistance mediated by AtGSTFs and AtGSTUs against the necrotrophic fungus Sclerotinia sclerotiorum

3．1 Abstract

3．2 Introduction

3．3 Materials and methods

3．3．1 Plant materials and growth conditions

3．3．2 Fungal strain and plant inoculation procedures

3．3．3 cis-acting regulatory element prediction

3．3．4 Gene expression analyses by RT-qPCR

3．3．5 Detection of ROS bursts

3．3．6 Statistical analysis

3．4 Results

3．4．1 GST mutants exhibited altered susceptibility to S.sclerotiorum infection

3．4．2 Expressional reponse of AtGST genes to S.sclerotiorum

3．4．3 cis-acting regulatory elements in AtGST gene promoters

3．4．4 GST expression pattern in Arabidopsis hormone related mutants constitutively and in response to S.sclerotiorum inoculation

3．4．5 Expression pattern of hormone related genes in AtGSTmutants constitutively and in response to S. sclerotiorum inoculation

3．4．6 Transcription factor-mediated modulation of AtGST gene expression

3．4．7 PAMP-triggered ROS accumulation in AtGST mutants

3．5 Discussion

3．5．1 Role of GSTs in disease resistance

3．5．2 Hormone-mediated regulation of Arabidopsis GST upon S. sclerotiorum infection

3．5．3 Transcription factor-mediated modulation of Arabidopsis GSTs in response to S.sclerotiorum

3．6 Conclusions

Chapter 4 Functional mechanisms of Arabidopsis GSTL2 in resistance against S.sclerotiorum

4．1 Abstract

4．2 Introduction

4．3 Materials and methods

4．3．1 Plant treatment and inoculation

4．3．2 Protein extraction for mass spectrometry analysis

4．3．3 Protein purification and digestion for mass spectrometry analysis

4．3．4 Mass spectrometry analysis

4．3．5 Protein identification

4．3．6 Differential analysis of identified proteins

4．3．7 Bioinformatics analysis

4．3．8 Statistical analyses

4．4 Results

4．4．1 Quercetin pre-treatment did not ameliorate Atgstl2 mutant resistance against S.sclerotiorum

4．4．2 AtGSTL2 gene knock out apparently deregulated SA pathway

4．4．3 AtGSTL2 expression pattern in Arabidopsis hormone related mutants constitutively and in response to S. sclerotiorum inoculation

4．4．4 Expression pattern of hormone related genes in Atgstl2 mutant constitutively and in response to S. sclerotiorum inoculation

4．4．5 Differential protein profile in Atgstl2 mutant

4．5 Discussion

4．5．1 Atgstl2 susceptibility to S.sclerotiorum is related with weakened defense systems

4．5．2 AtGSTL2 function is associated with diverse pathways

4．6 Conclusion

Chapter 5 General discussion and conclusions

5．1．General discussion

5．1．1 Distribution and phylogeny of nonplant GSTFs

5．1．2 Functional analyses of GSTs in Arabidopsis resistance to S. sclerotiorum

5．2．Conclusions

5．3 Significance of the study

References

Supplementary Tables and Figures

Appendix