大丽轮枝菌原生质体转化体系及FreB基因功能分析
发布时间：2021-10-31 15:39
大丽轮枝菌是一种土传、植物性病原真菌,可以引起多种重要经济作物的枯萎死亡。一旦植物被感染后,尚无有效的防治措施。目前,大量的研究工作集中于病原菌毒力及致病关键基因的筛查。尽管农杆菌介导的遗传转化方法普遍用于外源基因的转化,但是仍然需要一种更加快捷的转化方法。本研究中,我们获得了高质量的大丽轮枝菌原生质体用于外源基因的遗传转化。利用此体系,获得了针对铁还原酶跨膜元件前体3（FreB,VDAG06616）基因敲除突变体和回补体。随后,对Fre B基因在病原菌铁还原酶活性以及毒力方面的功能进行了深入的研究。具体研究结果如下:1.利用崩溃酶消化并获得了高质量的原生质体,在TB3液体培养基中再生效率可达65%。通过转化方式（电击和聚乙二醇）以及外源基因状态（线性DNA和质粒）对比发现:在PEG介导的遗传转化中,每微克线性GFP表达盒可得到600个转化子,每微克GFP质粒可得到250个转化子;电击遗传转化中,每微克线性化GFP表达盒可得到29个转化子,每微克GFP质粒可得到24个转化子。2.为探索小干扰RNA（siRNA）能否成功导入原生质体并发挥基因沉默作用,针对上述研究获...
【文章来源】：中国农业科学院北京市
【文章页数】：86 页
【学位级别】：博士
【文章目录】：
摘要
abstract
Abbreviations
CHAPTER I INTRODUCTION
1.1 Verticillium dahliae
1.1.1 Life cycle of Verticillium dahliae
1.1.2 Host range of Verticillium dahliae
1.1.3 Symptoms caused by Verticillium dahliae
1.2 Transformation of Verticillium dahliae
1.2.1 Biolistic transformation
1.2.2 Electroporation
1.2.3 Agrobacterium tumefaciens-mediated transformation (ATMT)
1.2.4 PEG-mediated transformation
1.3 Ferric reductase transmembrane component 3 precursor
1.4 Aims and objectives of the study
CHAPTER II MATERIALS AND METHODS
2.1 Fungal growth and spores’ collection
2.2 Protoplast isolation
2.3 Regeneration of protoplast
2.4 GFP plasmid and siRNAs
2.5 PEG-mediated transformation
2.6 Electroporation
2.7 siRNA inhibition assay for GFP and Vta2 genes
2.8 RNA extraction
2.9 First strand cDNA synthesis
2.10 qRT-PCR analysis of Vta2 expression level
2.11 Genomic DNA isolation
2.12 Phylogenetic analysis of FreB
2.13 Plasmid construction for mutants’ generation
2.13.1 PCR amplification of the respective fragments and genes
2.13.2 Gel purification of PCR products by Gel purification kit
2.13.3 Cloning the purified PCR product into T-vector
2.13.4 Transformation into Escherichia coli (heat shock method)
2.13.5 Construction of gene knockout fragment
2.13.6 Construction of GFP tagged plasmid
2.13.7 Construction of plasmid for complementation
2.13.8 Extraction of plasmid
2.14 PEG-mediated protoplast transformation for mutants’ generation
2.14.1 Generation of FreB knockout mutants (ΔFreB)
2.14.2 Generation of GFP disruption mutants
2.14.3 Generation of FreB complementary mutants (ΔFreB-C)
2.15 Growth of ΔFreB on different carbon sources
2.16 Analysis of growth on media with different iron sources
2.17 Ferric reductase assay
2.18 Oxidative stress assay
2.19 Pathogenicity assay
2.20 Disease index
2.21 Expression analysis of related genes
2.22 Composition of media and buffers
CHAPTER III Building an efficient transformation system for Verticillium dahliae
3.1 Introduction
3.2 Results
3.2.1 Isolation of protoplasts
3.2.2 Regeneration of protoplast
3.2.3 Observation of fluorescence from GFP expression
3.2.4 GFP transformants selection and stability of the transgene
3.2.5 Silencing of GFP gene in Vd-GFP strain with siRNAs
3.2.6 Silencing of Vta2 gene
3.3 Conclusion
CHAPTER IV Plasmid construction for gene deletion and complementation
4.1 Introduction
4.2 Results
4.2.1 Phylogenetic analysis
4.2.2 Generation of FreB mutants
4.2.3 Generation of GFP tagged mutants
4.2.4 Generation of FreB complementary mutants
4.3 Conclusion
CHAPTER V Analysis of ΔFreB mutants
5.1 Introduction
5.2 Results
5.2.1 Carbon sources utilization assay
5.2.2 Comparing the growth of mutants on different iron sources
5.2.3 Deletion of FreB gene impaired the surface ferric reductase activity
5.2.4 Disruption of FreB gene resulted in increased susceptibility to oxidative stress54
5.2.5 Deletion of FreB gene resulted in increased expression level of related genes..555.2.6 FreB deletion attenuates virulence of V. dahliae
5.2.6 FreB deletion attenuates virulence of V. dahliae
5.3 Conclusion
DISCUSSION
CONCLUSION
REFERENCES
ACKNOWLEDGEMENTS
CURRICULUM VITAE
【参考文献】：
期刊论文
[1]不同棉区黄萎病菌对4种常用杀菌剂的敏感性测定[J]. 李彩红,冯自力,李志芳,张志刚,师勇强,赵丽红,朱荷琴. 中国棉花. 2015(08)
[2]Functional Analysis of Autophagy Genes via Agrobacterium-Mediated Transformation in the Vascular Wilt Fungus Verticillium dahliae[J]. Lei Zhou,Jun Zhao,Wangzhen Guo,Tianzhen Zhang. Journal of Genetics and Genomics. 2013(08)
[3]Mapping and Quantitative Trait Loci Analysis of Verticillium Wilt Resistance Genes in Cotton[J]. Hong-Mei Wang1,2, Zhong-Xu Lin1, Xian-Long Zhang1, Wei Chen2, Xiao-Ping Guo1, Yi-Chun Nie1 and Yun-Hai Li2 (1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China; 2Key Laboratory of Cotton Genetic Improvement of the Ministry of Agriculture, Cotton Research Institute, Chinese Academy of Agricultural Sciences, Anyang 455112, China). Journal of Integrative Plant Biology. 2008(02)
本文编号：3468403
【文章来源】：中国农业科学院北京市
【文章页数】：86 页
【学位级别】：博士
【文章目录】：
摘要
abstract
Abbreviations
CHAPTER I INTRODUCTION
1.1 Verticillium dahliae
1.1.1 Life cycle of Verticillium dahliae
1.1.2 Host range of Verticillium dahliae
1.1.3 Symptoms caused by Verticillium dahliae
1.2 Transformation of Verticillium dahliae
1.2.1 Biolistic transformation
1.2.2 Electroporation
1.2.3 Agrobacterium tumefaciens-mediated transformation (ATMT)
1.2.4 PEG-mediated transformation
1.3 Ferric reductase transmembrane component 3 precursor
1.4 Aims and objectives of the study
CHAPTER II MATERIALS AND METHODS
2.1 Fungal growth and spores’ collection
2.2 Protoplast isolation
2.3 Regeneration of protoplast
2.4 GFP plasmid and siRNAs
2.5 PEG-mediated transformation
2.6 Electroporation
2.7 siRNA inhibition assay for GFP and Vta2 genes
2.8 RNA extraction
2.9 First strand cDNA synthesis
2.10 qRT-PCR analysis of Vta2 expression level
2.11 Genomic DNA isolation
2.12 Phylogenetic analysis of FreB
2.13 Plasmid construction for mutants’ generation
2.13.1 PCR amplification of the respective fragments and genes
2.13.2 Gel purification of PCR products by Gel purification kit
2.13.3 Cloning the purified PCR product into T-vector
2.13.4 Transformation into Escherichia coli (heat shock method)
2.13.5 Construction of gene knockout fragment
2.13.6 Construction of GFP tagged plasmid
2.13.7 Construction of plasmid for complementation
2.13.8 Extraction of plasmid
2.14 PEG-mediated protoplast transformation for mutants’ generation
2.14.1 Generation of FreB knockout mutants (ΔFreB)
2.14.2 Generation of GFP disruption mutants
2.14.3 Generation of FreB complementary mutants (ΔFreB-C)
2.15 Growth of ΔFreB on different carbon sources
2.16 Analysis of growth on media with different iron sources
2.17 Ferric reductase assay
2.18 Oxidative stress assay
2.19 Pathogenicity assay
2.20 Disease index
2.21 Expression analysis of related genes
2.22 Composition of media and buffers
CHAPTER III Building an efficient transformation system for Verticillium dahliae
3.1 Introduction
3.2 Results
3.2.1 Isolation of protoplasts
3.2.2 Regeneration of protoplast
3.2.3 Observation of fluorescence from GFP expression
3.2.4 GFP transformants selection and stability of the transgene
3.2.5 Silencing of GFP gene in Vd-GFP strain with siRNAs
3.2.6 Silencing of Vta2 gene
3.3 Conclusion
CHAPTER IV Plasmid construction for gene deletion and complementation
4.1 Introduction
4.2 Results
4.2.1 Phylogenetic analysis
4.2.2 Generation of FreB mutants
4.2.3 Generation of GFP tagged mutants
4.2.4 Generation of FreB complementary mutants
4.3 Conclusion
CHAPTER V Analysis of ΔFreB mutants
5.1 Introduction
5.2 Results
5.2.1 Carbon sources utilization assay
5.2.2 Comparing the growth of mutants on different iron sources
5.2.3 Deletion of FreB gene impaired the surface ferric reductase activity
5.2.4 Disruption of FreB gene resulted in increased susceptibility to oxidative stress54
5.2.5 Deletion of FreB gene resulted in increased expression level of related genes..555.2.6 FreB deletion attenuates virulence of V. dahliae
5.2.6 FreB deletion attenuates virulence of V. dahliae
5.3 Conclusion
DISCUSSION
CONCLUSION
REFERENCES
ACKNOWLEDGEMENTS
CURRICULUM VITAE
【参考文献】：
期刊论文
[1]不同棉区黄萎病菌对4种常用杀菌剂的敏感性测定[J]. 李彩红,冯自力,李志芳,张志刚,师勇强,赵丽红,朱荷琴. 中国棉花. 2015(08)
[2]Functional Analysis of Autophagy Genes via Agrobacterium-Mediated Transformation in the Vascular Wilt Fungus Verticillium dahliae[J]. Lei Zhou,Jun Zhao,Wangzhen Guo,Tianzhen Zhang. Journal of Genetics and Genomics. 2013(08)
[3]Mapping and Quantitative Trait Loci Analysis of Verticillium Wilt Resistance Genes in Cotton[J]. Hong-Mei Wang1,2, Zhong-Xu Lin1, Xian-Long Zhang1, Wei Chen2, Xiao-Ping Guo1, Yi-Chun Nie1 and Yun-Hai Li2 (1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China; 2Key Laboratory of Cotton Genetic Improvement of the Ministry of Agriculture, Cotton Research Institute, Chinese Academy of Agricultural Sciences, Anyang 455112, China). Journal of Integrative Plant Biology. 2008(02)
本文编号：3468403
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