烟草CBL家族基因NsylCBL4和NaylCBL6在非生物胁迫中的功能分析
发布时间：2024-07-07 06:28
钙离子是植物中的第二信使,它参与调节植物的生长发育以及生物和非生物胁迫反应。而类钙调神经素B亚基蛋白(CBL)被认为是植物特异性钙信号传导途径中至关重要的Ca2+传感器。目前为止,本课题组已从林烟草中鉴定到12个CBL蛋白。本研究构建了林烟草NsylCBL4、NsylCBL6的过表达材料并进一步探究了NsylCBL4和NsylCBL6两个基因的功能,主要获得以下结果:(1)蛋白序列分析:NsylCBL4的CDS序列编码234个氨基酸,其氨基酸序列中具有三个Ca2+结合基序的功能性EF手型结构。NsylCBL6的CDS序列编码211个氨基酸,其氨基酸序列中具有三个EF手型结构。另外,NsylCBL6蛋白序列与拟南芥AtCBL7蛋白序列有高度相似性,约71.35%,与杨树PeCBL3蛋白序列的相似性为72.51%。(2)基因过表达材料的获得:通过农杆菌介导的叶盘转化法将NsylCBL4转入烟草中,通过半定量PCR筛选出目标基因的低表达和高表达株系。在24个株系中,选择15号和24号株系的T2代材料进行功能分析。通过浸花法将NsylCBL6基因转入拟南芥中。通过基因组PCR检测T1代,通过半...
【文章页数】：101 页
【学位级别】：博士
【文章目录】：
摘要
abstract
Abbreviation
CHAPTER Ⅰ INTRODUCTION
1.1 STRUCTURE AND MECHANISM OF CBLS
1.2 FUNCTIONS OF CBLS IN PLANTS
1.3 MECHANISMS OF THE CBL-CIPK SIGNALLING NETWORK
1.3.1 Differential calcium response,expression,and localization of CBLs and CIPKs
1.3.2 Differential interaction and activation of CBL-CIPK complexes
1.3.3 Differential target activation by specific CBL-CIPK complexes
1.4 CBLS ARE INVOLVED IN RESPONSE TO BIOTIC AND ABIOTIC STRESS
1.4.1 Magnesium signalling
1.4.2 Sodium signalling
1.4.3 Potassium signalling
1.4.4 Nitrate signalling
1.4.5 Phosphorus signalling
1.5 PROSPECTIVE RESEARCH OF CBL GENES
CHAPTER Ⅱ THE FUNCTIONAL CHARACTERIZATION OF NSYLCBL4 IN ABIOTIC STRESS
2.1 RESEARCH AIMS AND OBJECTIVES
2.2 MATERIALS AND METHODS
2.2.1 Plant materials
2.2.2 Chemical reagents and kits used
2.2.3 Plant’s RNA extraction(Hot Phenol method)reagents
2.2.4 DNA plasmid extraction reagents
2.2.5 Agrobacterium suspension reagents
2.2.6 Others reagents
2.2.7 Antibiotics
2.2.8 Softwares and databases
2.2.9 Bioinformatic analysis
2.2.10 Primers
2.2.11 Construction of plasmids
2.2.12 Fragment purification procedures
2.2.13 Ligation of DNA fragment
2.2.14 Bacterial strains and vectors used
2.2.15 Instruments and equipments used
2.2.16 Plant growth requirements and stress treatments
2.2.17 Plant genomic DNA extraction(CTAB DNA method)
2.2.18 Identification of transgenic homozygous lines
2.2.19 Real-time fluorescence quantitative PCR reaction
2.2.20 PCR product recovery and purification
2.2.21 Screening and identification of positive bacterial colony for PCR
2.2.22 Double enzyme digestion
2.2.23 Transformation techniques of bacteria
2.2.24 Transformation in agrobacterium using the freeze-thaw method
2.2.25 Genetic transformation of tobacco
2.2.26 Transgenic screening analysis
2.2.26.1 Actin PCR
2.2.27 Quantitative real-time PCR(q RT-PCR)setting
2.2.28 Agrobacterium-mediated transient expression in tobacco
2.2.29 Plasmid construction for subcellular localization analysis
2.2.30 Histochemical GUS staining protocol
2.2.31 Plasmid construction for overexpression vector
2.2.32 Measurement of physiological parameters
2.3 RESULTS
2.3.1 Homological analysis of NsylCBL4
2.3.2 Identification of35S:NsylCBL4 transgenic tobacco
2.3.3 Functional analysis of NsylCBL4
2.3.4 Subcellular localization of NsylCBL4
2.3.5 NsylCBL4 proteins structure predictions
2.3.6 Effects of NsylCBL4 on leaf morphology in tobacco
2.3.7 Osmotic treatments to transgenic NsylCBL4,and its chlorophyll content determination
2.3.8 Effects of NsylCBL4 on leaf epidermal cell growth and light response curve
2.4 DISCUSSION
CHAPTER Ⅲ BIOINFORMATICS AND FUNCTIONAL ANALYSIS NSYLCBL6,AND ITS EFFECT ON THE ROOT SYSTEM UNDER LOW NITRATE IN ARABIDOPSIS
3.1 MATERIALS AND METHODS
3.1.1 Database search and Sequence analysis
3.1.2 Homological and structure prediction analysis
3.1.3 Homologue gene:AtCBL7
3.1.4 Molecular cloning
3.1.5 Plasmid constructions and plant transformation
3.1.6 Constructions of overexpression vectors
3.1.7 Plant transformation
3.1.8 Transient expression analysis
3.1.9 Gene expression analysis
3.1.10 Preparation of RNA and first-strand cDNA synthesis
3.1.11 Low NO3
- treatment stress assay
3.1.12 Chlorophyll content determination by suing the robust method via a microplate reader
3.1.13 Root morphological analysis and root hair microscopy
3.2 RESULTS
3.2.1 Nsyl CBL6 cloning and bioinformatics analysis
3.2.2 Classification and evolutionary analysis NsylCBL6
3.2.3 Nsyl CBL6 protein structure and functional analysis
3.2.4 Co-expression and predicted functional partners
3.2.5 Overexpression of NsylCBL6 gene from tobacco in transgenic Arabidopsis plants
3.2.6 Functional characterization of NsylCBL6 in Arabidopsis
3.2.7 Subcellular localization
3.2.7.1 Cytoplasm and nucleus location of NsylCBL6 protein
3.2.8 Effects of nitrate stress on chlorophyll fluorescence parameters and images of transgenic Arabidopsis
3.2.9 Low nitrate increase root hair density in NsylCBl6 in Arabidopsis
3.3 DISCUSSION
3.3.1 Structure and functional analysis
3.3.2 Subcellular localization of NsylCBL
3.3.3 Chlorophyll fluorescence
3.3.4 Effect of NsylCBL6 on the diversity of root system architecture in response to low NO3
-
CHAPTER Ⅳ CONCLUSIONS AND PERSPECTIVE
REFERENCES
ACKNOWLEDGMENTS
AUTHOR RESUME
本文编号：4003294
【文章页数】：101 页
【学位级别】：博士
【文章目录】：
摘要
abstract
Abbreviation
CHAPTER Ⅰ INTRODUCTION
1.1 STRUCTURE AND MECHANISM OF CBLS
1.2 FUNCTIONS OF CBLS IN PLANTS
1.3 MECHANISMS OF THE CBL-CIPK SIGNALLING NETWORK
1.3.1 Differential calcium response,expression,and localization of CBLs and CIPKs
1.3.2 Differential interaction and activation of CBL-CIPK complexes
1.3.3 Differential target activation by specific CBL-CIPK complexes
1.4 CBLS ARE INVOLVED IN RESPONSE TO BIOTIC AND ABIOTIC STRESS
1.4.1 Magnesium signalling
1.4.2 Sodium signalling
1.4.3 Potassium signalling
1.4.4 Nitrate signalling
1.4.5 Phosphorus signalling
1.5 PROSPECTIVE RESEARCH OF CBL GENES
CHAPTER Ⅱ THE FUNCTIONAL CHARACTERIZATION OF NSYLCBL4 IN ABIOTIC STRESS
2.1 RESEARCH AIMS AND OBJECTIVES
2.2 MATERIALS AND METHODS
2.2.1 Plant materials
2.2.2 Chemical reagents and kits used
2.2.3 Plant’s RNA extraction(Hot Phenol method)reagents
2.2.4 DNA plasmid extraction reagents
2.2.5 Agrobacterium suspension reagents
2.2.6 Others reagents
2.2.7 Antibiotics
2.2.8 Softwares and databases
2.2.9 Bioinformatic analysis
2.2.10 Primers
2.2.11 Construction of plasmids
2.2.12 Fragment purification procedures
2.2.13 Ligation of DNA fragment
2.2.14 Bacterial strains and vectors used
2.2.15 Instruments and equipments used
2.2.16 Plant growth requirements and stress treatments
2.2.17 Plant genomic DNA extraction(CTAB DNA method)
2.2.18 Identification of transgenic homozygous lines
2.2.19 Real-time fluorescence quantitative PCR reaction
2.2.20 PCR product recovery and purification
2.2.21 Screening and identification of positive bacterial colony for PCR
2.2.22 Double enzyme digestion
2.2.23 Transformation techniques of bacteria
2.2.24 Transformation in agrobacterium using the freeze-thaw method
2.2.25 Genetic transformation of tobacco
2.2.26 Transgenic screening analysis
2.2.26.1 Actin PCR
2.2.27 Quantitative real-time PCR(q RT-PCR)setting
2.2.28 Agrobacterium-mediated transient expression in tobacco
2.2.29 Plasmid construction for subcellular localization analysis
2.2.30 Histochemical GUS staining protocol
2.2.31 Plasmid construction for overexpression vector
2.2.32 Measurement of physiological parameters
2.3 RESULTS
2.3.1 Homological analysis of NsylCBL4
2.3.2 Identification of35S:NsylCBL4 transgenic tobacco
2.3.3 Functional analysis of NsylCBL4
2.3.4 Subcellular localization of NsylCBL4
2.3.5 NsylCBL4 proteins structure predictions
2.3.6 Effects of NsylCBL4 on leaf morphology in tobacco
2.3.7 Osmotic treatments to transgenic NsylCBL4,and its chlorophyll content determination
2.3.8 Effects of NsylCBL4 on leaf epidermal cell growth and light response curve
2.4 DISCUSSION
CHAPTER Ⅲ BIOINFORMATICS AND FUNCTIONAL ANALYSIS NSYLCBL6,AND ITS EFFECT ON THE ROOT SYSTEM UNDER LOW NITRATE IN ARABIDOPSIS
3.1 MATERIALS AND METHODS
3.1.1 Database search and Sequence analysis
3.1.2 Homological and structure prediction analysis
3.1.3 Homologue gene:AtCBL7
3.1.4 Molecular cloning
3.1.5 Plasmid constructions and plant transformation
3.1.6 Constructions of overexpression vectors
3.1.7 Plant transformation
3.1.8 Transient expression analysis
3.1.9 Gene expression analysis
3.1.10 Preparation of RNA and first-strand cDNA synthesis
3.1.11 Low NO3
- treatment stress assay
3.1.12 Chlorophyll content determination by suing the robust method via a microplate reader
3.1.13 Root morphological analysis and root hair microscopy
3.2 RESULTS
3.2.1 Nsyl CBL6 cloning and bioinformatics analysis
3.2.2 Classification and evolutionary analysis NsylCBL6
3.2.3 Nsyl CBL6 protein structure and functional analysis
3.2.4 Co-expression and predicted functional partners
3.2.5 Overexpression of NsylCBL6 gene from tobacco in transgenic Arabidopsis plants
3.2.6 Functional characterization of NsylCBL6 in Arabidopsis
3.2.7 Subcellular localization
3.2.7.1 Cytoplasm and nucleus location of NsylCBL6 protein
3.2.8 Effects of nitrate stress on chlorophyll fluorescence parameters and images of transgenic Arabidopsis
3.2.9 Low nitrate increase root hair density in NsylCBl6 in Arabidopsis
3.3 DISCUSSION
3.3.1 Structure and functional analysis
3.3.2 Subcellular localization of NsylCBL
3.3.3 Chlorophyll fluorescence
3.3.4 Effect of NsylCBL6 on the diversity of root system architecture in response to low NO3
-
REFERENCES
ACKNOWLEDGMENTS
AUTHOR RESUME
本文编号：4003294
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