煙草CBL家族基因NsylCBL4和NaylCBL6在非生物脅迫中的功能分析
發(fā)布時(shí)間：2024-07-07 06:28
鈣離子是植物中的第二信使,它參與調(diào)節(jié)植物的生長(zhǎng)發(fā)育以及生物和非生物脅迫反應(yīng)。而類(lèi)鈣調(diào)神經(jīng)素B亞基蛋白(CBL)被認(rèn)為是植物特異性鈣信號(hào)傳導(dǎo)途徑中至關(guān)重要的Ca2+傳感器。目前為止,本課題組已從林煙草中鑒定到12個(gè)CBL蛋白。本研究構(gòu)建了林煙草NsylCBL4、NsylCBL6的過(guò)表達(dá)材料并進(jìn)一步探究了NsylCBL4和NsylCBL6兩個(gè)基因的功能,主要獲得以下結(jié)果:(1)蛋白序列分析:NsylCBL4的CDS序列編碼234個(gè)氨基酸,其氨基酸序列中具有三個(gè)Ca2+結(jié)合基序的功能性EF手型結(jié)構(gòu)。NsylCBL6的CDS序列編碼211個(gè)氨基酸,其氨基酸序列中具有三個(gè)EF手型結(jié)構(gòu)。另外,NsylCBL6蛋白序列與擬南芥AtCBL7蛋白序列有高度相似性,約71.35%,與楊樹(shù)PeCBL3蛋白序列的相似性為72.51%。(2)基因過(guò)表達(dá)材料的獲得:通過(guò)農(nóng)桿菌介導(dǎo)的葉盤(pán)轉(zhuǎn)化法將NsylCBL4轉(zhuǎn)入煙草中,通過(guò)半定量PCR篩選出目標(biāo)基因的低表達(dá)和高表達(dá)株系。在24個(gè)株系中,選擇15號(hào)和24號(hào)株系的T2代材料進(jìn)行功能分析。通過(guò)浸花法將NsylCBL6基因轉(zhuǎn)入擬南芥中。通過(guò)基因組PCR檢測(cè)T1代,通過(guò)半...
【文章頁(yè)數(shù)】：101 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
本文編號(hào)：4003294
【文章頁(yè)數(shù)】：101 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
本文編號(hào)：4003294
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