NAC和WRKY轉(zhuǎn)錄因子(TFs)在環(huán)境脅迫中起著關(guān)鍵的作用,然而關(guān)于NAC和WRKY轉(zhuǎn)錄因子在竹子中的研究報道較少。慈竹是重要的經(jīng)濟竹種,其具有很強的生長能力,可以在各種生境中生長,在惡劣的環(huán)境條件下也能存活。本研究的主要目的是鑒定讓慈竹抵抗非生物脅迫環(huán)境相關(guān)的轉(zhuǎn)錄因子,并將其轉(zhuǎn)化到小麥中,研究其功能。本研究成功的從慈竹中克隆得到兩個新的脅迫相關(guān)的轉(zhuǎn)錄因子BeSNAC1”和“BeWRKY2”,并對其進(jìn)行生物信息學(xué)、組織表達(dá)、酵母單雜交和亞細(xì)胞定位分析。利用基因槍法對小麥“DN7742”的幼胚愈傷組織進(jìn)行遺傳轉(zhuǎn)化,并獲得轉(zhuǎn)基因植株,為BeSNAC1”和“BeWRKY2”功能的深入研究奠定基礎(chǔ),也為抗性小麥品種改良提供理論依據(jù)。主要研究結(jié)果如下:1.通過使用生物信息學(xué)方法和來自相應(yīng)植物的相應(yīng)數(shù)據(jù)庫的信息來分離基因。使用來自水稻的應(yīng)激響應(yīng)性SNAC1的氨基酸序列,通過同源克隆法克隆NAC TF。盡管為了分離WRKYTF,利用來自Phyllostachys雜環(huán)基因組數(shù)據(jù)庫和Bambusa Emeiensis轉(zhuǎn)錄組數(shù)據(jù)庫的信息。分離的TF分別命名為“BeSNAC1”和“BeWRKY2”。和“B... 

【文章來源】：西南科技大學(xué)四川省

【文章頁數(shù)】：146 頁

【學(xué)位級別】：碩士

【文章目錄】：
摘要
ABSTRACT
List of abbreviations
1 Introduction
    1.1 Research significance
        1.1.1 Wheat as a chief source of food
        1.1.2 Wheat production and demand
    1.2 Abiotic stresses
        1.2.1 Drought
        1.2.2 Salinity
    1.3 Research Background
        1.3.1 Transcription factors （TFs）
        1.3.2 NAC Transcription factors
        1.3.3 WRKY Transcription factors
    1.4 Research objectives
    1.5 Work layout
2 Cloning and bioinformatics analysis of BeSNACl and BeWRKY2 genesfrom Bambusa emeiensis
    2.1 Testing materials,main instruments and reagents
        2.1.1 Testing materials
        2.1.2 Main instruments for testing
        2.1.3 Main reagents and other materials for testing
        2.1.4 Reagents and medium preparation
    2.2 Testing Methods
        2.2.1 Selection and cloning of genes
        2.2.2 Total RNA extraction
        2.2.3 cDNA synthesis via RT-PCR
        2.2.4 PCR amplification of coding sequence of BeSNAC1 &BeWRKY2
        2.2.5 Validation of amplicons by Gel electrophoresis
        2.2.6 Purification of validated PCR amplicons
        2.2.7 Restriction digestion
        2.2.8 Vector and genes purification
        2.2.9 Ligation of purified amplicons to pMD19-T vector
        2.2.10 Transformation of recombinant plasmids intoEscherichia coli （DH5a）
        2.2.11 Blue-white screening
        2.2.12 PCR confirmation of target genes ligation
        2.2.13 Sequencing of genes
        2.2.14 Culture expansion and plasmids extraction
        2.2.15 Enzymatic validation of successful ligation
    2.3 Bioinformatics analysis of BeSNACl and BeWRKY2 genes
        2.3.1 The conserved motif analysis of BeSNACl and BeWRKY2
        2.3.2 Multiple protein sequence alignment of BeSNAC1 andBeWRKY2
        2.3.3 The phylogenetic analysis of BeSNAC1 and BeWRKY2
        2.3.4 The domain structure analysis of BeSNAC1 and BeWRKY2
    2.4 Results and Analysis
        2.4.1 Total RNA extraction
        2.4.2 PCR Amplification of BeSNACl and BeWRKY2
        2.4.3 The enzymatic validation of BeSNAC1 and BeWRKY2
        2.4.4 Sequencing results analysis
        2.4.5 Domain structure analysis of the BeSNAC1
        2.4.6 Domain structure analysis of the BeWRKY2
        2.4.7 Multiple protein Sequence alignment of BeSNAC1
        2.4.8 Multiple protein Sequence alignment of BeWRKY2
        2.4.9 Phylogenetic analysis of BeSNAC1
        2.4.10 Phylogenetic analysis of BeWRKY2
        2.4.11 The conserved domain structure of BeSNACl
        2.4.12 The conserved domain structure of BeWRKY2
3 Yeast one-hybrid screening of BeSNAC1 and BeWRKY2
    3.1 Testing materials, main instruments and reagents
        3.1.1 Materials for testing
        3.1.2 Main instruments for testing
        3.1.3 Reagents and other materials
        3.1.4 Main reagents preparation
    3.2 Testing methods
        3.2.1 PCR amplification of BeSNACl and BeWRKY2
        3.2.2 Gel electrophoresis validation of PCR amplicons
        3.2.3 Purification of validated PCR amplicons
        3.2.4 Restriction digestion
        3.2.5 Vector and genes purification
        3.2.6 Ligation of genes with pEG202
        3.2.7 Transformation of recombinant plasmids into Escherichiacoli （DH5α）
        3.2.8 Escherichia coli （DH5a） culture
        3.2.9 PCR confirmation of target gene ligation
        3.2.10 Culture expansion and plasmids extraction
        3.2.11 Enzymatic validation
        3.2.12 Yeast EGY-48 Competent cells Preparation and Yeasttransformation
        3.2.13 PCR validation of target genes
    3.3 Results and Analysis
        3.3.1 The PCR amplification of BeSNAC1 and Be WRKY2
        3.3.2 Restriction digestion and ligation
        3.3.3 Enzymatic validation
        3.3.4 RCR validation of target genes in yeast
        3.3.5 Transcriptional activity analysis of BeSNAC1 and BeWRKY2
4 Sub-Cellular localization analysis BeSNAC1 and BeWRKY2
    4.1 Testing materials, main instruments and reagents
        4.1.1 Materials for testing
        4.1.2 Main instruments for testing
        4.1.3 Main reagents and other materials
        4.1.4 Main reagents and medium preparation
    4.2 Testing methods
        4.2.1 PCR amplification of BeSNAC1 and BeWRKY2
        4.2.2 Gel electrophoresis validation of PCR amplicons
        4.2.3 Purification of validated PCR amplicons
        4.2.4 Restriction digestion
        4.2.5 Vector and genes purification
        4.2.6 Ligation of genes with pTEX-GFP
        4.2.7 Insertion of the vector into Escherichia coli （DH5α）
        4.2.8 Escherichia coli （DH5α） culture
        4.2.9 PCR confirmation of target genes ligation
        4.2.10 Culture expansion and plasmids extraction
        4.2.11 Enzymatic validation
        4.2.12 The onion epidermal cells preparation
        4.2.13 Gold preparation
        4.2.14 Coating plasmids with gold
        4.2.15 Transformation using particles delivery system
        4.2.16 Microscopic analysis
    4.3 Results and Analysis
        4.3.1 The gel electrophoresis validation of PCR amplicons
        4.3.2 Restriction digestion and ligation
        4.3.3 Enzymatic validation
        4.3.4 Subcellular localization of BeSNACl and BeWRKY2
5 The expression pattern analysis of BeSNAC1 and BeWRKY2 of Bambusaemeiensis
    5.1 Testing materials, main instruments and reagents
        5.1.1 Testing materials
        5.1.2 Main instruments for testing
        5.1.3 Main reagents and other materials for testing
        5.1.4 Reagents preparation
    5.2 Testing methods
        5.2.1 Plant materials and stress treatments
        5.2.2 Total RNA extraction
        5.2.3 cDNA synthesis via RT-PCR
        5.2.4 The qRT-PCR primers
        5.2.5 Real-time PCR Primers specificity detection
        5.2.6 Expression pattern analysis of BeSNACl and BeWRKY2 byQuantitative Real-time PCR （qRT-PCR）
        5.2.7 Statistical analysis
    5.3 Results and Analysis
        5.3.1 Gel electrophoresis analysis of Total RNA
        5.3.2 The qRT-PCR primers specificity detection
        5.3.3 Expression pattern analysis of BeSNACl via quantitativeReal-time PCR （qRT-PCR）
        5.3.4 Expression pattern analysis of BeWRKY2 via quantitativeReal-time PCR （qRT-PCR）
6 Construction of overexpression vector, wheat transformation andvalidation of overexpressed plants
    6.1 Testing materials, main instruments and reagents
        6.1.1 Testing materials
        6.1.2 Main instruments for testing
        6.1.3 Main reagents and other materials for testing
        6.1.4 Reagents and mediums preparation
    6.2 Testing methods
        6.2.1 Overexpression vector construction
        6.2.2 Ex-plant preparation and Callus induction
        6.2.3 Gold preparation
        6.2.4 Coating overexpression vectors with gold
        6.2.5 Osmotic treatment and transformation
        6.2.6 Validation and overexpression analysis of target genes intransformed plants
    6.3 Results and Analysis
        6.3.1 The gel electrophoresis validation of PCR amplicons
        6.3.2 Restriction digestion and ligation
        6.3.3 Enzymatic validation
        6.3.4 Callus induction and regeneration of seedlings bombardedwith BeSNACl
        6.3.5 Callus induction and regeneration of seedlings bombardedwith BeWRKY2
        6.3.6 Validation and overexpression analysis of BeSNACl intransformed plants
        6.3.7 Validation and Overexpression analysis of BeWRKY2 intransformed plants
        6.3.8 Ti generation of BeSNACl and BeWRKY2 overexpressedplants
7 Discussion and Conclusion
ACKNOWLEDGEMENT
DEDICATION
References
Academic papers and research findings



本文編號：2928615