蜡梅花香相关MYC2和bHLH13转录因子的克隆和功能鉴定
发布时间：2020-12-17 08:47
蜡梅（Chimonanthus praecox L.）是我国特有的传统植物,具有重要的观赏价值和经济价值,以其独特的开花时间和浓郁的花香而广为人知。因为缺乏基础的基因组和转录组数据,蜡梅的遗传和分子生物学研究较少。蜡梅芳香怡人,前期研究表明,蜡梅在盛花期释放大量的挥发成分,包括单萜类和倍半萜类,尤其以芳樟醇,β-罗勒烯和乙酸苄酯含量最为丰富。另外黄色和红色的花瓣中也含有黄酮醇和花青素。本研究以H29（Huazhong 29）蜡梅品系为材料,从5个不同花发育时期构建的转录组库中筛选到两个bHLH家族转录因子,分别命名为CpMYC2和CpbHLH13,序列分析表明CpMYC2含有1884 bp的开放阅读框,编码627个氨基酸;而CpbHLH13含有1779 bp的开放阅读框,编码593个氨基酸。两个基因序列都含有bHLH转录因子家族的保守结构域。实时定量表达分析显示这两个转录因子在盛花期表达最高,进而说明它们在开花过程中参与挥发性有机化合物和花色素的合成并具有重要的作用。系统进化分析表明,它们分别属于bHLH的MYC2和bHLH13/JAM2转录因子。将CpMYC2转录因子在拟南芥AtMY...
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：103 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
List of Abbreviations
1 INTRODUCTION
1.1 Floral scent and volatile organic compounds
1.2 Importance of terpenoids in plants
1.3 Terponoids biosynthesis in Arabidopsis thaliana
1.4 Role of Jasmonates（JAs）in volatiles production
1.5 Role of transcription factors in volatile production
1.6 The interaction of MYC2 transcription factor with Jasmonates（JAs）in volatiles production
1.7 Regulation of anthocyanin production in plants
1.8 Role of transcription factors in anthocyanin production
1.9 JAMs negatively regulates the responses of JA and the accumulation of anthocyanins
1.10 Wintersweet（Chimonanthus praecox L.）and its floral traits
1.11 Anthocyanin production in the inflorescence of wintersweet
Objectives of this study
2 MATERIAL AND METHODS
2.1 Plant Materials
2.1.1 Wintersweet（Chimonanthus praecox L.）
2.1.2 Arabidopsis（Arabidopsis thaliana）
2.1.3 Tobacco（Nicotiana tabacum）
2.2 Culture and growth of plants for transformation
2.2.1 Arabidopsis（Arabidopsis thaliana）
2.2.2 Tobacco（Nicotiana tabacum）
2.3 Selection and analysis of candidate genes for transformation
2.3.1 Protein structure analysis of the genes
2.3.2 Bioinformatics analysis of the candidate genes
2.4 Gene cloning and transformation
2.4.1 Primers designing for gene amplification
2.4.2 Plant genomic DNA extraction
2.4.3 Plant genomic RNA extraction
2.4.4 cDNA synthesis from RNA
2.4.5 Polymerase chain reaction（PCR）
2.4.6 Gel purification
2.4.7 Gene sequencing by TA cloning
2.4.8 Expression vector construction
2.4.9 Restriction endonuclease treatments
2.4.10 Culture and growth of bacteria
2.4.11 Gene transformation into plant materials
2.4.12 Screening for positive transformed clones
2.4.13 RNA extraction and real-time PCR
2.5 Volatile organic compounds collection and analysis
2.5.1 Volatile organic compounds collection from Tobacco plants
2.5.2 Volatile organic compounds collection from Arabidopsis plants
2.5.3 Volatile compounds analysis by GC-MS
2.6 Application of MeJA and GA3 to Arabidopsis plants
2.7 Analysis of total anthocyanin contents from Tobacco flowers
3 RESULTS AND ANALYSIS
3.1 Candidate genes selection from Wintersweet（Chimonanthus praecox）
3.2 Molecular cloning and functional characterization of CpMYC2 Wintersweet transcriptional factor
3.2.1 Transcriptomic expression pattern of CpMYC2 in Wintersweet H29 inflorescence
3.2.2 Bioinformatics analysis of the CpMYC2
3.2.3 Cloning and construction of the transfer vector
3.2.4 Transformation of CpMYC2 gene in plants
3.2.5 Functional analysis of CpMYC2 gene in transgenic plants
3.2.6 Transformation of CpMYC2 into Tobacco plants
3.3 Molecular cloning and functional characterization of CpbHLH13 from Wintersweet H
3.3.1 The quantitative expression pattern of CpbHLH13 in Wintersweet H29 inflorescence
3.3.2 Bioinformatics analysis of the CpbHLH13
3.3.3 Cloning and construction of transfer vectors
3.3.4 Transformation of CpbHLH13 into plants
3.3.5 Functional analysis of CpbHLH13 gene in transgenic plants
3.3.6 Transformation of CpbHLH13 into Tobacco plants
4 DISCUSSION
4.1 Motives behind the study of bHLH transcription factor
4.2 Gene selection and phylogenetic analysis revealed the gene characters
4.3 bHLH transcription factors are involved in VOC production
4.4 Over expressing of CpMYC2 and CpbHLH13 increases the volatiles production
4.5 MYC2 and its involvement in the regulation of crosstalk between JA and GA3 enhances terpene production
4.6 CpbHLH13 reduces the anthocyanin contents in Tobacco inflorescence
Summary
Future prospects and recommendations
REFERENCES
Acknowledgement
【参考文献】：
期刊论文
[1]不同品种百合花挥发性成分定性与定量分析[J]. 张辉秀,胡增辉,冷平生,王文和,徐芳,赵静. 中国农业科学. 2013(04)
[2]不同种姜花香气成分分析[J]. 范燕萍,王旭日,余让才,杨萍. 园艺学报. 2007(01)
[3]姜花挥发性成分的固相微萃取—气相色谱质谱分析[J]. 范燕萍,余让才,黄蕴,陈玉芬. 园艺学报. 2003(04)
本文编号：2921753
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：103 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
List of Abbreviations
1 INTRODUCTION
1.1 Floral scent and volatile organic compounds
1.2 Importance of terpenoids in plants
1.3 Terponoids biosynthesis in Arabidopsis thaliana
1.4 Role of Jasmonates（JAs）in volatiles production
1.5 Role of transcription factors in volatile production
1.6 The interaction of MYC2 transcription factor with Jasmonates（JAs）in volatiles production
1.7 Regulation of anthocyanin production in plants
1.8 Role of transcription factors in anthocyanin production
1.9 JAMs negatively regulates the responses of JA and the accumulation of anthocyanins
1.10 Wintersweet（Chimonanthus praecox L.）and its floral traits
1.11 Anthocyanin production in the inflorescence of wintersweet
Objectives of this study
2 MATERIAL AND METHODS
2.1 Plant Materials
2.1.1 Wintersweet（Chimonanthus praecox L.）
2.1.2 Arabidopsis（Arabidopsis thaliana）
2.1.3 Tobacco（Nicotiana tabacum）
2.2 Culture and growth of plants for transformation
2.2.1 Arabidopsis（Arabidopsis thaliana）
2.2.2 Tobacco（Nicotiana tabacum）
2.3 Selection and analysis of candidate genes for transformation
2.3.1 Protein structure analysis of the genes
2.3.2 Bioinformatics analysis of the candidate genes
2.4 Gene cloning and transformation
2.4.1 Primers designing for gene amplification
2.4.2 Plant genomic DNA extraction
2.4.3 Plant genomic RNA extraction
2.4.4 cDNA synthesis from RNA
2.4.5 Polymerase chain reaction（PCR）
2.4.6 Gel purification
2.4.7 Gene sequencing by TA cloning
2.4.8 Expression vector construction
2.4.9 Restriction endonuclease treatments
2.4.10 Culture and growth of bacteria
2.4.11 Gene transformation into plant materials
2.4.12 Screening for positive transformed clones
2.4.13 RNA extraction and real-time PCR
2.5 Volatile organic compounds collection and analysis
2.5.1 Volatile organic compounds collection from Tobacco plants
2.5.2 Volatile organic compounds collection from Arabidopsis plants
2.5.3 Volatile compounds analysis by GC-MS
2.6 Application of MeJA and GA3 to Arabidopsis plants
2.7 Analysis of total anthocyanin contents from Tobacco flowers
3 RESULTS AND ANALYSIS
3.1 Candidate genes selection from Wintersweet（Chimonanthus praecox）
3.2 Molecular cloning and functional characterization of CpMYC2 Wintersweet transcriptional factor
3.2.1 Transcriptomic expression pattern of CpMYC2 in Wintersweet H29 inflorescence
3.2.2 Bioinformatics analysis of the CpMYC2
3.2.3 Cloning and construction of the transfer vector
3.2.4 Transformation of CpMYC2 gene in plants
3.2.5 Functional analysis of CpMYC2 gene in transgenic plants
3.2.6 Transformation of CpMYC2 into Tobacco plants
3.3 Molecular cloning and functional characterization of CpbHLH13 from Wintersweet H
3.3.1 The quantitative expression pattern of CpbHLH13 in Wintersweet H29 inflorescence
3.3.2 Bioinformatics analysis of the CpbHLH13
3.3.3 Cloning and construction of transfer vectors
3.3.4 Transformation of CpbHLH13 into plants
3.3.5 Functional analysis of CpbHLH13 gene in transgenic plants
3.3.6 Transformation of CpbHLH13 into Tobacco plants
4 DISCUSSION
4.1 Motives behind the study of bHLH transcription factor
4.2 Gene selection and phylogenetic analysis revealed the gene characters
4.3 bHLH transcription factors are involved in VOC production
4.4 Over expressing of CpMYC2 and CpbHLH13 increases the volatiles production
4.5 MYC2 and its involvement in the regulation of crosstalk between JA and GA3 enhances terpene production
4.6 CpbHLH13 reduces the anthocyanin contents in Tobacco inflorescence
Summary
Future prospects and recommendations
REFERENCES
Acknowledgement
【参考文献】：
期刊论文
[1]不同品种百合花挥发性成分定性与定量分析[J]. 张辉秀,胡增辉,冷平生,王文和,徐芳,赵静. 中国农业科学. 2013(04)
[2]不同种姜花香气成分分析[J]. 范燕萍,王旭日,余让才,杨萍. 园艺学报. 2007(01)
[3]姜花挥发性成分的固相微萃取—气相色谱质谱分析[J]. 范燕萍,余让才,黄蕴,陈玉芬. 园艺学报. 2003(04)
本文编号：2921753
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