蠟梅花香相關(guān)MYC2和bHLH13轉(zhuǎn)錄因子的克隆和功能鑒定
發(fā)布時(shí)間：2020-12-17 08:47
蠟梅（Chimonanthus praecox L.）是我國(guó)特有的傳統(tǒng)植物,具有重要的觀賞價(jià)值和經(jīng)濟(jì)價(jià)值,以其獨(dú)特的開(kāi)花時(shí)間和濃郁的花香而廣為人知。因?yàn)槿狈A(chǔ)的基因組和轉(zhuǎn)錄組數(shù)據(jù),蠟梅的遺傳和分子生物學(xué)研究較少。蠟梅芳香怡人,前期研究表明,蠟梅在盛花期釋放大量的揮發(fā)成分,包括單萜類(lèi)和倍半萜類(lèi),尤其以芳樟醇,β-羅勒烯和乙酸芐酯含量最為豐富。另外黃色和紅色的花瓣中也含有黃酮醇和花青素。本研究以H29（Huazhong 29）蠟梅品系為材料,從5個(gè)不同花發(fā)育時(shí)期構(gòu)建的轉(zhuǎn)錄組庫(kù)中篩選到兩個(gè)bHLH家族轉(zhuǎn)錄因子,分別命名為CpMYC2和CpbHLH13,序列分析表明CpMYC2含有1884 bp的開(kāi)放閱讀框,編碼627個(gè)氨基酸;而CpbHLH13含有1779 bp的開(kāi)放閱讀框,編碼593個(gè)氨基酸。兩個(gè)基因序列都含有bHLH轉(zhuǎn)錄因子家族的保守結(jié)構(gòu)域。實(shí)時(shí)定量表達(dá)分析顯示這兩個(gè)轉(zhuǎn)錄因子在盛花期表達(dá)最高,進(jìn)而說(shuō)明它們?cè)陂_(kāi)花過(guò)程中參與揮發(fā)性有機(jī)化合物和花色素的合成并具有重要的作用。系統(tǒng)進(jìn)化分析表明,它們分別屬于bHLH的MYC2和bHLH13/JAM2轉(zhuǎn)錄因子。將CpMYC2轉(zhuǎn)錄因子在擬南芥AtMY...
【文章來(lái)源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校
【文章頁(yè)數(shù)】：103 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
【參考文獻(xiàn)】：
期刊論文
[1]不同品種百合花揮發(fā)性成分定性與定量分析[J]. 張輝秀,胡增輝,冷平生,王文和,徐芳,趙靜. 中國(guó)農(nóng)業(yè)科學(xué). 2013(04)
[2]不同種姜花香氣成分分析[J]. 范燕萍,王旭日,余讓才,楊萍. 園藝學(xué)報(bào). 2007(01)
[3]姜花揮發(fā)性成分的固相微萃取—?dú)庀嗌V質(zhì)譜分析[J]. 范燕萍,余讓才,黃蘊(yùn),陳玉芬. 園藝學(xué)報(bào). 2003(04)
本文編號(hào)：2921753
【文章來(lái)源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校
【文章頁(yè)數(shù)】：103 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
【參考文獻(xiàn)】：
期刊論文
[1]不同品種百合花揮發(fā)性成分定性與定量分析[J]. 張輝秀,胡增輝,冷平生,王文和,徐芳,趙靜. 中國(guó)農(nóng)業(yè)科學(xué). 2013(04)
[2]不同種姜花香氣成分分析[J]. 范燕萍,王旭日,余讓才,楊萍. 園藝學(xué)報(bào). 2007(01)
[3]姜花揮發(fā)性成分的固相微萃取—?dú)庀嗌V質(zhì)譜分析[J]. 范燕萍,余讓才,黃蘊(yùn),陳玉芬. 園藝學(xué)報(bào). 2003(04)
本文編號(hào)：2921753
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