不同温度下安吉白茶差异表达基因的分离、鉴定及CsPDS3基因的功能研究
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摘要
安吉白茶是一低温诱导白化的特异茶树品种,早春时节低温条件下其叶片具有阶段性返白-复绿现象。本文第一部分,通过人工模拟15℃、19℃、23℃三个不同环境温度培养安吉白茶,选取各温度下性状特征最明显的芽下第二叶为材料,研究温度对光合作用相关色素、叶绿体超微结构以及基因表达的影响；同时通过qRT-PCR技术,检测了不同温度下生长的安吉白茶叶片中质体发育早期阶段相关基因、光合作用系统相关基因以及类胡萝卜素生物合成途径中关键酶基因的表达水平。第二部分,应用Gateway技术体系构建了茶树类胡萝卜素合成途径中关键酶基因—八氢番茄红素脱氢酶3基因(Camellia Sinensis Phytoene Desaturase3,CsPDS3)的RNAi载体,并将其转入根癌农杆菌GV3101；探索了农杆菌渗入法(Agroinfiltration)在茶树基因功能研究中的可行性,并利用该方法初步研究了CsPDS3基因功能。本文主要研究结果如下：
1.选取不同温度下性状特征最明显的安吉白茶芽下第二叶为材料,以常规茶树品种福鼎大白茶作为对照,研究温度对光合作用相关色素及叶绿体超微结构的影响。结果表明,安吉白茶叶片叶绿素和p-胡萝卜素含量随着温度的上升而增加,叶绿体在15℃和19℃条件下发育异常。
2.应用cDNA-AFLP技术,结合银染方法,分析三个培养温度条件下安吉白茶差异表达基因。选取其中85个重复性好的差异条带,进行回收、测序及Blastx比对,结果有40条差异条带比对出同源序列。根据Blastx比对结果,可分为以下几类：物质及能量代谢,次生代谢,抗性相关,细胞结构蛋白及细胞周期,蛋白代谢,转录因子,信号转导,功能未知。另有45条TDFs未找到同源序列。
3.通过qRT-PCR技术,检测了不同温度下生长的安吉白茶叶片中质体发育早期阶段相关基因、光合作用系统相关基因以及类胡萝卜素生物合成途径中关键酶基因的表达水平。结果表明,质体发育早期阶段和儿茶素合成相关基因在19℃条件下表达最高,光合作用系统相关基因以及类胡萝卜素生物合成途径中关键酶基因大部分随着温度的升高而表达上调。
4.根据cDNA-AFLP分离得到的丝氨酸/苏氨酸蛋白磷酸酶基因(bsu-protein phosphatase)片段,采用Primer Premier5.0软件设计扩增两端的特异引物,并通过SMART-RACE技术分别扩增出其3’和5’末端序列,成功获得该基因全长cDNA序列(GenBank登录号JN255182),并命名为茶树CsBsul基因。所得序列全长3166bp,其开放阅读框编码888个氨基酸,蛋白分子量约为97358.3Da。该基因的氨基酸序列与葡萄、杨梅、蓖麻、马铃薯、番茄、拟南芥中的Bsul基因编码的氨基酸序列分别有88%、87%、86%、85%、85%、79%的同源性。茶树丝氨酸/苏氨酸蛋白磷酸酶基因的克隆为进一步了解茶树油菜素内酯(BRs)的信号转导奠定了基础。
5.根据CsPDS3(Camellia sinensis phytoene desaturase3, CsPDS3) cDNA片段(GenBank登录号KC955235)设计引物,通过SMART-RACE技术分别扩增出其3’和5’末端序列,成功获得该基因全长cDNA序列(GenBank登录号KC915039),并将其命名为CsPDS3基因。所得序列全长2326bp,其开放阅读框编码582个氨基酸,蛋白分子量约为64954.9Da。该基因推测的氨基酸序列与葡萄柚、柿子、菊花、葡萄、拟南芥、杏、苦瓜、木瓜、本氏烟、南瓜的相似性分别为87%、86%、85%、84%、84%、84%、82%、82%、82%、81%。CsPDS3基因的全长克隆为进一步研究安吉白茶的白化机理奠定了基础。
6.应用Gateway技术体系构建了茶树CsPDS3基因的RNAi载体,经PCR和DNA测序获得验证,并将其转入根癌农杆菌GV3101。
7.探索了农杆菌渗入法在茶树基因功能研究中的可行性,并通过该方法初步研究了类胡萝卜素合成途径中CsPDS3基因的功能,并成功得到转基因茶树白化叶片。
Tea cultivar Anji Baicha is a special green-revertible albino mutant in early spring. The present thesis was consisted of two parts. In the first part, the albino tea plants were placed in artificial climate incubators at15℃,19℃and23℃, respectively. The second leaf with significant feature was used for photosynthesis related pigments, chloroplast ultrastructures and gene expression. Besides, expression levels of genes involved in etioplast development in early stage, photosynthesis system, and key enzymes genes in carotenoid biosynthesis, in Anji Baicha leaf under different temperatures, which were detected by qRT-PCR. In the second part, the RNAi vector of CsPDS3, a key gene involved in carotenoid biosynthetic pathway, was constructed via Gateway cloning system, and it was transformed it to agrobacterium GV3101. The function of CsPDS3gene was characterized in vivio via agroinfiltration, which explored the feasibility of this method in the study of gene function in tea plant. The results of this thesis were listed as follows:
1. In order to study the impact of temperature on photosynthesis related pigments and chloroplast ultrastructures, the second leaf with significant feature of Anji baicha under different temperatures was selected for further analysis by using common cultivar Fudingdabai as control. The results indicated that chlorphyll and β-carotene increase with temperature rise, and chlorplast abnormal in15℃and19℃.
2. In the present study, the gene expression profiling of a green-revertible albino mutant Anji Baicha at different temperatures was analyzed by using cDNA-AFLP. A total of85differentially expressed genes were isolated. Bioinfomation analysis revealed that differentially expressed genes could be grouped into9functions covering material and energy metabolism, secondary metabolism, defence/disease, cell structure protein and cell cycle, protein metabolism, transcription factor, signal transduction, function unknown and no hits, respectively.
3. Expression levels of genes involved in etioplast development in early stage, photosynthesis system, key enzymes genes in carotenoid biosynthesis, in Anji Baicha leaf under different temperatures, which were detected by qRT-PCR. The results demonstrated that genes involved in etioplast development in early stage and catechin biosynthesis related genes were high expression in19℃, while most genes involved in photosynthesis system and carotenoid biosynthesis were up-regulated expression with risen temperature.
4. According to cDNA-AFLP analysis, TDF Bsul was obtaineed. The specific primers were designed by Primer Premier5.0. Based on the fragment, the full-length of serine/threonine-protein phosphatase, Bsul with3166bp (GenBank Accession No. JN255182) cDNA was obtained via rapid amplification of cDNA ends (RACE), named as CsBsul gene. It contained an open reading frame (ORF) encoding a protein of888amino acid residues with a predicable molecular mass of97358.3Da. The deduced amino acid sequence of CsBsul showed88%,87%,86%,85%,85%,79%homology with serine/threonine-protein phosphatase, Bsul genes from Vitis vinifera, Morella rubra, Ricinus communis, Solanum tuberosum, Solanum lycopersicum, Arabidopsis thaliana, respectively. The cloning and sequencing analysis of CsBsul gene established a solid foundation for further study on the signal transduction of brassinosteroids (BRs) in tea plant.
5. According to partial cDNA of CsPDS3(Genebank Accession No. KC955235), we designed primers for amplification. Based on the fragment, the full-length of CsPDS3with2326bp (GenBank Accession No. KC915039) cDNA was obtained via rapid amplification of cDNA ends (RACE), named CsPDS3gene. It contained an open reading frame (ORF) encoding a polypeptide of582amino acid residues with a predicable molecular mass of64954.9Da. The deduced amino acid sequence showed87%,86%,85%,84%,84%,84%,82%,82%,82%,81%homology with CsPDS3from Citrus x paradise, Diospyros kaki, Chrysanthemum, Vitis vinifera, Arabidopsis thaliana, Prunus armeniaca, Momordica charantia, Carica papaya, Nicotiana benthamiana and Cucurbita moschata, respectively. Cloning full-length cDNA of CsPDS3gene establishes a good foundation for further study on the abino mechanism in Anji Baicha.
6. The CsPDS3gene RNAi vector were constructed via Gateway cloning system, which RNAi vector were confirmed by PCR and DNA sequencing, and transformed it to agrobacterium G V3101.
7. The function of CsPDS3, a gene involved in carotenoid biosynthethtic pathway, was analyzed via agroinfiltration, which exploring the feasibility of this method in the study of gene function in tea plant and obtaining transgenic albino tea leaf.
1.选取不同温度下性状特征最明显的安吉白茶芽下第二叶为材料,以常规茶树品种福鼎大白茶作为对照,研究温度对光合作用相关色素及叶绿体超微结构的影响。结果表明,安吉白茶叶片叶绿素和p-胡萝卜素含量随着温度的上升而增加,叶绿体在15℃和19℃条件下发育异常。
2.应用cDNA-AFLP技术,结合银染方法,分析三个培养温度条件下安吉白茶差异表达基因。选取其中85个重复性好的差异条带,进行回收、测序及Blastx比对,结果有40条差异条带比对出同源序列。根据Blastx比对结果,可分为以下几类：物质及能量代谢,次生代谢,抗性相关,细胞结构蛋白及细胞周期,蛋白代谢,转录因子,信号转导,功能未知。另有45条TDFs未找到同源序列。
3.通过qRT-PCR技术,检测了不同温度下生长的安吉白茶叶片中质体发育早期阶段相关基因、光合作用系统相关基因以及类胡萝卜素生物合成途径中关键酶基因的表达水平。结果表明,质体发育早期阶段和儿茶素合成相关基因在19℃条件下表达最高,光合作用系统相关基因以及类胡萝卜素生物合成途径中关键酶基因大部分随着温度的升高而表达上调。
4.根据cDNA-AFLP分离得到的丝氨酸/苏氨酸蛋白磷酸酶基因(bsu-protein phosphatase)片段,采用Primer Premier5.0软件设计扩增两端的特异引物,并通过SMART-RACE技术分别扩增出其3’和5’末端序列,成功获得该基因全长cDNA序列(GenBank登录号JN255182),并命名为茶树CsBsul基因。所得序列全长3166bp,其开放阅读框编码888个氨基酸,蛋白分子量约为97358.3Da。该基因的氨基酸序列与葡萄、杨梅、蓖麻、马铃薯、番茄、拟南芥中的Bsul基因编码的氨基酸序列分别有88%、87%、86%、85%、85%、79%的同源性。茶树丝氨酸/苏氨酸蛋白磷酸酶基因的克隆为进一步了解茶树油菜素内酯(BRs)的信号转导奠定了基础。
5.根据CsPDS3(Camellia sinensis phytoene desaturase3, CsPDS3) cDNA片段(GenBank登录号KC955235)设计引物,通过SMART-RACE技术分别扩增出其3’和5’末端序列,成功获得该基因全长cDNA序列(GenBank登录号KC915039),并将其命名为CsPDS3基因。所得序列全长2326bp,其开放阅读框编码582个氨基酸,蛋白分子量约为64954.9Da。该基因推测的氨基酸序列与葡萄柚、柿子、菊花、葡萄、拟南芥、杏、苦瓜、木瓜、本氏烟、南瓜的相似性分别为87%、86%、85%、84%、84%、84%、82%、82%、82%、81%。CsPDS3基因的全长克隆为进一步研究安吉白茶的白化机理奠定了基础。
6.应用Gateway技术体系构建了茶树CsPDS3基因的RNAi载体,经PCR和DNA测序获得验证,并将其转入根癌农杆菌GV3101。
7.探索了农杆菌渗入法在茶树基因功能研究中的可行性,并通过该方法初步研究了类胡萝卜素合成途径中CsPDS3基因的功能,并成功得到转基因茶树白化叶片。
Tea cultivar Anji Baicha is a special green-revertible albino mutant in early spring. The present thesis was consisted of two parts. In the first part, the albino tea plants were placed in artificial climate incubators at15℃,19℃and23℃, respectively. The second leaf with significant feature was used for photosynthesis related pigments, chloroplast ultrastructures and gene expression. Besides, expression levels of genes involved in etioplast development in early stage, photosynthesis system, and key enzymes genes in carotenoid biosynthesis, in Anji Baicha leaf under different temperatures, which were detected by qRT-PCR. In the second part, the RNAi vector of CsPDS3, a key gene involved in carotenoid biosynthetic pathway, was constructed via Gateway cloning system, and it was transformed it to agrobacterium GV3101. The function of CsPDS3gene was characterized in vivio via agroinfiltration, which explored the feasibility of this method in the study of gene function in tea plant. The results of this thesis were listed as follows:
1. In order to study the impact of temperature on photosynthesis related pigments and chloroplast ultrastructures, the second leaf with significant feature of Anji baicha under different temperatures was selected for further analysis by using common cultivar Fudingdabai as control. The results indicated that chlorphyll and β-carotene increase with temperature rise, and chlorplast abnormal in15℃and19℃.
2. In the present study, the gene expression profiling of a green-revertible albino mutant Anji Baicha at different temperatures was analyzed by using cDNA-AFLP. A total of85differentially expressed genes were isolated. Bioinfomation analysis revealed that differentially expressed genes could be grouped into9functions covering material and energy metabolism, secondary metabolism, defence/disease, cell structure protein and cell cycle, protein metabolism, transcription factor, signal transduction, function unknown and no hits, respectively.
3. Expression levels of genes involved in etioplast development in early stage, photosynthesis system, key enzymes genes in carotenoid biosynthesis, in Anji Baicha leaf under different temperatures, which were detected by qRT-PCR. The results demonstrated that genes involved in etioplast development in early stage and catechin biosynthesis related genes were high expression in19℃, while most genes involved in photosynthesis system and carotenoid biosynthesis were up-regulated expression with risen temperature.
4. According to cDNA-AFLP analysis, TDF Bsul was obtaineed. The specific primers were designed by Primer Premier5.0. Based on the fragment, the full-length of serine/threonine-protein phosphatase, Bsul with3166bp (GenBank Accession No. JN255182) cDNA was obtained via rapid amplification of cDNA ends (RACE), named as CsBsul gene. It contained an open reading frame (ORF) encoding a protein of888amino acid residues with a predicable molecular mass of97358.3Da. The deduced amino acid sequence of CsBsul showed88%,87%,86%,85%,85%,79%homology with serine/threonine-protein phosphatase, Bsul genes from Vitis vinifera, Morella rubra, Ricinus communis, Solanum tuberosum, Solanum lycopersicum, Arabidopsis thaliana, respectively. The cloning and sequencing analysis of CsBsul gene established a solid foundation for further study on the signal transduction of brassinosteroids (BRs) in tea plant.
5. According to partial cDNA of CsPDS3(Genebank Accession No. KC955235), we designed primers for amplification. Based on the fragment, the full-length of CsPDS3with2326bp (GenBank Accession No. KC915039) cDNA was obtained via rapid amplification of cDNA ends (RACE), named CsPDS3gene. It contained an open reading frame (ORF) encoding a polypeptide of582amino acid residues with a predicable molecular mass of64954.9Da. The deduced amino acid sequence showed87%,86%,85%,84%,84%,84%,82%,82%,82%,81%homology with CsPDS3from Citrus x paradise, Diospyros kaki, Chrysanthemum, Vitis vinifera, Arabidopsis thaliana, Prunus armeniaca, Momordica charantia, Carica papaya, Nicotiana benthamiana and Cucurbita moschata, respectively. Cloning full-length cDNA of CsPDS3gene establishes a good foundation for further study on the abino mechanism in Anji Baicha.
6. The CsPDS3gene RNAi vector were constructed via Gateway cloning system, which RNAi vector were confirmed by PCR and DNA sequencing, and transformed it to agrobacterium G V3101.
7. The function of CsPDS3, a gene involved in carotenoid biosynthethtic pathway, was analyzed via agroinfiltration, which exploring the feasibility of this method in the study of gene function in tea plant and obtaining transgenic albino tea leaf.
引文
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