短日照诱导甘菊成花转变的分子机理
发布时间：2018-08-06 14:32
【摘要】：植物在特定时间开花是长期适应环境的结果,开花品质既是观赏性状又是栽培特性。菊花(Chrysanthemum×morifolium Ramat)为我国的传统名花,大多数传统菊花品种为秋菊和寒菊,为典型的短日植物。对长日和短日植物成花调控机理研究发现,昼夜节律钟基因感受日照信号长短并将日长信号转变为开花信号。通过有效操纵昼夜节律钟及下游成花关键基因,可以培育出自然花期适合周年产业化生产的具有自主知识产权的菊花新品种。由于菊花遗传背景复杂,因此针对菊花开花调控机理的研究进展缓慢。本文作者以参与菊花起源物种之一的二倍体植物甘菊[C. lavandulifolium (Fisch. ex Trautv.) Makino]作为研究的模式植物材料,在对光周期诱导甘菊开花特性进行充分研究的基础上,利用甘菊转录组及表达谱数据分析的结果,对昼夜节律钟及下游CO和FT基因进行分离并对其表达规律进行研究,在此基础上对关键基因进行转基因研究,以期获得短日照诱导甘菊成花转变的分子机理,为菊花花期改良的分子育种奠定理论基础。 本研究获得了以下主要结果： (1)通过对光周期诱导甘菊开花特性进行研究发现甘菊为典型的短日植物,临界光周期为13h光照/11h黑暗。甘菊幼龄期结束时叶龄为14片真叶。甘菊接受22d短日照(12h光照/12h黑暗)处理后即使随后处于长日照条件(16h光照/8h黑暗)下也能顺利完成开花过程,因此甘菊限界性光周期为22d短日照。当光周期的暗期长度达到12h(16h光照/12h黑暗和8h光照/12h黑暗)时,无论光照时间延长还是缩短,甘菊均能在14.25-16.25个循环处理后顺利现蕾,38.45-40.25个循环处理后开花。因此甘菊是严格的短日植物,暗期长度和短日照处理时间是影响其顺利成花的关键因素。 (2)通过对甘菊短日照诱导条件数字表达谱中表达相对稳定的9个内参候选基因采用实时荧光定量PCR技术,分析其在甘菊不同发育时期不同组织及不同光周期处理条件下叶片中的表达稳定性,筛选得到甘菊不同发育时期不同组织中表达最为稳定的SAND基因和不同光周期处理条件下叶片中表达最为稳定的MTP基因,为后续相关基因表达模式分析奠定了基础。 (3)通过对甘菊转录组数据库进行分析,结合RACE技术共分离得到11个甘菊昼夜节律钟相关同源基因。通过对其在不同组织中的表达规律进行研究发现,除ClELF3、ClPRR1和ClPRR73基因外,大多数昼夜节律钟基因均在叶片中高表达。通过对长日照(16h光照/8h黑暗)和短日照(12h光照/12h黑暗)条件下昼夜节律钟基因表达规律进行研究,发现其昼夜节律表达模式与拟南芥中昼夜节律钟基因表达模式类似。甘菊中昼夜节律钟基因在持续光照条件下基本能维持昼夜节律振荡的特性,但振荡周期有所改变,振幅降低,表明昼夜节律钟基因还受外界环境条件如光照的调节。暗中断处理条件(12h光照/12h黑暗的暗期中间阶段给予2h白光处理)下,ClGIs基因在光照后2h表达高峰完全消失,ClFKF1基因表达量下降,而在非诱导的8h光照/8h黑暗条件下CIFKF1基因表达高峰完全消失,而CIGIs基因的表达量没有明显改变,表明ClFKF1基因与ClGIs基因或单独发挥作用或形成复合物影响甘菊开花时间。对昼夜节律钟输出基因ClGI-1进行转基因研究发现,过表达ClGI-1基因的拟南芥转基因植株开花提前,并且CO和FT基因的表达水平上升,推测ClGI-1基因在甘菊中可能通过促进CO和FT相关同源基因的表达促进成花。 (4)利用甘菊转录组数据库信息,结合RACE技术共分离得到11个甘菊CO同源基因,分别命名为ClCOL1-11基因。组织特异性表达结果表明,ClCOL1-5和ClCOL10-11基因均在叶片和茎尖中高量表达,短日照诱导条件下ClCOL4-5基因和ClCOL7-8基因的表达水平显著高于长日照条件下的表达水平。通过对CICOL1基因进行功能研究发现,其不仅可以促进开花,而且在控制株型、株高和开花持续期方面发挥作用。ClCOL5基因仅能促进拟南芥成花,并且其促进成花作用比ClCOL1基因更明显。这一结果表明CICOL5基因与甘菊成花诱导过程密切相关,在甘菊中发挥促进成花的作用,而ClCOL1基因可能冗余地与ClCOL5基因发挥促进开花的功能。 (5)利用甘菊转录组数据库信息,结合RACE技术共分离得到2个FT同源基因,分别命名为ClFT1基因和ClFT2基因。组织特异性表达分析表明ClFT1基因在叶片中的表达量高于茎尖,ClFT2基因在茎尖中的表达量高于叶片。短日照诱导条件下ClFT1基因持续上升,ClFT2基因持续下降。转基因研究结果表明,ClFT1基因促进拟南芥成花转变,ClFT2基因抑制拟南芥成花转变,表明其在甘菊成花转变过程中发挥相反的功能。 综合上述研究结果我们得出了本研究的主要结论：甘菊为严格短日植物,暗期长度和短日照处理时间决定其成花与否。光暗的昼夜交替使甘菊昼夜节律钟基因表达保持稳定的昼夜节律,当暗期长度超过其临界夜长时,ClGIs基因在光照的开始阶段高量表达进而激活ClCOL4/5基因和ClFT1基因表达,而ClFKF1或与ClGIs基因形成复合物间接抑制ClFT2基因表达,当ClFT1基因表达量远远超过ClFT2基因时,甘菊即可启动成花转变过程。本研究为研究菊科植物测量日长的分子机制奠定了重要的理论基础,同时也为通过人工操纵昼夜节律钟基因及下游开花基因进而改变菊花花期提供了新思路。
[Abstract]:The flowering quality of plants is the result of long-term adaptation to the environment, and the quality of flowering is both ornamental and cultivated. Chrysanthemum x morifolium Ramat is a traditional flower in China. Most of the traditional chrysanthemum varieties are chrysanthemum and chrysanthemum, which are typical short daily plants. The circadian clock gene feels the length of the sunshine signal and changes the day length signal to the flowering signal. By effectively manipulating the circadian clock and the key genes in the lower reaches of the flower, a new chrysanthemum variety with independent intellectual property right for the annual industrial production can be cultivated. The research on the mechanism of flower regulation is slow. The author uses the [C. lavandulifolium (Fisch. ex Trautv.) Makino], one of the diploid plants of the chrysanthemum, as a model plant material. On the basis of the full study of the flowering characteristics of the photoperiod induced chamomile, the data of the chrysanthemum transcriptome and the expression profiles are used. As a result, the genes of the circadian clock and the downstream CO and FT genes were separated and their expression rules were studied. On the basis of this, the key genes were studied in order to obtain the molecular mechanism of the transformation of flower formation by short day illumination, and lay a theoretical foundation for the molecular breeding of chrysanthemum flower flowering stage.
The main results of this study are as follows:
(1) through the study of the flowering characteristics of chamomile induced by photoperiod, it is found that the chrysanthemum is a typical short day plant, and the critical photoperiod is 13h light /11h dark. The leaf age of chamomile is 14 true leaves at the end of the young age. The chrysanthemum is treated with 22d short sunshine (12h light /12h dark) treatment even after the long sunshine conditions (16h light /8h dark) can also be smooth. When the flowering process is completed, the limited Photoperiod of the chamomile is 22d short day. When the length of the dark period of the photoperiod reaches 12h (16h light /12h dark and 8h light /12h dark), no matter the duration or shortening of the illumination time, Gan Jujun can be successfully buds after the 14.25-16.25 cycle treatment and the 38.45-40.25 cycle is treated to blossom. Therefore, the chamomile is strict. Dark days and Short Daylight treatment time were the key factors affecting the smooth flowering of short day plants.
(2) through the real-time fluorescence quantitative PCR technique, the expression stability of the leaves in different tissues and different photoperiod treatments of chamomile at different developmental stages was analyzed by real-time fluorescent quantitative PCR technique, and the table was screened for different tissues in different developmental stages of chamomile. The most stable MTP gene was expressed in the leaves of the most stable SAND gene and different photoperiod, which laid the foundation for the analysis of subsequent related gene expression patterns.
(3) through the analysis of the database of the chamomile transcriptional group, 11 diurnal circadian clock related homologous genes were isolated by RACE technology. Through the study of its expression in different tissues, it was found that most of the circadian clock genes were highly expressed in leaves except ClELF3, ClPRR1 and ClPRR73. The expression pattern of circadian clock gene in 16h light /8h dark) and short sunshine (12h light /12h dark) was studied. It was found that the circadian rhythm expression pattern was similar to that of the circadian clock gene expression pattern in Arabidopsis. The circadian clock gene is also regulated by the ambient conditions, such as light, the circadian clock gene is also regulated by the ambient conditions, such as light, and the 2H expression peak of the ClGIs gene completely disappeared and the ClFKF1 gene expression decreased, and the non induced 8h light /8h was found in the dark interruption treatment conditions (the middle stage of dark dark period of 12h light /12h). The expression peak of CIFKF1 gene expression was completely disappeared under the dark condition, but the expression of CIGIs gene was not significantly changed, indicating that the ClFKF1 gene and the ClGIs gene played a role or formed a complex to influence the flowering time of the chrysanthemum. Transgene study on the output gene ClGI-1 of the circadian clock found that the transgenic Arabidopsis transgene expressed by the ClGI-1 gene was genetically modified. The plant flowering was ahead of time, and the expression level of CO and FT genes increased. It is presumed that the ClGI-1 gene may promote flower formation by promoting the expression of CO and FT related homologous genes in chamomile.
(4) 11 homologous genes of chamomile CO were isolated by RACE technology by using the database information of chamomile transcriptional group and named ClCOL1-11 gene respectively. The tissue specific expression results showed that both ClCOL1-5 and ClCOL10-11 genes were expressed in the leaf and stem apex, and the expression level of ClCOL4-5 gene and ClCOL7-8 gene under the condition of short sunshine induction was obvious. The functional study of CICOL1 gene shows that it not only promotes flowering, but also plays a role in controlling plant type, plant height and flowering duration with.ClCOL5 gene only promoting Arabidopsis flower formation, and it promotes flowering to be more obvious than ClCOL1 gene. This result indicates that CICOL5 The gene is closely related to the induction process of flower formation in chamomile, which plays a role in promoting flower formation in chamomile, and the ClCOL1 gene may be redundant with the ClCOL5 gene to promote the function of flowering.
(5) 2 FT homologous genes were isolated by RACE technology by using the database information of chamomile transcriptional group and named ClFT1 gene and ClFT2 gene respectively. Tissue specific expression analysis showed that the expression of ClFT1 gene in leaf was higher than that of stem tip, and the expression of ClFT2 gene in stem tip was higher than that of leaf. ClFT1 gene was held under short sunshine induction condition. The ClFT2 gene continued to decline. The results of the transgenic study showed that the ClFT1 gene promoted the transformation of Arabidopsis flower formation, and the ClFT2 gene inhibited the transformation of Arabidopsis flower formation, indicating that it played the opposite function in the process of flowering transformation of chamomile.
The main conclusions of the study are as follows: chrysanthemum is a strict short day plant, the length of dark period and the time of short day treatment determine whether the flower is the flower or not. The day and night alternation of light and dark causes the stable circadian rhythm of the gene expression of the circadian clock in the chamomile. When the length of the dark period exceeds its critical night length, the ClGIs gene is illuminated. At the beginning stage, high expression then activates the expression of ClCOL4/5 gene and ClFT1 gene, and the complex of ClFKF1 or ClGIs gene can inhibit the expression of ClFT2 gene indirectly. When the expression of ClFT1 gene is far beyond the ClFT2 gene, the chamomile can start the process of flower transformation. This study lays the foundation for the molecular mechanism of measuring the day length of the chrysanthemum plants. The important theoretical basis also provides a new idea for altering chrysanthemum flowering stage by manipulating circadian clock genes and downstream flowering genes.
【学位授予单位】：北京林业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：S682.11
本文编号：2168032
[Abstract]:The flowering quality of plants is the result of long-term adaptation to the environment, and the quality of flowering is both ornamental and cultivated. Chrysanthemum x morifolium Ramat is a traditional flower in China. Most of the traditional chrysanthemum varieties are chrysanthemum and chrysanthemum, which are typical short daily plants. The circadian clock gene feels the length of the sunshine signal and changes the day length signal to the flowering signal. By effectively manipulating the circadian clock and the key genes in the lower reaches of the flower, a new chrysanthemum variety with independent intellectual property right for the annual industrial production can be cultivated. The research on the mechanism of flower regulation is slow. The author uses the [C. lavandulifolium (Fisch. ex Trautv.) Makino], one of the diploid plants of the chrysanthemum, as a model plant material. On the basis of the full study of the flowering characteristics of the photoperiod induced chamomile, the data of the chrysanthemum transcriptome and the expression profiles are used. As a result, the genes of the circadian clock and the downstream CO and FT genes were separated and their expression rules were studied. On the basis of this, the key genes were studied in order to obtain the molecular mechanism of the transformation of flower formation by short day illumination, and lay a theoretical foundation for the molecular breeding of chrysanthemum flower flowering stage.
The main results of this study are as follows:
(1) through the study of the flowering characteristics of chamomile induced by photoperiod, it is found that the chrysanthemum is a typical short day plant, and the critical photoperiod is 13h light /11h dark. The leaf age of chamomile is 14 true leaves at the end of the young age. The chrysanthemum is treated with 22d short sunshine (12h light /12h dark) treatment even after the long sunshine conditions (16h light /8h dark) can also be smooth. When the flowering process is completed, the limited Photoperiod of the chamomile is 22d short day. When the length of the dark period of the photoperiod reaches 12h (16h light /12h dark and 8h light /12h dark), no matter the duration or shortening of the illumination time, Gan Jujun can be successfully buds after the 14.25-16.25 cycle treatment and the 38.45-40.25 cycle is treated to blossom. Therefore, the chamomile is strict. Dark days and Short Daylight treatment time were the key factors affecting the smooth flowering of short day plants.
(2) through the real-time fluorescence quantitative PCR technique, the expression stability of the leaves in different tissues and different photoperiod treatments of chamomile at different developmental stages was analyzed by real-time fluorescent quantitative PCR technique, and the table was screened for different tissues in different developmental stages of chamomile. The most stable MTP gene was expressed in the leaves of the most stable SAND gene and different photoperiod, which laid the foundation for the analysis of subsequent related gene expression patterns.
(3) through the analysis of the database of the chamomile transcriptional group, 11 diurnal circadian clock related homologous genes were isolated by RACE technology. Through the study of its expression in different tissues, it was found that most of the circadian clock genes were highly expressed in leaves except ClELF3, ClPRR1 and ClPRR73. The expression pattern of circadian clock gene in 16h light /8h dark) and short sunshine (12h light /12h dark) was studied. It was found that the circadian rhythm expression pattern was similar to that of the circadian clock gene expression pattern in Arabidopsis. The circadian clock gene is also regulated by the ambient conditions, such as light, the circadian clock gene is also regulated by the ambient conditions, such as light, and the 2H expression peak of the ClGIs gene completely disappeared and the ClFKF1 gene expression decreased, and the non induced 8h light /8h was found in the dark interruption treatment conditions (the middle stage of dark dark period of 12h light /12h). The expression peak of CIFKF1 gene expression was completely disappeared under the dark condition, but the expression of CIGIs gene was not significantly changed, indicating that the ClFKF1 gene and the ClGIs gene played a role or formed a complex to influence the flowering time of the chrysanthemum. Transgene study on the output gene ClGI-1 of the circadian clock found that the transgenic Arabidopsis transgene expressed by the ClGI-1 gene was genetically modified. The plant flowering was ahead of time, and the expression level of CO and FT genes increased. It is presumed that the ClGI-1 gene may promote flower formation by promoting the expression of CO and FT related homologous genes in chamomile.
(4) 11 homologous genes of chamomile CO were isolated by RACE technology by using the database information of chamomile transcriptional group and named ClCOL1-11 gene respectively. The tissue specific expression results showed that both ClCOL1-5 and ClCOL10-11 genes were expressed in the leaf and stem apex, and the expression level of ClCOL4-5 gene and ClCOL7-8 gene under the condition of short sunshine induction was obvious. The functional study of CICOL1 gene shows that it not only promotes flowering, but also plays a role in controlling plant type, plant height and flowering duration with.ClCOL5 gene only promoting Arabidopsis flower formation, and it promotes flowering to be more obvious than ClCOL1 gene. This result indicates that CICOL5 The gene is closely related to the induction process of flower formation in chamomile, which plays a role in promoting flower formation in chamomile, and the ClCOL1 gene may be redundant with the ClCOL5 gene to promote the function of flowering.
(5) 2 FT homologous genes were isolated by RACE technology by using the database information of chamomile transcriptional group and named ClFT1 gene and ClFT2 gene respectively. Tissue specific expression analysis showed that the expression of ClFT1 gene in leaf was higher than that of stem tip, and the expression of ClFT2 gene in stem tip was higher than that of leaf. ClFT1 gene was held under short sunshine induction condition. The ClFT2 gene continued to decline. The results of the transgenic study showed that the ClFT1 gene promoted the transformation of Arabidopsis flower formation, and the ClFT2 gene inhibited the transformation of Arabidopsis flower formation, indicating that it played the opposite function in the process of flowering transformation of chamomile.
The main conclusions of the study are as follows: chrysanthemum is a strict short day plant, the length of dark period and the time of short day treatment determine whether the flower is the flower or not. The day and night alternation of light and dark causes the stable circadian rhythm of the gene expression of the circadian clock in the chamomile. When the length of the dark period exceeds its critical night length, the ClGIs gene is illuminated. At the beginning stage, high expression then activates the expression of ClCOL4/5 gene and ClFT1 gene, and the complex of ClFKF1 or ClGIs gene can inhibit the expression of ClFT2 gene indirectly. When the expression of ClFT1 gene is far beyond the ClFT2 gene, the chamomile can start the process of flower transformation. This study lays the foundation for the molecular mechanism of measuring the day length of the chrysanthemum plants. The important theoretical basis also provides a new idea for altering chrysanthemum flowering stage by manipulating circadian clock genes and downstream flowering genes.
【学位授予单位】：北京林业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：S682.11
【参考文献】
相关期刊论文 前10条
1 付建新;张超;王翊;戴思兰;;甘菊下胚轴离体再生体系的建立[J];北京林业大学学报;2012年03期
2 付建新;王翊;戴思兰;;高等植物CO基因研究进展[J];分子植物育种;2010年05期
3 郭春晓;田素波;郑成淑;王文莉;孙宪芝;;光周期途径植物开花决定关键基因FT[J];基因组学与应用生物学;2009年03期
4 亓帅;付建新;王翊;杨立文;戴思兰;;甘菊下胚轴遗传转化体系的建立[J];分子植物育种;2014年02期
5 李钱峰;蒋美艳;于恒秀;辛世文;顾铭洪;刘巧泉;;水稻胚乳RNA定量RT-PCR分析中参照基因选择[J];扬州大学学报(农业与生命科学版);2008年02期
6 雍伟东,谭克辉,许智宏,朱至清,种康;高等植物开花时间决定的基因调控研究[J];科学通报;2000年05期
7 胡惠蓉;胡晓龙;狄文伟;包满珠;;‘幻想’矮牵牛幼龄期和限界性诱导光周期的研究[J];园艺学报;2007年01期
8 潘才博;张启翔;潘会堂;孙明;;菊花FT类似基因的克隆与表达分析[J];园艺学报;2010年05期
9 田素波;林桂玉;郑成淑;孙霞;任洪艳;温立柱;;菊花花发育基因CmCO和CmFT的克隆与表达分析[J];园艺学报;2011年06期
10 侯维海;孙鹏;陈全家;李先恩;;地黄实时定量PCR内参基因的筛选[J];中国农学通报;2011年17期
相关博士学位论文 前2条
1 丁焱;DFL基因在甘菊中的转化与表达研究[D];北京林业大学;2009年
2 胡可;花青素苷合成途径中结构基因的表达对菊花和瓜叶菊花色的影响[D];北京林业大学;2010年
，本文编号：2168032
本文链接：https://www.wllwen.com/falvlunwen/zhishichanquanfa/2168032.html
