短日照誘導(dǎo)甘菊成花轉(zhuǎn)變的分子機(jī)理
發(fā)布時(shí)間：2018-08-06 14:32
【摘要】：植物在特定時(shí)間開花是長(zhǎng)期適應(yīng)環(huán)境的結(jié)果,開花品質(zhì)既是觀賞性狀又是栽培特性。菊花(Chrysanthemum×morifolium Ramat)為我國(guó)的傳統(tǒng)名花,大多數(shù)傳統(tǒng)菊花品種為秋菊和寒菊,為典型的短日植物。對(duì)長(zhǎng)日和短日植物成花調(diào)控機(jī)理研究發(fā)現(xiàn),晝夜節(jié)律鐘基因感受日照信號(hào)長(zhǎng)短并將日長(zhǎng)信號(hào)轉(zhuǎn)變?yōu)殚_花信號(hào)。通過有效操縱晝夜節(jié)律鐘及下游成花關(guān)鍵基因,可以培育出自然花期適合周年產(chǎn)業(yè)化生產(chǎn)的具有自主知識(shí)產(chǎn)權(quán)的菊花新品種。由于菊花遺傳背景復(fù)雜,因此針對(duì)菊花開花調(diào)控機(jī)理的研究進(jìn)展緩慢。本文作者以參與菊花起源物種之一的二倍體植物甘菊[C. lavandulifolium (Fisch. ex Trautv.) Makino]作為研究的模式植物材料,在對(duì)光周期誘導(dǎo)甘菊開花特性進(jìn)行充分研究的基礎(chǔ)上,利用甘菊轉(zhuǎn)錄組及表達(dá)譜數(shù)據(jù)分析的結(jié)果,對(duì)晝夜節(jié)律鐘及下游CO和FT基因進(jìn)行分離并對(duì)其表達(dá)規(guī)律進(jìn)行研究,在此基礎(chǔ)上對(duì)關(guān)鍵基因進(jìn)行轉(zhuǎn)基因研究,以期獲得短日照誘導(dǎo)甘菊成花轉(zhuǎn)變的分子機(jī)理,為菊花花期改良的分子育種奠定理論基礎(chǔ)。 本研究獲得了以下主要結(jié)果： (1)通過對(duì)光周期誘導(dǎo)甘菊開花特性進(jìn)行研究發(fā)現(xiàn)甘菊為典型的短日植物,臨界光周期為13h光照/11h黑暗。甘菊幼齡期結(jié)束時(shí)葉齡為14片真葉。甘菊接受22d短日照(12h光照/12h黑暗)處理后即使隨后處于長(zhǎng)日照條件(16h光照/8h黑暗)下也能順利完成開花過程,因此甘菊限界性光周期為22d短日照。當(dāng)光周期的暗期長(zhǎng)度達(dá)到12h(16h光照/12h黑暗和8h光照/12h黑暗)時(shí),無論光照時(shí)間延長(zhǎng)還是縮短,甘菊均能在14.25-16.25個(gè)循環(huán)處理后順利現(xiàn)蕾,38.45-40.25個(gè)循環(huán)處理后開花。因此甘菊是嚴(yán)格的短日植物,暗期長(zhǎng)度和短日照處理時(shí)間是影響其順利成花的關(guān)鍵因素。 (2)通過對(duì)甘菊短日照誘導(dǎo)條件數(shù)字表達(dá)譜中表達(dá)相對(duì)穩(wěn)定的9個(gè)內(nèi)參候選基因采用實(shí)時(shí)熒光定量PCR技術(shù),分析其在甘菊不同發(fā)育時(shí)期不同組織及不同光周期處理?xiàng)l件下葉片中的表達(dá)穩(wěn)定性,篩選得到甘菊不同發(fā)育時(shí)期不同組織中表達(dá)最為穩(wěn)定的SAND基因和不同光周期處理?xiàng)l件下葉片中表達(dá)最為穩(wěn)定的MTP基因,為后續(xù)相關(guān)基因表達(dá)模式分析奠定了基礎(chǔ)。 (3)通過對(duì)甘菊轉(zhuǎn)錄組數(shù)據(jù)庫進(jìn)行分析,結(jié)合RACE技術(shù)共分離得到11個(gè)甘菊晝夜節(jié)律鐘相關(guān)同源基因。通過對(duì)其在不同組織中的表達(dá)規(guī)律進(jìn)行研究發(fā)現(xiàn),除ClELF3、ClPRR1和ClPRR73基因外,大多數(shù)晝夜節(jié)律鐘基因均在葉片中高表達(dá)。通過對(duì)長(zhǎng)日照(16h光照/8h黑暗)和短日照(12h光照/12h黑暗)條件下晝夜節(jié)律鐘基因表達(dá)規(guī)律進(jìn)行研究,發(fā)現(xiàn)其晝夜節(jié)律表達(dá)模式與擬南芥中晝夜節(jié)律鐘基因表達(dá)模式類似。甘菊中晝夜節(jié)律鐘基因在持續(xù)光照條件下基本能維持晝夜節(jié)律振蕩的特性,但振蕩周期有所改變,振幅降低,表明晝夜節(jié)律鐘基因還受外界環(huán)境條件如光照的調(diào)節(jié)。暗中斷處理?xiàng)l件(12h光照/12h黑暗的暗期中間階段給予2h白光處理)下,ClGIs基因在光照后2h表達(dá)高峰完全消失,ClFKF1基因表達(dá)量下降,而在非誘導(dǎo)的8h光照/8h黑暗條件下CIFKF1基因表達(dá)高峰完全消失,而CIGIs基因的表達(dá)量沒有明顯改變,表明ClFKF1基因與ClGIs基因或單獨(dú)發(fā)揮作用或形成復(fù)合物影響甘菊開花時(shí)間。對(duì)晝夜節(jié)律鐘輸出基因ClGI-1進(jìn)行轉(zhuǎn)基因研究發(fā)現(xiàn),過表達(dá)ClGI-1基因的擬南芥轉(zhuǎn)基因植株開花提前,并且CO和FT基因的表達(dá)水平上升,推測(cè)ClGI-1基因在甘菊中可能通過促進(jìn)CO和FT相關(guān)同源基因的表達(dá)促進(jìn)成花。 (4)利用甘菊轉(zhuǎn)錄組數(shù)據(jù)庫信息,結(jié)合RACE技術(shù)共分離得到11個(gè)甘菊CO同源基因,分別命名為ClCOL1-11基因。組織特異性表達(dá)結(jié)果表明,ClCOL1-5和ClCOL10-11基因均在葉片和莖尖中高量表達(dá),短日照誘導(dǎo)條件下ClCOL4-5基因和ClCOL7-8基因的表達(dá)水平顯著高于長(zhǎng)日照條件下的表達(dá)水平。通過對(duì)CICOL1基因進(jìn)行功能研究發(fā)現(xiàn),其不僅可以促進(jìn)開花,而且在控制株型、株高和開花持續(xù)期方面發(fā)揮作用。ClCOL5基因僅能促進(jìn)擬南芥成花,并且其促進(jìn)成花作用比ClCOL1基因更明顯。這一結(jié)果表明CICOL5基因與甘菊成花誘導(dǎo)過程密切相關(guān),在甘菊中發(fā)揮促進(jìn)成花的作用,而ClCOL1基因可能冗余地與ClCOL5基因發(fā)揮促進(jìn)開花的功能。 (5)利用甘菊轉(zhuǎn)錄組數(shù)據(jù)庫信息,結(jié)合RACE技術(shù)共分離得到2個(gè)FT同源基因,分別命名為ClFT1基因和ClFT2基因。組織特異性表達(dá)分析表明ClFT1基因在葉片中的表達(dá)量高于莖尖,ClFT2基因在莖尖中的表達(dá)量高于葉片。短日照誘導(dǎo)條件下ClFT1基因持續(xù)上升,ClFT2基因持續(xù)下降。轉(zhuǎn)基因研究結(jié)果表明,ClFT1基因促進(jìn)擬南芥成花轉(zhuǎn)變,ClFT2基因抑制擬南芥成花轉(zhuǎn)變,表明其在甘菊成花轉(zhuǎn)變過程中發(fā)揮相反的功能。 綜合上述研究結(jié)果我們得出了本研究的主要結(jié)論：甘菊為嚴(yán)格短日植物,暗期長(zhǎng)度和短日照處理時(shí)間決定其成花與否。光暗的晝夜交替使甘菊晝夜節(jié)律鐘基因表達(dá)保持穩(wěn)定的晝夜節(jié)律,當(dāng)暗期長(zhǎng)度超過其臨界夜長(zhǎng)時(shí),ClGIs基因在光照的開始階段高量表達(dá)進(jìn)而激活ClCOL4/5基因和ClFT1基因表達(dá),而ClFKF1或與ClGIs基因形成復(fù)合物間接抑制ClFT2基因表達(dá),當(dāng)ClFT1基因表達(dá)量遠(yuǎn)遠(yuǎn)超過ClFT2基因時(shí),甘菊即可啟動(dòng)成花轉(zhuǎn)變過程。本研究為研究菊科植物測(cè)量日長(zhǎng)的分子機(jī)制奠定了重要的理論基礎(chǔ),同時(shí)也為通過人工操縱晝夜節(jié)律鐘基因及下游開花基因進(jìn)而改變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.
【學(xué)位授予單位】：北京林業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：S682.11
本文編號(hào)：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.
【學(xué)位授予單位】：北京林業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：S682.11
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