金花茶花色相关基因的克隆及其功能研究
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摘要
金花茶（Camellia nitidissima）属于山茶属金花茶组，花色金黄，是我国珍稀濒危植物，亦是世界名贵的观赏植物之一，有“茶族皇后”之美誉，是培育黄色山茶花的珍稀遗传资源。尽管关于金花茶杂交育种的研究也屡见报导，但多年的杂交育种工作却很少获得像金花茶花色那么金黄的杂交后代。其原因可能在于研究者对其花色的遗传变异规律以及花色素合成的分子机理不甚了解，未能很好地利用金花茶所特有的种质资源进行黄色山茶花的育种工作。因此，本研究对金花茶花色的形成变化规律以及若干关键基因的功能进行了初步探索，对开展黄色山茶花的分子育种具有重要的理论意义和应用价值。
利用RT-PCR (Reverse transcription polymerase chain reaction)方法和RACE（RapidAmplification of cDNA Ends）技术从金花茶花瓣中分离出6个与类黄酮色素Quercetin（Qu）合成相关基因的cDNA全长，1个与类胡萝卜素新黄质合成相关基因的cDNA全长，分别命名为金花茶查尔酮合成酶基因（CnCHS）、金花茶查尔酮异构酶基因（CnCHI）、金花茶黄烷酮3-羟化酶基因（CnF3H）、金花茶类黄酮3’-羟化酶基因（CnF3’H）、金花茶黄酮醇合成酶基因（CnFLS）、金花茶二氢黄酮醇还原酶基因（CnDFR）和金花茶新黄质合成酶基因（CnNSY）。对各基因的生物信息学分析表明，这些基因为所克隆的目的基因。将这些基因在Genbank中登录，其登录号码分别是：HQ269804，HQ269805，HQ290517，HQ290518，JF343560，JF440957，CnDFR尚未登录。
高效液相色谱（High-performance Liquid Chromatography，HPLC）分析与花瓣表皮细胞色素观察结果表明，在金花茶花瓣呈绿色时，花瓣中类黄酮成份槲皮素-7-O-β-D-葡萄糖苷（Quertecin-7-O-β-D-glucoside， Qu7G）、槲皮素-3-O-β-D-葡萄糖苷（Quertecin-3-O-β-D-glucoside，Qu3G）、槲皮素（Quercetin，Qu）含量以及类胡萝素成份新黄质（neoxanthin）、叶黄素（xanthophyll）、紫黄质（violaxanthin）等含量均很低，显微镜下观测不到细胞中有色体的积累；在花色微黄色时，花瓣中QU7G、Qu3G含量最高，类胡萝卜素只在少数细胞中积累；在花瓣金黄色时，Qu7G、Qu3G含量比花色微黄时有所下降，类胡萝卜素有色体体积增大，每个细胞均呈黄色。这些结果表明Qu7G、Qu3G等类黄酮成份对金花茶花色起一定的辅助作用，花瓣中含有大量淡黄至近于无色的黄酮醇苷而不含有色的花青素苷，为类胡萝卜素成份的有效显色提供了必要条件，类胡萝卜素含量的升高并在细胞中不断积累是金花茶花瓣显色的最重要因素。
荧光定量PCR表明，CnCHS、CnCHI、CnF3H、CnF3’H主要在花瓣绿色至淡黄色时期表达，在此时期，Qu7G、Qu3G含量不断升高并达最大值。CnFLS主要在初蕾期表达，而CnDFR基因的表达主要在花色金黄的半开期，二者表达时间的差异使DFR酶与FLS酶避免了对同一底物DHQ的竞争，进而抑制了花青素的合成，促使合成途径朝着Qu的方向进行，为Qu3G、Qu7G的合成提供充足的底物。
利用pCAMBIA1300表达载体分别构建了CnFLS与CnNSY基因的正义表达载体及干扰表达载体共4个，并获得每个载体的转基因植株各10株。从每个基因的10株转化株中随机挑选5株，经PCR、Southern blotting鉴定出阳性植株各3株，将这些阳性植株扩繁为株系，每个株系6株。实时荧光定量PCR分析表明，与对照相比，转CnFLS正义基因烟草株系中FLS表达量显著提高，达8-13倍，而转CnFLS干扰基因的株系其表达量显著下降，仅为对照的0.1-0.5倍。转CnNSY基因的烟草株系其NSY基因的表达量显著提高，为对照的3-8倍，而转CnNSY干扰基因的烟草植株其表达量则明显下降，仅为对照的0.3-0.5倍。表明CnFLS、CnNSY正义基因与干扰基因均能在转基因烟草植株中正常表达，并具有明显的促进与抑制作用。
观察转CnFLS正义基因烟草阳性株系的花色表型发现，3个转CnFLS正义基因烟草株系花色由对照株的粉红色变为淡红色或淡黄白色，花色产生了明显变化。HPLC分析表明，花色显淡黄白色烟草株系TF1花瓣中主要色素成份花青素-3-O-鼠李糖苷（Cyanidin-3-O-rutinoside，Cy3R）含量最低，约为对照的0.1倍；Qu3G、Qu7G含量明显升高，Qu3G含量与对照相比增加了约3倍，Qu7G约增加了1倍。转CnFLS干扰基因株系花由对照株的粉红色变为深红色，花色成份中Cy3R含量明显增加，花色最深的株系Tf23其花瓣中Cy3R含量最高，约为对照的3倍；3个CnFLS干扰株系中Qu3G、Qu7G含量均略有降低。表明CnFLS基因的超表达可促进黄酮醇合成而使花色变浅，而FLS的表达被抑制后则可促进花青素的合成使花色加深。
对转CnNSY正义基因植株的表型观测发现，3个转基因烟草株系花色由对照株的粉红色变为淡红色或淡黄色。HPLC结果表明，花色显淡黄烟草株系TN10花瓣Cy3R含量最低，约为对照的5%；新黄质、紫黄质、叶黄素含量与对照相比均增加了约1倍。而转CnNSY干扰基因株系花色由粉红变为浅橙红色，3个转CnNSY干扰基因株系中Cy3R含量约为对照的0.6倍，新黄质、紫黄质含量略有下降，叶黄素含量略有上升。表明CnNSY基因具有调控类胡萝卜素合成的作用，其过量表达能够促进新黄质、紫黄质的合成而使花色变浅呈现黄色的趋势，干扰NSY的表达对类胡萝卜素合成影响不大。
通过本研究，我们认为金花茶CnCHS、CnCHI、CnF3H、CnF3’H、CnFLS、CnDFR基因的表达特性对花瓣中只含黄酮醇（苷）而无花青素成份具有决定性作用，花瓣中淡黄或近于无色的黄酮醇（苷）为金花茶花瓣中类胡萝卜素有色体呈现金黄色创造了必要条件，对金花茶花色起决定作用的则是类胡萝卜素及其在花瓣中不断累积所形成的有色体。因此，我们认为在山茶花的分子育种中应采取转入CnFLS正义基因或CnDFR干扰基因以抑制花青素合成的同时，转入相关的类胡萝卜素合成基因如CnNSY等以提高类胡萝卜素含量的方法，才能育成更好的黄色茶花品种。
Camellia nitidissima, a famous ornamental plant of the world, which has golden flowersand known as “Queen of camellias” is an important parent to breed yellow camellias. Theresearches of cross breeding with C.nitidssima are reported constantly while breeders didn’tgain the hybids which have goledn yellow flowers by many years’ work. The reason of thisresult may be that the reasearchers don’t uderstand the regularity and biosynthesis pathway ofthe pigmentions. Based on this point, this research was carried out to search the transformatinmechanism and some relative key genes to provide the theory support for breeding the yellowcamellias.
To investigate the biosynthesis pathway of the pigments,7genes’ cDNA sequences werecloned from the petals of C. nitidissima by the methods of RT-PCR(Reverse transcriptionpolymerase chain reaction) and RACE(Rapid Amplification of cDNA Ends), named CnCHS,CnCHI, CnF3H, CnF3’H, CnFLS, CnDFR and CnNSY respectively. The analysis of thesegenes’ bioinformations showed that these are the goal genes we want.The Genbank loadedcodes are HQ269804, HQ269805, HQ290517, HQ290518, JF343560and JF440957, CnDFR isnot loaded.
The results of HPLC(High-performance Liquid Chromatography) and the pigmentobservation in epidermal cell of petals showed that the contents of Quercetin(Qu), Quercetin-7-O-β-D-glucoside(Qu7G), Quercetin-3-O-β-D-glucoside(Qu3G), and carotenoids are low andthe accumulation of chromoplasts can’t be found in the stage of petal uncolored；during theperiod of petals yellowish, the content of Qu3G and Qu7G are high, the chromoplastsaccumulated in serveal cells; when the petals golden yellow, the contents of Qu7G and Qu3Gare lower than before but the chromoplasts are bigger and in almost all the cells. The resultsindicated that the high amounts of colorless flavonols and no anthocyanidin in the petlas provide the necessary condition for the carotenoids to show its color, and the accumulation oftotal carotenoids in the cells is the determinant factor of the petal color.
The results of quantitative Real-time PCR showed that the highest expression quantity ofCnCHS, CnCHI, CnF3H, CnF3’H appears during the period of uncolor petals to yellowish, inwhich the contents of Qu7G and Qu3G reach to highest level; the coperation of these genesmake the production of CnCHS transform to dihydroquercetion,which is the substrate of Qu7Gand Qu3G. The expression of CnFLS is mainly in the stage of uncolored petal and the CnDFRis during the time of full yellow colored indicate that the difference of the two genes makethem avoid competing the same substrate of dihydroquercetin, which restrains the combinationof anthocyanidin and provide enough substrates for the synthsis of Qu7G and Qu3G.
We successfully constructed the sense and RNAi expression vectors of CnFLS and CnNSYrespectively by using the expression vector pCAMBIA1300and then the four genes weretransferred to tobacco by the Agrobacterium EHA105, finally,10lines of transgene plants ofevery gene were gained.5were selected in every10transgene plants, after the identifion ofPCR and Southern blotting,3lines transgene tobaccos were obtained of every gene. The resultsof quantitative Real-time PCR showed that the expression of FLS in trans-sense CnFLS is8-13times to the wild types and0.1-0.5times copmared to wild types in trans-interference CnFLSlines. The expression of NSY in trans-sense CnNSY is3-8times to the wild types and0.3-0.5times copmared to wild types in trans-interference CnNSY lines. This results show that theCnFLS and CnNSY can express in the transgens lines normally.
The flower color of3lines of trans-sense CnLFS tobaccos appeared shallow yellow-white to shallow pink compared with the color of wild type tobacco. The results of HPLCshowed that the content of Cy3R in the petals of TF1which shows yellowish white color wasalmost0.1times of that of wild tobacco, the content of Qu3G is4times and Qu7G is2times ofthat of wild types. The color of trans-interference CnFLS tobaccos became more red than wildtype, the content of Cy3R in Tf23which shows the reddest color is3times of that of wild type,but the content of Qu3G、Qu7G are lower than that of wild type. These results indicates that the overexpression of CnFLS can make the flower color shallow and repression of FLS willcause the flower color deeper.
The flower color of3lines of trans-sense CnNSY tobaccos appeared yellowish to shallowpink compared with the color of wild type tobacco. The results of HPLC showed that thecontent of Cy3R in the petals of TN10which shows yellowish white color was almost0.05times of that of wild tobacco, the contents of neoxanthin, violaxanthin, xanthophyll are2timesof that of wild types. The color of trans-interference CnNSY tobaccos became shallow orangered, the content of Cy3R in the3lines is about0.6times of that of wild type, and the contentsof neoxanthin, violaxanthin, xanthophyll are a little lower than that of wild type. These resultsindicates that the overexpression of CnNSY can promot the synthesis of carotenoids and makethe flower color shallow and repression of NSY will not affect the synthesis of caroteniods butthe overexpression of CnNSY might affect the synthesis of Cy3R.
From this research we draw the conclusion that the expression characters of CnCHS,CnCHI, CnF3H, CnF3’H, CnFLS,CnDFR and CnFLS play the decisive role in the synthesis offlavonol and no synthesis of anthocyanidin. The exists of colorless flavonol provide a essentialcondition that the chromoplasts of carotenoids can show its golden color naturally,but the it isthe chromoplasts of carotenoids which accumulated in cells constantly rather than flavonoldecided the color of C.nitidissima. So, we believe that we can obtain the yellow camellias byusing the methods of trans-sense CnFLS or trans-interference CnDFR to decrease the contentof anthocyanidin ans increase the content of carotenoids by the method of trans-sense somecarotenoids synthsis genes such as CnNSY and so on.
利用RT-PCR (Reverse transcription polymerase chain reaction)方法和RACE（RapidAmplification of cDNA Ends）技术从金花茶花瓣中分离出6个与类黄酮色素Quercetin（Qu）合成相关基因的cDNA全长，1个与类胡萝卜素新黄质合成相关基因的cDNA全长，分别命名为金花茶查尔酮合成酶基因（CnCHS）、金花茶查尔酮异构酶基因（CnCHI）、金花茶黄烷酮3-羟化酶基因（CnF3H）、金花茶类黄酮3’-羟化酶基因（CnF3’H）、金花茶黄酮醇合成酶基因（CnFLS）、金花茶二氢黄酮醇还原酶基因（CnDFR）和金花茶新黄质合成酶基因（CnNSY）。对各基因的生物信息学分析表明，这些基因为所克隆的目的基因。将这些基因在Genbank中登录，其登录号码分别是：HQ269804，HQ269805，HQ290517，HQ290518，JF343560，JF440957，CnDFR尚未登录。
高效液相色谱（High-performance Liquid Chromatography，HPLC）分析与花瓣表皮细胞色素观察结果表明，在金花茶花瓣呈绿色时，花瓣中类黄酮成份槲皮素-7-O-β-D-葡萄糖苷（Quertecin-7-O-β-D-glucoside， Qu7G）、槲皮素-3-O-β-D-葡萄糖苷（Quertecin-3-O-β-D-glucoside，Qu3G）、槲皮素（Quercetin，Qu）含量以及类胡萝素成份新黄质（neoxanthin）、叶黄素（xanthophyll）、紫黄质（violaxanthin）等含量均很低，显微镜下观测不到细胞中有色体的积累；在花色微黄色时，花瓣中QU7G、Qu3G含量最高，类胡萝卜素只在少数细胞中积累；在花瓣金黄色时，Qu7G、Qu3G含量比花色微黄时有所下降，类胡萝卜素有色体体积增大，每个细胞均呈黄色。这些结果表明Qu7G、Qu3G等类黄酮成份对金花茶花色起一定的辅助作用，花瓣中含有大量淡黄至近于无色的黄酮醇苷而不含有色的花青素苷，为类胡萝卜素成份的有效显色提供了必要条件，类胡萝卜素含量的升高并在细胞中不断积累是金花茶花瓣显色的最重要因素。
荧光定量PCR表明，CnCHS、CnCHI、CnF3H、CnF3’H主要在花瓣绿色至淡黄色时期表达，在此时期，Qu7G、Qu3G含量不断升高并达最大值。CnFLS主要在初蕾期表达，而CnDFR基因的表达主要在花色金黄的半开期，二者表达时间的差异使DFR酶与FLS酶避免了对同一底物DHQ的竞争，进而抑制了花青素的合成，促使合成途径朝着Qu的方向进行，为Qu3G、Qu7G的合成提供充足的底物。
利用pCAMBIA1300表达载体分别构建了CnFLS与CnNSY基因的正义表达载体及干扰表达载体共4个，并获得每个载体的转基因植株各10株。从每个基因的10株转化株中随机挑选5株，经PCR、Southern blotting鉴定出阳性植株各3株，将这些阳性植株扩繁为株系，每个株系6株。实时荧光定量PCR分析表明，与对照相比，转CnFLS正义基因烟草株系中FLS表达量显著提高，达8-13倍，而转CnFLS干扰基因的株系其表达量显著下降，仅为对照的0.1-0.5倍。转CnNSY基因的烟草株系其NSY基因的表达量显著提高，为对照的3-8倍，而转CnNSY干扰基因的烟草植株其表达量则明显下降，仅为对照的0.3-0.5倍。表明CnFLS、CnNSY正义基因与干扰基因均能在转基因烟草植株中正常表达，并具有明显的促进与抑制作用。
观察转CnFLS正义基因烟草阳性株系的花色表型发现，3个转CnFLS正义基因烟草株系花色由对照株的粉红色变为淡红色或淡黄白色，花色产生了明显变化。HPLC分析表明，花色显淡黄白色烟草株系TF1花瓣中主要色素成份花青素-3-O-鼠李糖苷（Cyanidin-3-O-rutinoside，Cy3R）含量最低，约为对照的0.1倍；Qu3G、Qu7G含量明显升高，Qu3G含量与对照相比增加了约3倍，Qu7G约增加了1倍。转CnFLS干扰基因株系花由对照株的粉红色变为深红色，花色成份中Cy3R含量明显增加，花色最深的株系Tf23其花瓣中Cy3R含量最高，约为对照的3倍；3个CnFLS干扰株系中Qu3G、Qu7G含量均略有降低。表明CnFLS基因的超表达可促进黄酮醇合成而使花色变浅，而FLS的表达被抑制后则可促进花青素的合成使花色加深。
对转CnNSY正义基因植株的表型观测发现，3个转基因烟草株系花色由对照株的粉红色变为淡红色或淡黄色。HPLC结果表明，花色显淡黄烟草株系TN10花瓣Cy3R含量最低，约为对照的5%；新黄质、紫黄质、叶黄素含量与对照相比均增加了约1倍。而转CnNSY干扰基因株系花色由粉红变为浅橙红色，3个转CnNSY干扰基因株系中Cy3R含量约为对照的0.6倍，新黄质、紫黄质含量略有下降，叶黄素含量略有上升。表明CnNSY基因具有调控类胡萝卜素合成的作用，其过量表达能够促进新黄质、紫黄质的合成而使花色变浅呈现黄色的趋势，干扰NSY的表达对类胡萝卜素合成影响不大。
通过本研究，我们认为金花茶CnCHS、CnCHI、CnF3H、CnF3’H、CnFLS、CnDFR基因的表达特性对花瓣中只含黄酮醇（苷）而无花青素成份具有决定性作用，花瓣中淡黄或近于无色的黄酮醇（苷）为金花茶花瓣中类胡萝卜素有色体呈现金黄色创造了必要条件，对金花茶花色起决定作用的则是类胡萝卜素及其在花瓣中不断累积所形成的有色体。因此，我们认为在山茶花的分子育种中应采取转入CnFLS正义基因或CnDFR干扰基因以抑制花青素合成的同时，转入相关的类胡萝卜素合成基因如CnNSY等以提高类胡萝卜素含量的方法，才能育成更好的黄色茶花品种。
Camellia nitidissima, a famous ornamental plant of the world, which has golden flowersand known as “Queen of camellias” is an important parent to breed yellow camellias. Theresearches of cross breeding with C.nitidssima are reported constantly while breeders didn’tgain the hybids which have goledn yellow flowers by many years’ work. The reason of thisresult may be that the reasearchers don’t uderstand the regularity and biosynthesis pathway ofthe pigmentions. Based on this point, this research was carried out to search the transformatinmechanism and some relative key genes to provide the theory support for breeding the yellowcamellias.
To investigate the biosynthesis pathway of the pigments,7genes’ cDNA sequences werecloned from the petals of C. nitidissima by the methods of RT-PCR(Reverse transcriptionpolymerase chain reaction) and RACE(Rapid Amplification of cDNA Ends), named CnCHS,CnCHI, CnF3H, CnF3’H, CnFLS, CnDFR and CnNSY respectively. The analysis of thesegenes’ bioinformations showed that these are the goal genes we want.The Genbank loadedcodes are HQ269804, HQ269805, HQ290517, HQ290518, JF343560and JF440957, CnDFR isnot loaded.
The results of HPLC(High-performance Liquid Chromatography) and the pigmentobservation in epidermal cell of petals showed that the contents of Quercetin(Qu), Quercetin-7-O-β-D-glucoside(Qu7G), Quercetin-3-O-β-D-glucoside(Qu3G), and carotenoids are low andthe accumulation of chromoplasts can’t be found in the stage of petal uncolored；during theperiod of petals yellowish, the content of Qu3G and Qu7G are high, the chromoplastsaccumulated in serveal cells; when the petals golden yellow, the contents of Qu7G and Qu3Gare lower than before but the chromoplasts are bigger and in almost all the cells. The resultsindicated that the high amounts of colorless flavonols and no anthocyanidin in the petlas provide the necessary condition for the carotenoids to show its color, and the accumulation oftotal carotenoids in the cells is the determinant factor of the petal color.
The results of quantitative Real-time PCR showed that the highest expression quantity ofCnCHS, CnCHI, CnF3H, CnF3’H appears during the period of uncolor petals to yellowish, inwhich the contents of Qu7G and Qu3G reach to highest level; the coperation of these genesmake the production of CnCHS transform to dihydroquercetion,which is the substrate of Qu7Gand Qu3G. The expression of CnFLS is mainly in the stage of uncolored petal and the CnDFRis during the time of full yellow colored indicate that the difference of the two genes makethem avoid competing the same substrate of dihydroquercetin, which restrains the combinationof anthocyanidin and provide enough substrates for the synthsis of Qu7G and Qu3G.
We successfully constructed the sense and RNAi expression vectors of CnFLS and CnNSYrespectively by using the expression vector pCAMBIA1300and then the four genes weretransferred to tobacco by the Agrobacterium EHA105, finally,10lines of transgene plants ofevery gene were gained.5were selected in every10transgene plants, after the identifion ofPCR and Southern blotting,3lines transgene tobaccos were obtained of every gene. The resultsof quantitative Real-time PCR showed that the expression of FLS in trans-sense CnFLS is8-13times to the wild types and0.1-0.5times copmared to wild types in trans-interference CnFLSlines. The expression of NSY in trans-sense CnNSY is3-8times to the wild types and0.3-0.5times copmared to wild types in trans-interference CnNSY lines. This results show that theCnFLS and CnNSY can express in the transgens lines normally.
The flower color of3lines of trans-sense CnLFS tobaccos appeared shallow yellow-white to shallow pink compared with the color of wild type tobacco. The results of HPLCshowed that the content of Cy3R in the petals of TF1which shows yellowish white color wasalmost0.1times of that of wild tobacco, the content of Qu3G is4times and Qu7G is2times ofthat of wild types. The color of trans-interference CnFLS tobaccos became more red than wildtype, the content of Cy3R in Tf23which shows the reddest color is3times of that of wild type,but the content of Qu3G、Qu7G are lower than that of wild type. These results indicates that the overexpression of CnFLS can make the flower color shallow and repression of FLS willcause the flower color deeper.
The flower color of3lines of trans-sense CnNSY tobaccos appeared yellowish to shallowpink compared with the color of wild type tobacco. The results of HPLC showed that thecontent of Cy3R in the petals of TN10which shows yellowish white color was almost0.05times of that of wild tobacco, the contents of neoxanthin, violaxanthin, xanthophyll are2timesof that of wild types. The color of trans-interference CnNSY tobaccos became shallow orangered, the content of Cy3R in the3lines is about0.6times of that of wild type, and the contentsof neoxanthin, violaxanthin, xanthophyll are a little lower than that of wild type. These resultsindicates that the overexpression of CnNSY can promot the synthesis of carotenoids and makethe flower color shallow and repression of NSY will not affect the synthesis of caroteniods butthe overexpression of CnNSY might affect the synthesis of Cy3R.
From this research we draw the conclusion that the expression characters of CnCHS,CnCHI, CnF3H, CnF3’H, CnFLS,CnDFR and CnFLS play the decisive role in the synthesis offlavonol and no synthesis of anthocyanidin. The exists of colorless flavonol provide a essentialcondition that the chromoplasts of carotenoids can show its golden color naturally,but the it isthe chromoplasts of carotenoids which accumulated in cells constantly rather than flavonoldecided the color of C.nitidissima. So, we believe that we can obtain the yellow camellias byusing the methods of trans-sense CnFLS or trans-interference CnDFR to decrease the contentof anthocyanidin ans increase the content of carotenoids by the method of trans-sense somecarotenoids synthsis genes such as CnNSY and so on.
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