彩色棉GhF3'H与GhF3'5'H基因的克隆、鉴定及四种类黄酮物质的HPLC分析
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摘要
棉花是世界上重要的经济作物,棉纤维是纺织工业中天然纤维的主要来源。彩色棉是一种棉纤维具有天然色泽的棉花类型,由于其加工过程无须人工染色而博得了“生态棉”的美誉,具有非常广阔的市场前景,但是彩色棉纤维品质差、色泽单一,制约的彩色棉产业的发展。
本研究以彩色棉为研究对象,对彩色棉纤维中色素物质生物合成途径的分子调控和生物化学等方面进行了初步探索,为丰富彩色棉纤维的色彩,增强纤维品质提供一定理论依据。本研究从彩棉纤维细胞中克隆了参与类黄酮生物合成的两个重要分支酶基因,采用生物信息学分析、分子杂交、Real Time-PCR表达分析和在烟草中转基因表达等方法系统分析了它们的分子特征、表达特性和功能活性等。主要结果如下：
1、利用cDNA-AFLP差异显示技术,比较天彩5号(棕色),天彩7号(绿色)与白棉开花后18天的纤维细胞的基因表达,寻找与棉纤维色素基因表达相关的片段。结果表明：64对引物组合检测到约3800条TDF片段条带,平均每一对选择性引物可以获得25条左右,挑取明显差异片段60条,进行反northern杂交,获得17个阳性片段。通过BlastX比对,获得类黄酮3’羟基化酶与类黄酮3’5’羟基化酶,两个与色素相关的差异片段,一个蛋白激酶,一个顺乌头归酸酶,一个与老化相关酶,一个苹果酸脱氢酶,其余无法比对。
2、利用RACE-PCR技术获得F3’5'H基因全长cDNA,1876bp,含有一个编码509个氨基酸残基的开放阅读框。Blast分析表明该基因的编码产物为类黄酮3’5’羟基化酶,命名为GhF3'5'H, GenBank登录号为GU062184；同时获得F3’H基因的全长cDNA,1873bp,含有一个编码510个氨基酸残基的开放阅读框。Blast分析表明该基因的编码产物为类黄酮3’羟基化酶,命名为GhF3'H, GenBank登录号为GU062185。通过保守结构域分析,GhF3'5'H与GhF3'H基因属于P450细胞色素家族基因。
3、通过RT-PCR与实时荧光定量RT-PCR,对白色棉,绿色棉,棕色棉品种中的根、茎、叶、花和棉纤维细胞中GhF3'5'H与GhF3'H基因的转录水平进行检测和比较。结果显示：GhF3'5'H与GhF3'H在彩色棉不同组织中的表达量均显著高于其近等基因系白色棉,且主要在纤维细胞中表达。在纤维发育过程中,GhF3'5'H与GhF3'H基因在纤维发育的早期表达量最高,并随纤维发育逐渐降低。推测这两个基因可能在彩色棉纤维色素前体物质的形成中发挥作用。
4、类黄酮合成途径中关键酶的底物专一性研究,对于揭示花色的合成途径和分子改良的技术路线有着重要的意义。构建pET28a-GhF3'H与pET28a-GhF3'5'H蛋白表达载体,通过不同表达温度和时间发现,加入IPTG诱导后,在大肠杆菌中可以诱导出目的蛋白,能够检测到较高量的目的蛋白表达,纯化发现pET28a-GhF3'H与pET28a-GhF3'5'H蛋白均以包涵体形式存在,这为验证GhF3'H和GhF3'5'H基因的功能和其编码酶的活性研究提供了基础。
5、分别构建GhCHS基因的超量表达载体,GhCHS基因RNAi干扰载体,用于烟草、棉花等转化研究。通过PCR检测分析,获得转GhCHS基因的阳性烟草10株,转基因烟草与野生型相比生长缓慢,植株较小；对转基因烟草植株与野生型烟草进行蔗糖非生物胁迫处理,发现随着胁迫时间的增加,叶片总黄酮含量在增加,并且转基因烟草中总黄酮的含量高于野生型中总黄酮的含量；推测增加碳源可以促进类黄酮物质的合成。
6、运用HPLC技术,建立了同时检测四种类黄酮物质的方法,对棉纤维发育过程中的类黄酮物质(柑橘素,杨梅素,槲皮素,山奈素)进行检测并分析。结果发现：(1)白色与彩色棉花纤维中均存在多种类黄酮物质,包括柑橘素、山奈素、槲皮素和杨梅素等色素前体物质。(2)柑橘素在棉纤维发育的早期积累,随着棉纤维的发育该物质累积下降。(3)杨梅素与槲皮素在彩色棉纤维中积累量远高于同时期白色棉中累积量,且差异显著。(4)山奈素在棕色与绿色棉纤维快速伸长期极少量积累。结果表明：在棉花纤维发育过程中有大量活跃复杂的色素物质合成并转化,柑橘素、山奈素、槲皮素和杨梅素等色素前体物质对彩色棉纤维色素形成有一定作用。
Cotton is one of the most globally important cash crops, and cotton fiber is the main natural fiber in textile industry. Color cotton fiber is a kind of cotton with natural color in its fiber. Color cotton is healthy and profitable for mankind for it no dye printing in processing. Due to the advantage, it has caught tremendous attention and named "ecological fiber" highly praised by consumers. However, there are still some weak points that block its further development, only a few color series, instability of pigment, etc. which hinde the development of cotton industry.
This research regarded colored cotton as materials and focused on fiber biochemistry and molecular biolosy. Cloning of genes related to color formation study will help thoroughly understand pigment synthesis pathway and molecular mechanism, and perform genetic engineering. Two important genes related to flavonoids bio-synthesis were cloned, and their characteristics, expression profiles and functional activity were studied by bioinformatics analysis, blot, Real Time-PCR, and expression in tobacco. The results are as follow:
1. The study was carried out to search the different expressing genes in the fiber of brown, green and white color cotton though complementary DNA amplified fragment lenth polymorphism-(cDNA-AFLP) analysis. The result showed that sixty-four pairs of primers were used to amplify products for analysis. About 3800 bands were detected and the average number of bands per pair of primers amplified was 25. Using cDNA as probe,60 special framents were analyzed by reverse Northern blot, and got 17. BLASTx analysis showed that 6 TDFs had the homologous sequences with other sequences in GenBank, we got two framents which relatived to pigment formation. They are flavoionds 3'hydroxylase in brown color cotton fiber and flavoionds 3'5'hydroxylase in green color cotton fiber.
2. Using rapid amplification of cDNA ends (RACE), the complete cDNA sequence of GhF3'5'H gene was isolated from green cotton fiber. The full-length of this cDNA colne was 1876bp, and its open reading frame encoded 509 amino acids. The gene was designated as GhF3'5'H (GenBank accession:GU062184). By the same way, we also got the complete cDNA sequence from brown cotton fiber and named GhF3'H (GenBank accession:GU062185). The full-length of this gene have 1873bp, and its open reading frame encoded 510 amino acids. Both GhF3'5'H and GhF3'H genes are belong to P450 family by the analysis of their conservation structure region.
3. RT-PCR and Fluorescence real time quantitative PCR technology were used to study the expression of GhF3'5'H and GhF3'H genes in different development stages of fiber and organs. Real-time PCR analysis showed that GhF3'5'H and GhF3'5'H specially expressed in naturally color cotton fibers prior to white cotton fibers, and there expression levels are much higher in color cotton than in white cotton. They shrink in proportion to fiber development. Suggesting that GhF3'5'H and GhF3'H genes may involve in the precursor sythesis and play an important role in pigment formation during natural green and brown cotton fibers development.
4. In the flavnoid sythesis pathway, studying on constructed enzyme choosing substrate is most important to reveal flower pigment formation. We constructed pET28a-GhF3'H and pET28a-GhF3'5'H protein vector at same time, which were induced by the IPTG in different conditions. We could found induced aim albume in the gel. Both pET28a-GhF3'H and pET28a-GhF3'5'H albume were presence in inclusion body which is had to pure.
5. We constructed GhCHS gene expression vector and GhCHS RNAi vector repectively, for the tobacco and cotton transformation research. Got ten transformed tobacco plants which detected by PCR technique. By the assay, we found that tranformed plants grow more slowly and smaller than wild plants. With sucrose streesing, total flavonoid in transformed and wild tobacco planta is increasing as the time goes by, moreover, transformed plants have much more flavonoid than wilds'.we have a conclusion that sucrose might promote flavonoid proucts sythesis.
6. We established a determination method which could detect four effective flavonoids in cotton fiber at the same time. During the fiber developing, we detected myricetin、quercetin、naringenin and kaempferol four flavonoids in the fiber through HPLC. We got following results:(1) There are many different flavonoids in both white and colour cotton fiber; (2) Naringenin accumulated in the early time and decreased by the time; (3) Myricetin and quercetin accumulated much more in colour cotton than white cotton fibers, and difference remarkable; (4) Kaempferol accumulated little in the fast elongation stage. These results demonsted that numerous actively and complicated flavonoids sythesis and transtfored in the process of cotton fiber development. Myricetin、quercetin naringenin and kaempferol are useful to precursor pigment in the color fiber.
本研究以彩色棉为研究对象,对彩色棉纤维中色素物质生物合成途径的分子调控和生物化学等方面进行了初步探索,为丰富彩色棉纤维的色彩,增强纤维品质提供一定理论依据。本研究从彩棉纤维细胞中克隆了参与类黄酮生物合成的两个重要分支酶基因,采用生物信息学分析、分子杂交、Real Time-PCR表达分析和在烟草中转基因表达等方法系统分析了它们的分子特征、表达特性和功能活性等。主要结果如下：
1、利用cDNA-AFLP差异显示技术,比较天彩5号(棕色),天彩7号(绿色)与白棉开花后18天的纤维细胞的基因表达,寻找与棉纤维色素基因表达相关的片段。结果表明：64对引物组合检测到约3800条TDF片段条带,平均每一对选择性引物可以获得25条左右,挑取明显差异片段60条,进行反northern杂交,获得17个阳性片段。通过BlastX比对,获得类黄酮3’羟基化酶与类黄酮3’5’羟基化酶,两个与色素相关的差异片段,一个蛋白激酶,一个顺乌头归酸酶,一个与老化相关酶,一个苹果酸脱氢酶,其余无法比对。
2、利用RACE-PCR技术获得F3’5'H基因全长cDNA,1876bp,含有一个编码509个氨基酸残基的开放阅读框。Blast分析表明该基因的编码产物为类黄酮3’5’羟基化酶,命名为GhF3'5'H, GenBank登录号为GU062184；同时获得F3’H基因的全长cDNA,1873bp,含有一个编码510个氨基酸残基的开放阅读框。Blast分析表明该基因的编码产物为类黄酮3’羟基化酶,命名为GhF3'H, GenBank登录号为GU062185。通过保守结构域分析,GhF3'5'H与GhF3'H基因属于P450细胞色素家族基因。
3、通过RT-PCR与实时荧光定量RT-PCR,对白色棉,绿色棉,棕色棉品种中的根、茎、叶、花和棉纤维细胞中GhF3'5'H与GhF3'H基因的转录水平进行检测和比较。结果显示：GhF3'5'H与GhF3'H在彩色棉不同组织中的表达量均显著高于其近等基因系白色棉,且主要在纤维细胞中表达。在纤维发育过程中,GhF3'5'H与GhF3'H基因在纤维发育的早期表达量最高,并随纤维发育逐渐降低。推测这两个基因可能在彩色棉纤维色素前体物质的形成中发挥作用。
4、类黄酮合成途径中关键酶的底物专一性研究,对于揭示花色的合成途径和分子改良的技术路线有着重要的意义。构建pET28a-GhF3'H与pET28a-GhF3'5'H蛋白表达载体,通过不同表达温度和时间发现,加入IPTG诱导后,在大肠杆菌中可以诱导出目的蛋白,能够检测到较高量的目的蛋白表达,纯化发现pET28a-GhF3'H与pET28a-GhF3'5'H蛋白均以包涵体形式存在,这为验证GhF3'H和GhF3'5'H基因的功能和其编码酶的活性研究提供了基础。
5、分别构建GhCHS基因的超量表达载体,GhCHS基因RNAi干扰载体,用于烟草、棉花等转化研究。通过PCR检测分析,获得转GhCHS基因的阳性烟草10株,转基因烟草与野生型相比生长缓慢,植株较小；对转基因烟草植株与野生型烟草进行蔗糖非生物胁迫处理,发现随着胁迫时间的增加,叶片总黄酮含量在增加,并且转基因烟草中总黄酮的含量高于野生型中总黄酮的含量；推测增加碳源可以促进类黄酮物质的合成。
6、运用HPLC技术,建立了同时检测四种类黄酮物质的方法,对棉纤维发育过程中的类黄酮物质(柑橘素,杨梅素,槲皮素,山奈素)进行检测并分析。结果发现：(1)白色与彩色棉花纤维中均存在多种类黄酮物质,包括柑橘素、山奈素、槲皮素和杨梅素等色素前体物质。(2)柑橘素在棉纤维发育的早期积累,随着棉纤维的发育该物质累积下降。(3)杨梅素与槲皮素在彩色棉纤维中积累量远高于同时期白色棉中累积量,且差异显著。(4)山奈素在棕色与绿色棉纤维快速伸长期极少量积累。结果表明：在棉花纤维发育过程中有大量活跃复杂的色素物质合成并转化,柑橘素、山奈素、槲皮素和杨梅素等色素前体物质对彩色棉纤维色素形成有一定作用。
Cotton is one of the most globally important cash crops, and cotton fiber is the main natural fiber in textile industry. Color cotton fiber is a kind of cotton with natural color in its fiber. Color cotton is healthy and profitable for mankind for it no dye printing in processing. Due to the advantage, it has caught tremendous attention and named "ecological fiber" highly praised by consumers. However, there are still some weak points that block its further development, only a few color series, instability of pigment, etc. which hinde the development of cotton industry.
This research regarded colored cotton as materials and focused on fiber biochemistry and molecular biolosy. Cloning of genes related to color formation study will help thoroughly understand pigment synthesis pathway and molecular mechanism, and perform genetic engineering. Two important genes related to flavonoids bio-synthesis were cloned, and their characteristics, expression profiles and functional activity were studied by bioinformatics analysis, blot, Real Time-PCR, and expression in tobacco. The results are as follow:
1. The study was carried out to search the different expressing genes in the fiber of brown, green and white color cotton though complementary DNA amplified fragment lenth polymorphism-(cDNA-AFLP) analysis. The result showed that sixty-four pairs of primers were used to amplify products for analysis. About 3800 bands were detected and the average number of bands per pair of primers amplified was 25. Using cDNA as probe,60 special framents were analyzed by reverse Northern blot, and got 17. BLASTx analysis showed that 6 TDFs had the homologous sequences with other sequences in GenBank, we got two framents which relatived to pigment formation. They are flavoionds 3'hydroxylase in brown color cotton fiber and flavoionds 3'5'hydroxylase in green color cotton fiber.
2. Using rapid amplification of cDNA ends (RACE), the complete cDNA sequence of GhF3'5'H gene was isolated from green cotton fiber. The full-length of this cDNA colne was 1876bp, and its open reading frame encoded 509 amino acids. The gene was designated as GhF3'5'H (GenBank accession:GU062184). By the same way, we also got the complete cDNA sequence from brown cotton fiber and named GhF3'H (GenBank accession:GU062185). The full-length of this gene have 1873bp, and its open reading frame encoded 510 amino acids. Both GhF3'5'H and GhF3'H genes are belong to P450 family by the analysis of their conservation structure region.
3. RT-PCR and Fluorescence real time quantitative PCR technology were used to study the expression of GhF3'5'H and GhF3'H genes in different development stages of fiber and organs. Real-time PCR analysis showed that GhF3'5'H and GhF3'5'H specially expressed in naturally color cotton fibers prior to white cotton fibers, and there expression levels are much higher in color cotton than in white cotton. They shrink in proportion to fiber development. Suggesting that GhF3'5'H and GhF3'H genes may involve in the precursor sythesis and play an important role in pigment formation during natural green and brown cotton fibers development.
4. In the flavnoid sythesis pathway, studying on constructed enzyme choosing substrate is most important to reveal flower pigment formation. We constructed pET28a-GhF3'H and pET28a-GhF3'5'H protein vector at same time, which were induced by the IPTG in different conditions. We could found induced aim albume in the gel. Both pET28a-GhF3'H and pET28a-GhF3'5'H albume were presence in inclusion body which is had to pure.
5. We constructed GhCHS gene expression vector and GhCHS RNAi vector repectively, for the tobacco and cotton transformation research. Got ten transformed tobacco plants which detected by PCR technique. By the assay, we found that tranformed plants grow more slowly and smaller than wild plants. With sucrose streesing, total flavonoid in transformed and wild tobacco planta is increasing as the time goes by, moreover, transformed plants have much more flavonoid than wilds'.we have a conclusion that sucrose might promote flavonoid proucts sythesis.
6. We established a determination method which could detect four effective flavonoids in cotton fiber at the same time. During the fiber developing, we detected myricetin、quercetin、naringenin and kaempferol four flavonoids in the fiber through HPLC. We got following results:(1) There are many different flavonoids in both white and colour cotton fiber; (2) Naringenin accumulated in the early time and decreased by the time; (3) Myricetin and quercetin accumulated much more in colour cotton than white cotton fibers, and difference remarkable; (4) Kaempferol accumulated little in the fast elongation stage. These results demonsted that numerous actively and complicated flavonoids sythesis and transtfored in the process of cotton fiber development. Myricetin、quercetin naringenin and kaempferol are useful to precursor pigment in the color fiber.
引文
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