油菜柠檬酸合酶与柠檬酸裂解酶基因克隆及功能研究
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摘要
油菜是世界重要的油料作物,也是我国具有传统优势的重要油料作物。研究油菜高含油量的机理,有重要的理论价值和广阔的应用前景。柠檬酸合酶与柠檬酸裂解酶都是糖代谢的关键酶,其作用底物和产物是糖代谢中的关键中间产物,并可以作为脂肪酸合成的底物,因此,在植物的代谢中与脂肪酸合成紧密相关。研究柠檬酸合酶与柠檬酸裂解酶对脂肪酸合成的影响,以及对种子含油量的贡献,具有一定的理论意义。
根据已知的EST序列设计引物,利用现有的油菜cDNA文库及油菜cDNA,本实验成功克隆了油菜柠檬酸合酶、柠檬酸裂解酶A亚基、柠檬酸裂解酶B亚基的序列。分析表明,克隆到的序列含油菜柠檬酸合酶、柠檬酸裂解酶A亚基、柠檬酸裂解酶B亚基的完整开放阅读框。聚类分析表明,三个基因的序列在植物内高度同源。
根据克隆的柠檬酸裂解酶A亚基、B亚基和柠檬酸合酶基因(ACLA,ACLB和CS)的cDNA序列,共设计了16对EST引物,其中ACLA 5对,ACLB 5对,CS 6对,用于高低油F2群体和不同含油量品种群体标记。将这些引物用于高低油F2群体,最终有5对引物在群体中有差异条带。将16对引物应用于15个高含油量和15个低含油量的油菜品种群体,有4对引物在群体中有差异条带。实验结果证明设计的EST引物可以用于油菜分子标记。
采用半定量PCR和Real-time PCR的方法,检测柠檬酸合酶和柠檬酸裂解酶的表达。结果表明,在含油量不同的油菜品种中,其叶片内柠檬酸合酶表达量有差异,高油品种内表达量较高;在油菜的不同器官和组织中,柠檬酸合酶和柠檬酸裂解酶的表达强度不同,在角果发育早期表达量都较高,随着胚珠的成熟,表达量逐渐下降。在不同的胁迫条件下,油菜叶片中柠檬酸合酶和柠檬酸裂解酶的表达呈现不同的表达规律。柠檬酸合酶在水渍、干旱、高温和低温胁迫下, IAA和6-BA、ABA处理后表达量在特定时期均有上升,同样,柠檬酸裂解酶在水渍、低、强光和ABA处理后在表达量在特定时期均有上升。本实验结果表明,柠檬酸合酶在不同含油量油菜品种叶片中表达强度存在差异。柠檬酸合酶和柠檬酸裂解酶在油菜各器官中广泛表达,而且表达强度存在差异,说明这两个酶是组织和器官发育和执行生理功能不可缺少的。
根据克隆的油菜柠檬酸合酶基因,分别构建了油菜柠檬酸合酶基因的顺式植物表达质粒pMD-tCS,pMN-tCS,和反式植物表达质粒pMD-rCS、pMN-rCS,另外成功构建了柠檬酸裂解酶A亚基基因的顺式表达载体Gate-tACLA、反式表达质粒pMD-rACLA、pMN-rACLA。将构建好的植物表达载体转化拟南芥后,经抗性筛选和PCR检测后,除质粒pMN-rCS外,其余得到不同数量的阳性株系。而质粒pMN-rACLA和Gate-tACLA植株尚未进行性状检测。
在拟南芥中正义和反义表达油菜柠檬酸合酶基因,均造成了表型的变化。正义表达的植株中,侧枝的生长比野生型旺盛,种子和叶片中含油量均有上升,并且在叶片中尤为明显,此外,种子和叶片的脂肪酸组分也发生了一定的变化。而在反义表达的植株中,出现了矮化、叶片变小、果荚缩短等表型,种子和叶片中含油量降低,组分发生了变化。因此,实验结果表明,柠檬酸合酶在植物的正常生长发育中起重要作用,过量表达和降低表达均会造成植株发育的变化;另外,在植物的脂肪酸合成中,是重要的调控酶,该酶表达的上升会造成脂肪酸合成的增加,反义造成脂肪酸合成的降低。而在拟南芥中,反义表达柠檬酸裂解酶A亚基基因,同样会造成矮化、叶片变小、果荚缩短等表型。反义表达柠檬酸裂解酶,也同样造成了植物脂肪酸合成的降低,说明柠檬酸裂解酶同样是脂肪酸合成的调控酶。
Brassica napus is one of major oil crops in China and whole world. The mechanism of high oil content in Brassica napus is a very important subject for theoritic and applied studies. As the key enzymes of sugar metabolism, citrate synthase and ATP-citrate lyase are related to fatty acid synthesis by the fact that their production can be used to synthesize fatty acid. It has significant value theoretically to discover the function of citrate synthase and ATP-citrate lyase in oil content accumulation in seed.
In this study, we cloned cDNA of citrate synthase, ATP-citrate lyase A and ATP-citrate lyase B from cDNA library or cDNA in Brassica napus. Those products had full open reading frames which were confirmed by sequencing. Alignment analysis indicated that those genes were highly conserved in plants.
We designed 16 pairs of EST primers by cDNA sequences of citrate synthase, ATP-citrate lyase A and ATP-citrate lyase B, including 5 pairs of ALCA, 5 pairs of ALCB and 6 pairs of CS, and used them in an oil content segregation F2 population and a different oil content strain population. There were 5 pair of polymorphic primers found in oil content segregation F2 population and 4 pair of polymorphic primers found in different oil content strain population. Correlation analysis between markers and oil content indicated that there was no marker linked to oil content.
We detected the expression of citrate synthase and ATP-citrate lyase by semi-quantity PCR and Real-time PCR, and found that the expression of citrate synthase was different in different oil content rapeseed leaves. The expression of citrate synthase was higher in the leaf of high oil content strain than in the leaf of low oil content strain. It suggested that the expression of citrate synthase maybe be related to oil content. And the expression of citrate synthase and ATP-citrate lyase was diverse in organs, so that those enzymes might be indispensable for normal physiological function. The expression levels of those two enzymes were high in early days and digressive latterly in ovule. This fact indicated that they were important in early development of ovule. The expression of citrate synthase gene was increased at different time in treatments of water logging, drought, low or high temperature, IAA, 6-BA, and ABA., and the expression of ATP-citrate lyase gene was increased at different time in treatments of water logging, low temperature, high illumination, and ABA. The expressing analysis of enzymes under stresses with various pattern proved they took a part in these metabolic control and responding to some environmental changes.
Then we constructed over expressing vectors and repressive expressing vectors for citrate synthase gene and ATP-citrate lyase A gene, named pMD-tCS, pMN-tCS, Gate-tACLA, pMD-rCS, pMN-rCS, pMD-rACLA and pMN-rACLA. After transfoming above plasmids to Arabidopsis thaliana, we otained a respective transgenic plants of diffenent plasmids except pMN-rCS. The successful transformation was confirmed by fastness filtration and PCR. The properties of transgenic plants of plasmid Gate-tACLA and pMN-rACLA were not been analyzed in this study.
Both over or repressive expression of citrate synthase gene made phenotypes changed in transgenic plants. It had thicker side shoots, higher oil content in leaf and seed, and different fatty acid composition in over expression plants compared with wild type. Repressive expression plants were dwarf with miniature leaf, short silique and low oil content. It indicated that citrate synthase played an important role in plant growth, as its expression variety could affect development. And it was one of main regulative enzyme in fatty acid synthesis in plant, because of up or down regulation of its expression resulted in oil content increased or decreased respectively. Furthermore, repressive expression of ATP-citrate lyase A gene led to phenotypes such as dwarfing, leaf miniaturization, shorten silique and oil content decreasing, sugesst that ATP-citrate lyase was also a regulative enzyme in oil synthesis.
根据已知的EST序列设计引物,利用现有的油菜cDNA文库及油菜cDNA,本实验成功克隆了油菜柠檬酸合酶、柠檬酸裂解酶A亚基、柠檬酸裂解酶B亚基的序列。分析表明,克隆到的序列含油菜柠檬酸合酶、柠檬酸裂解酶A亚基、柠檬酸裂解酶B亚基的完整开放阅读框。聚类分析表明,三个基因的序列在植物内高度同源。
根据克隆的柠檬酸裂解酶A亚基、B亚基和柠檬酸合酶基因(ACLA,ACLB和CS)的cDNA序列,共设计了16对EST引物,其中ACLA 5对,ACLB 5对,CS 6对,用于高低油F2群体和不同含油量品种群体标记。将这些引物用于高低油F2群体,最终有5对引物在群体中有差异条带。将16对引物应用于15个高含油量和15个低含油量的油菜品种群体,有4对引物在群体中有差异条带。实验结果证明设计的EST引物可以用于油菜分子标记。
采用半定量PCR和Real-time PCR的方法,检测柠檬酸合酶和柠檬酸裂解酶的表达。结果表明,在含油量不同的油菜品种中,其叶片内柠檬酸合酶表达量有差异,高油品种内表达量较高;在油菜的不同器官和组织中,柠檬酸合酶和柠檬酸裂解酶的表达强度不同,在角果发育早期表达量都较高,随着胚珠的成熟,表达量逐渐下降。在不同的胁迫条件下,油菜叶片中柠檬酸合酶和柠檬酸裂解酶的表达呈现不同的表达规律。柠檬酸合酶在水渍、干旱、高温和低温胁迫下, IAA和6-BA、ABA处理后表达量在特定时期均有上升,同样,柠檬酸裂解酶在水渍、低、强光和ABA处理后在表达量在特定时期均有上升。本实验结果表明,柠檬酸合酶在不同含油量油菜品种叶片中表达强度存在差异。柠檬酸合酶和柠檬酸裂解酶在油菜各器官中广泛表达,而且表达强度存在差异,说明这两个酶是组织和器官发育和执行生理功能不可缺少的。
根据克隆的油菜柠檬酸合酶基因,分别构建了油菜柠檬酸合酶基因的顺式植物表达质粒pMD-tCS,pMN-tCS,和反式植物表达质粒pMD-rCS、pMN-rCS,另外成功构建了柠檬酸裂解酶A亚基基因的顺式表达载体Gate-tACLA、反式表达质粒pMD-rACLA、pMN-rACLA。将构建好的植物表达载体转化拟南芥后,经抗性筛选和PCR检测后,除质粒pMN-rCS外,其余得到不同数量的阳性株系。而质粒pMN-rACLA和Gate-tACLA植株尚未进行性状检测。
在拟南芥中正义和反义表达油菜柠檬酸合酶基因,均造成了表型的变化。正义表达的植株中,侧枝的生长比野生型旺盛,种子和叶片中含油量均有上升,并且在叶片中尤为明显,此外,种子和叶片的脂肪酸组分也发生了一定的变化。而在反义表达的植株中,出现了矮化、叶片变小、果荚缩短等表型,种子和叶片中含油量降低,组分发生了变化。因此,实验结果表明,柠檬酸合酶在植物的正常生长发育中起重要作用,过量表达和降低表达均会造成植株发育的变化;另外,在植物的脂肪酸合成中,是重要的调控酶,该酶表达的上升会造成脂肪酸合成的增加,反义造成脂肪酸合成的降低。而在拟南芥中,反义表达柠檬酸裂解酶A亚基基因,同样会造成矮化、叶片变小、果荚缩短等表型。反义表达柠檬酸裂解酶,也同样造成了植物脂肪酸合成的降低,说明柠檬酸裂解酶同样是脂肪酸合成的调控酶。
Brassica napus is one of major oil crops in China and whole world. The mechanism of high oil content in Brassica napus is a very important subject for theoritic and applied studies. As the key enzymes of sugar metabolism, citrate synthase and ATP-citrate lyase are related to fatty acid synthesis by the fact that their production can be used to synthesize fatty acid. It has significant value theoretically to discover the function of citrate synthase and ATP-citrate lyase in oil content accumulation in seed.
In this study, we cloned cDNA of citrate synthase, ATP-citrate lyase A and ATP-citrate lyase B from cDNA library or cDNA in Brassica napus. Those products had full open reading frames which were confirmed by sequencing. Alignment analysis indicated that those genes were highly conserved in plants.
We designed 16 pairs of EST primers by cDNA sequences of citrate synthase, ATP-citrate lyase A and ATP-citrate lyase B, including 5 pairs of ALCA, 5 pairs of ALCB and 6 pairs of CS, and used them in an oil content segregation F2 population and a different oil content strain population. There were 5 pair of polymorphic primers found in oil content segregation F2 population and 4 pair of polymorphic primers found in different oil content strain population. Correlation analysis between markers and oil content indicated that there was no marker linked to oil content.
We detected the expression of citrate synthase and ATP-citrate lyase by semi-quantity PCR and Real-time PCR, and found that the expression of citrate synthase was different in different oil content rapeseed leaves. The expression of citrate synthase was higher in the leaf of high oil content strain than in the leaf of low oil content strain. It suggested that the expression of citrate synthase maybe be related to oil content. And the expression of citrate synthase and ATP-citrate lyase was diverse in organs, so that those enzymes might be indispensable for normal physiological function. The expression levels of those two enzymes were high in early days and digressive latterly in ovule. This fact indicated that they were important in early development of ovule. The expression of citrate synthase gene was increased at different time in treatments of water logging, drought, low or high temperature, IAA, 6-BA, and ABA., and the expression of ATP-citrate lyase gene was increased at different time in treatments of water logging, low temperature, high illumination, and ABA. The expressing analysis of enzymes under stresses with various pattern proved they took a part in these metabolic control and responding to some environmental changes.
Then we constructed over expressing vectors and repressive expressing vectors for citrate synthase gene and ATP-citrate lyase A gene, named pMD-tCS, pMN-tCS, Gate-tACLA, pMD-rCS, pMN-rCS, pMD-rACLA and pMN-rACLA. After transfoming above plasmids to Arabidopsis thaliana, we otained a respective transgenic plants of diffenent plasmids except pMN-rCS. The successful transformation was confirmed by fastness filtration and PCR. The properties of transgenic plants of plasmid Gate-tACLA and pMN-rACLA were not been analyzed in this study.
Both over or repressive expression of citrate synthase gene made phenotypes changed in transgenic plants. It had thicker side shoots, higher oil content in leaf and seed, and different fatty acid composition in over expression plants compared with wild type. Repressive expression plants were dwarf with miniature leaf, short silique and low oil content. It indicated that citrate synthase played an important role in plant growth, as its expression variety could affect development. And it was one of main regulative enzyme in fatty acid synthesis in plant, because of up or down regulation of its expression resulted in oil content increased or decreased respectively. Furthermore, repressive expression of ATP-citrate lyase A gene led to phenotypes such as dwarfing, leaf miniaturization, shorten silique and oil content decreasing, sugesst that ATP-citrate lyase was also a regulative enzyme in oil synthesis.
引文
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