大花君子兰查尔酮合酶和黄烷酮-3-羟化酶基因的克隆及其功能验证
发布时间：2018-09-19 14:14
【摘要】：大花君子兰(Clivia miniata),为石蒜科君子兰属多年生草本观赏植物,原产于南部非洲,后经德国和日本等地传入中国,是长春市市花;花大且花期长,是十分重要的观赏花卉;但因其花色单一,严重影响了其观赏价值和经济价值;因此研究其花色形成机制显得十分有意义。类黄酮、类胡萝卜素及甜菜色素的含量和种类决定了植物花色的鲜艳度。其中类黄酮是影响花色的主要因素,查尔酮合酶(CHS)和黄烷酮-3-羟化酶(F3H)都是类黄酮代谢的关键酶。大量研究表明,这两种酶对植物花色代谢起着十分重要的作用。本论文采用RACE-PCR技术,从君子兰花中成功克隆得到CmCHS和CmF3H全长cDNA序列;大小分别为1173bp和1128bp;分别编码390和375个氨基酸。生物信息学分析预测其功能,并构建了真核表达载体转拟南芥突变体和构建了原核表达载体制备蛋白进行酶活反应鉴定基因功能,以期为君子兰花色改良及花色机制研究奠定基础。本实验主要结果如下:1、君子兰花色苷成分分析利用高效液相质谱联用技术分析了君子兰橘红色花瓣花色苷组成并检测了花瓣不同颜色部位(分别为:橘红色部位、黄色部位和白色部位)花色苷含量;同时也分析了紫色叶基花色苷组成。结果显示君子兰橘红色花瓣中只存在天竺葵色素,并且不同颜色部位含量为:橘红色部位多于黄色部位、黄色部位多于白色部位;紫色叶基中存在飞燕草色素。2、君子兰查尔酮合酶(CmCHS)和黄烷酮-3-羟化酶(CmF3H)基因的克隆根据NCBI数据库,查找与君子兰同种、属或科等亲缘关系较近物种的查尔酮合酶(CmCHS)和黄烷酮-3-羟化酶(CmF3H)的氨基酸序列,以其保守区域设计简并引物,获得中间片段;通过RACE技术成功扩增得到了CmCHS和CmF3H基因的cDNA全长序列。3、君子兰查尔酮合酶(CmCHS)和黄烷酮-3-羟化酶(CmF3H)基因的表达量分析根据CmCHS和CmF3H基因cDNA全长序列,设计特异性引物;利用半定量RT-PCR方法对CmCHS和CmF3H基因在主要着色组织和不同开花时期进行了表达量分析。结果显示CmCHS和CmF3H基因表达量随着着色组织颜色变化而变化,且大致呈现正相关趋势。4、载体构建成功构建了真核表达载体pBI121-CmCHS和pBI121-CmF3H和原核表达载体pET28-CmCHS;用于CmCHS和CmF3H从真核和原核两个方面进行功能验证。5、转拟南芥突变体验证基因功能CmCHS和CmF3H两个基因都能使相应的拟南芥突变体表型部分恢复,主要体现为T2代种子的种皮颜色明显恢复,经3%蔗糖1/2MS培养基诱导3天后幼苗生长点处变紫,并利用HPLC技术对转基因幼苗进行了花色苷和黄酮醇的检测,检测结果显示:转基因幼苗中花色苷和黄酮醇的合成能力均得到恢复,证明了CmCHS和CmF3H基因与花色现成有关。6、体外酶活反应验证基因功能成功得到了可溶性重组蛋白CmCHS,以香豆酰辅酶A(p-Coumaroyl-CoA)与丙二酰辅酶A(Malonyl-CoA)为底物对其进行底物特异性反应,结果显示CmCHS能够催化这两种底物形成柚皮素(Naringenir),说明原核表达制备的CmCHS蛋白具有查尔酮合酶的催化功能,进一步证明我们分离的基因是查尔酮合酶基因。
[Abstract]:Clivia miniata, a perennial herbaceous ornamental plant of the genus Clematis of Lycoridaceae, originated in southern Africa, was introduced to China by Germany and Japan. It is a very important ornamental flower with large flowers and long flowering period, but its ornamental value and economic value are seriously affected because of its single flower color. Flavonoids, carotenoids and betaine pigments determine the brightness of flower colors. Flavonoids are the main factors affecting flower colors, and chalcone synthase (CHS) and flavanone-3-hydroxylase (F3H) are key enzymes in flavonoid metabolism. Numerous studies have shown that these two enzymes are important for plant coloring. The full-length cDNA sequences of CmCHS and CmF3H were cloned successfully from Gentiana by RACE-PCR, which were 1173 BP and 1128 BP in size, encoding 390 and 375 amino acids, respectively. A prokaryotic expression vector was constructed to prepare proteins for identification of gene function by enzyme activity reaction. The main results were as follows: 1. The composition of anthocyanins in the red and orange petals of Gentiana were analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS). The anthocyanin content in different color parts (orange-red part, yellow part and white part) and the composition of anthocyanin in purple leaves were also analyzed. Cloning of CmCHS and CmF3H genes from the purple leaf base was carried out according to the NCBI database. The amino acid sequences of CmCHS and CmF3H from similar species, genus or family of Gentiana and Gentianae were searched for. The full-length sequences of CmCHS and CMF3H genes were amplified by RACE. 3, CmCHS and flavanone-3-hydroxylase (CmF3H) genes were analyzed by semi-quantitative RT-PCR, and the specific primers were designed according to the full-length sequences of CmCHS and CMF3H genes. The results showed that the expression of CmCHS and C mF3H genes changed with the color of colored tissues and showed a positive correlation trend. 4. Eukaryotic expression vectors pBI121-CmCHS and pBI121-C mF3H and prokaryotic expression vectors pET28-CmCHS were constructed successfully. MCHS and CMF3H were used to verify their functions from eukaryotic and prokaryotic aspects. 5. Transgenic Arabidopsis mutants showed that CmCHS and CMF3H genes could partly restore the phenotype of the corresponding Arabidopsis mutants, mainly reflected in the obvious restoration of seed coat color of T2 generation seeds, and the seedling growth point became violet after 3 days induction with 3% sucrose 1/2MS medium. The anthocyanins and flavonols in transgenic seedlings were detected by HPLC. The results showed that the synthesis ability of anthocyanins and flavonols in transgenic seedlings was restored. It was proved that CmCHS and CMF3H genes were related to the ready-made flower color. 6. The soluble recombinant protein CmCHS was successfully obtained by enzyme activity in vitro. Coumarioyl-CoA (p-Coumaroyl-CoA) and Malonyl-CoA (Malonyl-CoA) were used as substrates for substrate-specific reactions. The results showed that CmCHS could catalyze the formation of naringin (Naringenir) from these two substrates, indicating that the CmCHS protein prepared by prokaryotic expression had the catalytic function of chalcone synthase, which further proved that the gene we isolated was a chalcone synthase. The gene of ketone synthase.
【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S682.13
本文编号：2250369
[Abstract]:Clivia miniata, a perennial herbaceous ornamental plant of the genus Clematis of Lycoridaceae, originated in southern Africa, was introduced to China by Germany and Japan. It is a very important ornamental flower with large flowers and long flowering period, but its ornamental value and economic value are seriously affected because of its single flower color. Flavonoids, carotenoids and betaine pigments determine the brightness of flower colors. Flavonoids are the main factors affecting flower colors, and chalcone synthase (CHS) and flavanone-3-hydroxylase (F3H) are key enzymes in flavonoid metabolism. Numerous studies have shown that these two enzymes are important for plant coloring. The full-length cDNA sequences of CmCHS and CmF3H were cloned successfully from Gentiana by RACE-PCR, which were 1173 BP and 1128 BP in size, encoding 390 and 375 amino acids, respectively. A prokaryotic expression vector was constructed to prepare proteins for identification of gene function by enzyme activity reaction. The main results were as follows: 1. The composition of anthocyanins in the red and orange petals of Gentiana were analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS). The anthocyanin content in different color parts (orange-red part, yellow part and white part) and the composition of anthocyanin in purple leaves were also analyzed. Cloning of CmCHS and CmF3H genes from the purple leaf base was carried out according to the NCBI database. The amino acid sequences of CmCHS and CmF3H from similar species, genus or family of Gentiana and Gentianae were searched for. The full-length sequences of CmCHS and CMF3H genes were amplified by RACE. 3, CmCHS and flavanone-3-hydroxylase (CmF3H) genes were analyzed by semi-quantitative RT-PCR, and the specific primers were designed according to the full-length sequences of CmCHS and CMF3H genes. The results showed that the expression of CmCHS and C mF3H genes changed with the color of colored tissues and showed a positive correlation trend. 4. Eukaryotic expression vectors pBI121-CmCHS and pBI121-C mF3H and prokaryotic expression vectors pET28-CmCHS were constructed successfully. MCHS and CMF3H were used to verify their functions from eukaryotic and prokaryotic aspects. 5. Transgenic Arabidopsis mutants showed that CmCHS and CMF3H genes could partly restore the phenotype of the corresponding Arabidopsis mutants, mainly reflected in the obvious restoration of seed coat color of T2 generation seeds, and the seedling growth point became violet after 3 days induction with 3% sucrose 1/2MS medium. The anthocyanins and flavonols in transgenic seedlings were detected by HPLC. The results showed that the synthesis ability of anthocyanins and flavonols in transgenic seedlings was restored. It was proved that CmCHS and CMF3H genes were related to the ready-made flower color. 6. The soluble recombinant protein CmCHS was successfully obtained by enzyme activity in vitro. Coumarioyl-CoA (p-Coumaroyl-CoA) and Malonyl-CoA (Malonyl-CoA) were used as substrates for substrate-specific reactions. The results showed that CmCHS could catalyze the formation of naringin (Naringenir) from these two substrates, indicating that the CmCHS protein prepared by prokaryotic expression had the catalytic function of chalcone synthase, which further proved that the gene we isolated was a chalcone synthase. The gene of ketone synthase.
【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S682.13
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