大花君子蘭查爾酮合酶和黃烷酮-3-羥化酶基因的克隆及其功能驗(yàn)證
發(fā)布時(shí)間：2018-09-19 14:14
【摘要】：大花君子蘭(Clivia miniata),為石蒜科君子蘭屬多年生草本觀賞植物,原產(chǎn)于南部非洲,后經(jīng)德國和日本等地傳入中國,是長春市市花;花大且花期長,是十分重要的觀賞花卉;但因其花色單一,嚴(yán)重影響了其觀賞價(jià)值和經(jīng)濟(jì)價(jià)值;因此研究其花色形成機(jī)制顯得十分有意義。類黃酮、類胡蘿卜素及甜菜色素的含量和種類決定了植物花色的鮮艷度。其中類黃酮是影響花色的主要因素,查爾酮合酶(CHS)和黃烷酮-3-羥化酶(F3H)都是類黃酮代謝的關(guān)鍵酶。大量研究表明,這兩種酶對植物花色代謝起著十分重要的作用。本論文采用RACE-PCR技術(shù),從君子蘭花中成功克隆得到CmCHS和CmF3H全長cDNA序列;大小分別為1173bp和1128bp;分別編碼390和375個(gè)氨基酸。生物信息學(xué)分析預(yù)測其功能,并構(gòu)建了真核表達(dá)載體轉(zhuǎn)擬南芥突變體和構(gòu)建了原核表達(dá)載體制備蛋白進(jìn)行酶活反應(yīng)鑒定基因功能,以期為君子蘭花色改良及花色機(jī)制研究奠定基礎(chǔ)。本實(shí)驗(yàn)主要結(jié)果如下:1、君子蘭花色苷成分分析利用高效液相質(zhì)譜聯(lián)用技術(shù)分析了君子蘭橘紅色花瓣花色苷組成并檢測了花瓣不同顏色部位(分別為:橘紅色部位、黃色部位和白色部位)花色苷含量;同時(shí)也分析了紫色葉基花色苷組成。結(jié)果顯示君子蘭橘紅色花瓣中只存在天竺葵色素,并且不同顏色部位含量為:橘紅色部位多于黃色部位、黃色部位多于白色部位;紫色葉基中存在飛燕草色素。2、君子蘭查爾酮合酶(CmCHS)和黃烷酮-3-羥化酶(CmF3H)基因的克隆根據(jù)NCBI數(shù)據(jù)庫,查找與君子蘭同種、屬或科等親緣關(guān)系較近物種的查爾酮合酶(CmCHS)和黃烷酮-3-羥化酶(CmF3H)的氨基酸序列,以其保守區(qū)域設(shè)計(jì)簡并引物,獲得中間片段;通過RACE技術(shù)成功擴(kuò)增得到了CmCHS和CmF3H基因的cDNA全長序列。3、君子蘭查爾酮合酶(CmCHS)和黃烷酮-3-羥化酶(CmF3H)基因的表達(dá)量分析根據(jù)CmCHS和CmF3H基因cDNA全長序列,設(shè)計(jì)特異性引物;利用半定量RT-PCR方法對CmCHS和CmF3H基因在主要著色組織和不同開花時(shí)期進(jìn)行了表達(dá)量分析。結(jié)果顯示CmCHS和CmF3H基因表達(dá)量隨著著色組織顏色變化而變化,且大致呈現(xiàn)正相關(guān)趨勢。4、載體構(gòu)建成功構(gòu)建了真核表達(dá)載體pBI121-CmCHS和pBI121-CmF3H和原核表達(dá)載體pET28-CmCHS;用于CmCHS和CmF3H從真核和原核兩個(gè)方面進(jìn)行功能驗(yàn)證。5、轉(zhuǎn)擬南芥突變體驗(yàn)證基因功能CmCHS和CmF3H兩個(gè)基因都能使相應(yīng)的擬南芥突變體表型部分恢復(fù),主要體現(xiàn)為T2代種子的種皮顏色明顯恢復(fù),經(jīng)3%蔗糖1/2MS培養(yǎng)基誘導(dǎo)3天后幼苗生長點(diǎn)處變紫,并利用HPLC技術(shù)對轉(zhuǎn)基因幼苗進(jìn)行了花色苷和黃酮醇的檢測,檢測結(jié)果顯示:轉(zhuǎn)基因幼苗中花色苷和黃酮醇的合成能力均得到恢復(fù),證明了CmCHS和CmF3H基因與花色現(xiàn)成有關(guān)。6、體外酶活反應(yīng)驗(yàn)證基因功能成功得到了可溶性重組蛋白CmCHS,以香豆酰輔酶A(p-Coumaroyl-CoA)與丙二酰輔酶A(Malonyl-CoA)為底物對其進(jìn)行底物特異性反應(yīng),結(jié)果顯示CmCHS能夠催化這兩種底物形成柚皮素(Naringenir),說明原核表達(dá)制備的CmCHS蛋白具有查爾酮合酶的催化功能,進(jìn)一步證明我們分離的基因是查爾酮合酶基因。
[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.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S682.13
本文編號：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.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S682.13
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