發(fā)布時間：2018-03-02 23:07

  本文選題：葛根素　切入點：異黃酮　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：葛根是一種藥用植物,根部可作為藥材,具有很高的藥用價值,廣泛用于治療心腦血管疾病、糖尿病、肝病等疾病。葛根中的主要活性物質(zhì)為葛根素、大豆苷等異黃酮類化合物,該類化合物具有具有抗炎、抗癌、抗氧化等特性。糖基轉(zhuǎn)移酶UGT(glycosyltransferases)能將UDP活化的糖分子轉(zhuǎn)移到生物大分子上,是自然界最重要的修飾基因之一。異黃酮類化合物是植物中分布最廣泛的此生代謝產(chǎn)物之一。在葛根中,UGT對黃酮類化合物進(jìn)行糖基化修飾,合成異黃酮糖苷,在黃酮的合成、轉(zhuǎn)運、存儲等方面發(fā)揮了重要的作用。葛根素結(jié)構(gòu)的特殊性在于葡糖糖基以C-C鍵與母環(huán)相連,而絕大多數(shù)異黃酮糖苷都以C-O鍵與甘元母環(huán)相連。葛根中存在2類對應(yīng)的UGT,即碳苷糖基轉(zhuǎn)移酶(CGT)和氧苷糖基轉(zhuǎn)移酶(OGT)。為了挖掘葛根中的UGT基因,本課題通過轉(zhuǎn)錄組測序找到了140多個UGT基因序列。通過與已知CGT和OGT進(jìn)行序列比對分析,選定8個目標(biāo)基因,分別命名為GT1、GT2、GT3、GT4、GT5、GT6、GT7、GT8,成功克隆基因,并通過蛋白原核表達(dá)及轉(zhuǎn)基因大豆發(fā)根誘導(dǎo)技術(shù)初步驗證基因功能。將目標(biāo)基因構(gòu)建到原核表達(dá)載體上,將載體轉(zhuǎn)入原核表達(dá)菌株Rosetta(DE3)中,用IPTG誘導(dǎo)蛋白表達(dá)。基因GT1、GT2、GT3、GT4、GT5、GT7、GT8均成功誘導(dǎo)出目標(biāo)蛋白(51KD左右),用超聲破碎法提取蛋白,發(fā)現(xiàn)蛋白大部分出現(xiàn)在沉淀中,上清液中含量極少。在配好的反應(yīng)體系中加入提取的GT蛋白上清液,發(fā)現(xiàn)GT4具有很強的糖基轉(zhuǎn)移酶活性,能夠催化大豆甘元、染料木素、芒柄花素、黃豆黃素合成對應(yīng)的7-O-葡糖糖苷,且轉(zhuǎn)化率很高,接近完全轉(zhuǎn)化;GT4對甘草素和異甘草素?zé)o反應(yīng)。由此說明GT4是特異性識別異黃酮并高效催化其合成7-O-葡萄糖苷的糖基轉(zhuǎn)移酶基因。此外,發(fā)現(xiàn)GT1可催化芒柄花素合成芒柄花苷,但是活性很低。其他GT基因無糖基轉(zhuǎn)移酶活性。將目標(biāo)基因構(gòu)建到超表達(dá)載體pB7WG2D,轉(zhuǎn)入發(fā)根農(nóng)桿菌K599并侵染大豆子葉,誘導(dǎo)轉(zhuǎn)基因發(fā)根產(chǎn)生。提取發(fā)根異黃酮類化合物,經(jīng)過HPLC檢測,結(jié)果發(fā)現(xiàn)與對照相比,超表達(dá)目標(biāo)基因的發(fā)根中無葛根素,且異黃酮糖苷的含量也無顯著差異。本實驗未能成功克隆催化葛根素合成的CGT,但是鑒定出一個新的糖基糖基轉(zhuǎn)移酶基因,該基因具有很強的活性,可為后續(xù)研究及生產(chǎn)應(yīng)用提供借鑒。
[Abstract]:Pueraria lobata is a kind of medicinal plant, the root can be used as medicinal material, it has high medicinal value, and is widely used in the treatment of cardiovascular and cerebrovascular diseases, diabetes, liver diseases, etc. The main active substance in Pueraria lobata is puerarin, the main active substance in Pueraria lobata is puerarin. Isoflavones, such as daidzein, have anti-inflammatory, anticancer and antioxidant properties. Glycosyltransferases (UGTG) can transfer carbohydrates activated by UDP onto biomolecules. Isoflavones are one of the most widely distributed metabolites in plants. UGT glycosylation of flavonoids in Pueraria lobata, synthesis of isoflavone glycosides, synthesis of flavonoids, The special structure of puerarin is that the glucosyl group is linked to the parent ring by C-C bond. However, most isoflavone glycosides are linked to the Ganyuan maternal ring by C-O bond. There are two types of UGTs in Pueraria lobata, that is, CGT) and Oxyglycosyltransferase (OGTT). In order to explore the UGT gene in Pueraria lobata, In this study, more than 140 UGT gene sequences were identified by transcriptome sequencing. Through sequence alignment analysis with known CGT and OGT, eight target genes were selected, named GT1, GT2, GT3, GT3, GT5, GT6, GT7 and GT8, respectively. The target gene was constructed into prokaryotic expression vector and transferred into prokaryotic expression strain Rosetta-DE3). Protein expression was induced by IPTG. The target protein was successfully induced by the gene GT1, GT1, GT2, GT3, GT4, GT5, GT7, GT8. The protein was extracted by ultrasonic fragmentation, and most of the proteins appeared in the precipitate. The supernatant contained very little in the supernatant. Adding the supernatant of GT protein into the reaction system, it was found that GT4 had strong activity of glycosyltransferase and could catalyze glycosyltransferase activity of soybean, genistein and amaranthin. The corresponding 7-Oglucoside was synthesized by daidzein, and the conversion rate was very high. Nearly complete transformation of GT4 did not react with glycyrrhizin and isoglycyrrhizin, which indicated that GT4 is a glycosyltransferase gene that can specifically recognize isoflavones and efficiently catalyze the synthesis of 7-O- glucoside. In addition, it was found that GT1 can catalyze the synthesis of Acanthopanthin. But the activity is very low. The other GT genes have no glycosyltransferase activity. The target gene was constructed into pB7WG2D. the target gene was transformed into Agrobacterium rhizogenes K599 and infected with soybean cotyledons to induce the production of transgenic hairy roots. The results of HPLC analysis showed that there was no puerarin in the hair root of the overexpression target gene compared with the control. There was no significant difference in the content of isoflavone glucosides. In this study, the CGT catalyzed puerarin synthesis was not successfully cloned, but a new glycosyltransferase gene was identified, which has strong activity. It can be used for reference for follow-up research and production application.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S567.19

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 楊旭東;王愛勤;何龍飛;;葛根種質(zhì)資源及其開發(fā)利用研究進(jìn)展[J];中國農(nóng)學(xué)通報;2014年24期

2 李超;劉艾林;杜冠華;;天然異黃酮類化合物的藥理學(xué)研究進(jìn)展[J];中國新藥雜志;2013年12期

3 尹麗紅;李艷楓;孟繁琳;;葛根的化學(xué)成分、藥理作用和臨床應(yīng)用[J];黑龍江醫(yī)藥;2010年03期

4 董英;徐斌;林琳;查青;;葛根的化學(xué)成分研究[J];食品與機械;2005年06期

5 顧志平,連文琰,陳碧珠,陳四保,王慎保;中藥葛根資源的調(diào)查研究[J];中藥材;1993年08期

，

本文編號：1558485