本文選題：黃花蒿 + 青蒿素��； 參考：《湖南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：黃花蒿(Artemisia annua L.)為一年生草本植物,其主要活性成分青蒿素是目前國際上治療瘧疾的首選藥物。近年發(fā)現(xiàn)單一青蒿素治療容易使瘧原蟲產(chǎn)生耐藥性,而黃花蒿中黃酮類物質(zhì)能協(xié)同青蒿素的吸收,減少耐藥性的產(chǎn)生。3-羥基-3-甲基戊二酰輔酶A還原酶(HMGR)是青蒿素生物合成的第一個(gè)關(guān)鍵酶基因,過表達(dá)該基因能顯著提高青蒿素的含量,但關(guān)于該啟動(dòng)子的活性分析和核心序列篩選的研究還未見報(bào)道；同時(shí),黃烷酮3-羥化酶(AaF3H)和黃酮醇合酶(AaFLS)是黃花蒿黃酮醇生物合成的關(guān)鍵酶基因,其體外生物功能雖已得到驗(yàn)證,但亦未見其真核表達(dá)研究。為此,本研究采用基因克隆、GUS基因標(biāo)記及農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化煙草的方法,對(duì)HMGR啟動(dòng)子在高溫、低溫和脫水條件下的生物活性及核心序列進(jìn)行了分析,并在分析AaF3H cDNA全長序列的基礎(chǔ)上,研究了AaF3H和AaFLS基因在煙草中的過表達(dá),以為黃花蒿青蒿素與黃酮類化合物的生物合成及其協(xié)同抗瘧的后續(xù)研究提供參考。主要研究結(jié)果如下：1)以黃花蒿葉片為材料,提取DNA,采用5'端系列缺失的方法克隆了黃花蒿HMGR啟動(dòng)子至表達(dá)載體PCX-GUS-P,分別轉(zhuǎn)入植物表達(dá)載體農(nóng)桿菌GV3101,經(jīng)農(nóng)桿菌侵染法導(dǎo)入煙草DNA并GUS染色,結(jié)果發(fā)現(xiàn)轉(zhuǎn)HMGR啟動(dòng)子煙草的根、莖、葉、花、果莢均出現(xiàn)藍(lán)色,脫水和高溫脅迫條件下藍(lán)色變淺,轉(zhuǎn)入啟動(dòng)子片段P-HMGR-1、 P-HMGR-2和P-HMGR-3的煙草葉片變藍(lán),其中P-HMGR-2藍(lán)色深淺與P-HMGR-1相近,P-HMGR-3藍(lán)色比P-HMGR-1淺,而P-HMGR-4、P-HMGR-5和對(duì)照煙草葉片不變藍(lán),說明HMGR啟動(dòng)子在煙草整個(gè)生長期內(nèi)均能夠穩(wěn)定表達(dá),且對(duì)高溫和脫水條件敏感而對(duì)低溫不敏感,同時(shí)該啟動(dòng)子的核心序列在P-HMGR-3區(qū)域,而P-HMGR-2和P-HMGR-3的非重疊區(qū)則存在與HMGR啟動(dòng)子活性強(qiáng)弱相關(guān)的調(diào)控元件。2)通過SMART-RACE技術(shù)獲得黃花蒿AaF3H基因全長cDNA序列,該基因全長為2136 bp。序列分析表明,該序列包含一個(gè)完整的編碼區(qū),共1092 bp,編碼364個(gè)氨基酸組成的蛋白。該蛋白的分子量為41.18(KDa),理論等電點(diǎn)(pI)為5.67,是一種穩(wěn)定的親水性蛋白,且為非分泌和非跨膜蛋白,共有13個(gè)磷酸化位點(diǎn),屬于典型的20G-FeⅡ-OXY加氧酶超家族,二級(jí)結(jié)構(gòu)以無規(guī)則卷曲為主,其次是α-螺旋和延伸鏈,與菊花F3H蛋白的一致性高達(dá)97%。3)以黃花蒿幼嫩芽葉為材料,提取總RNA,并逆轉(zhuǎn)錄得到cDNA,克隆了黃花蒿中AaF3H基因和AaFLS基因,通過將其與過表達(dá)載體PCXSN整合并導(dǎo)入農(nóng)桿菌GV3101中,構(gòu)建植物表達(dá)載體PCXSN-AaF3H和PCXSN-AaFLS,經(jīng)農(nóng)桿菌侵染,成功將PCXSN-AaF3H和PCXSN-AaFLS表達(dá)載體分別轉(zhuǎn)入煙草DNA中,結(jié)果表明,過表達(dá)AaF3H和AaFLS基因都能顯著轉(zhuǎn)化柚皮素,且過表達(dá)AaF3H轉(zhuǎn)化柚皮素的能力較強(qiáng)。
[Abstract]:Artemisia annua L. Artemisinin, an annual herb, is the first choice drug for malaria treatment in the world. In recent years, it has been found that single artemisinin treatment makes Plasmodium falciparum resistant and flavonoids in Artemisia annua synergize with artemisinin absorption. HMGR) is the first key enzyme gene in artemisinin biosynthesis, and its overexpression can significantly increase the content of artemisinin. However, studies on the activity analysis and core sequence screening of the promoter have not been reported. Meanwhile, flavanone 3-hydroxylase (AaF3H) and flavonol synthase (AaFLS) are the key enzyme genes for flavonol biosynthesis of Artemisia annua. Although its biological function has been verified in vitro, it has not been studied in eukaryotic expression. Therefore, the biological activity and core sequence of HMGR promoter under high temperature, low temperature and dehydration were analyzed by means of gene cloning Gus marker and Agrobacterium tumefaciens mediated transformation of tobacco. The overexpression of AaF3H and AaFLS genes in tobacco was studied on the basis of analyzing the full-length cDNA sequence of AaF3H, which provided a reference for the biosynthesis of artemisinin and flavonoids from Artemisia annua and its synergistic antimalarial studies. The main results are as follows: (1) the leaves of Artemisia annua were used as materials. The HMGR promoter of Artemisia annua L. was cloned into the expression vector PCX-GUS-Pby 5 '-terminal series deletion method, and transferred into Agrobacterium tumefaciens GV3101, respectively. The HMGR promoter was introduced into tobacco DNA and stained with Gus by Agrobacterium tumefaciens. The results showed that the HMGR promoter was transformed into roots and stems of tobacco. The leaves, flowers and fruit pods were all blue, and the blue became light under dehydration and high temperature stress. The tobacco leaves transferred to the promoter fragments P-HMGR-1, P-HMGR-2 and P-HMGR-3 turned blue. The blue depth of P-HMGR-2 was lighter than that of P-HMGR-1, but P-HMGR-4P-HMGR-5 was the same blue as the control. The results showed that HMGR promoter was stably expressed during the whole growth period of tobacco, and was sensitive to high temperature and dehydration conditions, but not sensitive to low temperature, and the core sequence of HMGR promoter was in the region of P-HMGR-3. However, the non-overlapping region of P-HMGR-2 and P-HMGR-3 contained a regulatory element. 2) the full-length cDNA sequence of AaF3H gene of Artemisia annua was obtained by SMART-RACE, which was 2136 BP. Sequence analysis showed that the sequence contained a complete coding region of 1092 BP, encoding 364 amino acids. The molecular weight of the protein is 41.18kDa, and the theoretical isoelectric point (Pi) is 5.67. It is a stable hydrophilic protein, and it is a non-secretory and non-transmembrane protein with 13 phosphorylation sites, belonging to the typical superfamily of 20G-Fe 鈪，

本文編號(hào)：2011300