黄花蒿抗疟成分基因克隆与功能分析

发布时间：2018-06-12 22:27

  本文选题：黄花蒿 + 青蒿素　； 参考：《湖南农业大学》2016年硕士论文

【摘要】：黄花蒿(Artemisia annua L.)为一年生草本植物,其主要活性成分青蒿素是目前国际上治疗疟疾的首选药物。近年发现单一青蒿素治疗容易使疟原虫产生耐药性,而黄花蒿中黄酮类物质能协同青蒿素的吸收,减少耐药性的产生。3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)是青蒿素生物合成的第一个关键酶基因,过表达该基因能显著提高青蒿素的含量,但关于该启动子的活性分析和核心序列筛选的研究还未见报道；同时,黄烷酮3-羟化酶(AaF3H)和黄酮醇合酶(AaFLS)是黄花蒿黄酮醇生物合成的关键酶基因,其体外生物功能虽已得到验证,但亦未见其真核表达研究。为此,本研究采用基因克隆、GUS基因标记及农杆菌介导法转化烟草的方法,对HMGR启动子在高温、低温和脱水条件下的生物活性及核心序列进行了分析,并在分析AaF3H cDNA全长序列的基础上,研究了AaF3H和AaFLS基因在烟草中的过表达,以为黄花蒿青蒿素与黄酮类化合物的生物合成及其协同抗疟的后续研究提供参考。主要研究结果如下：1)以黄花蒿叶片为材料,提取DNA,采用5'端系列缺失的方法克隆了黄花蒿HMGR启动子至表达载体PCX-GUS-P,分别转入植物表达载体农杆菌GV3101,经农杆菌侵染法导入烟草DNA并GUS染色,结果发现转HMGR启动子烟草的根、茎、叶、花、果荚均出现蓝色,脱水和高温胁迫条件下蓝色变浅,转入启动子片段P-HMGR-1、 P-HMGR-2和P-HMGR-3的烟草叶片变蓝,其中P-HMGR-2蓝色深浅与P-HMGR-1相近,P-HMGR-3蓝色比P-HMGR-1浅,而P-HMGR-4、P-HMGR-5和对照烟草叶片不变蓝,说明HMGR启动子在烟草整个生长期内均能够稳定表达,且对高温和脱水条件敏感而对低温不敏感,同时该启动子的核心序列在P-HMGR-3区域,而P-HMGR-2和P-HMGR-3的非重叠区则存在与HMGR启动子活性强弱相关的调控元件。2)通过SMART-RACE技术获得黄花蒿AaF3H基因全长cDNA序列,该基因全长为2136 bp。序列分析表明,该序列包含一个完整的编码区,共1092 bp,编码364个氨基酸组成的蛋白。该蛋白的分子量为41.18(KDa),理论等电点(pI)为5.67,是一种稳定的亲水性蛋白,且为非分泌和非跨膜蛋白,共有13个磷酸化位点,属于典型的20G-FeⅡ-OXY加氧酶超家族,二级结构以无规则卷曲为主,其次是α-螺旋和延伸链,与菊花F3H蛋白的一致性高达97%。3)以黄花蒿幼嫩芽叶为材料,提取总RNA,并逆转录得到cDNA,克隆了黄花蒿中AaF3H基因和AaFLS基因,通过将其与过表达载体PCXSN整合并导入农杆菌GV3101中,构建植物表达载体PCXSN-AaF3H和PCXSN-AaFLS,经农杆菌侵染,成功将PCXSN-AaF3H和PCXSN-AaFLS表达载体分别转入烟草DNA中,结果表明,过表达AaF3H和AaFLS基因都能显著转化柚皮素,且过表达AaF3H转化柚皮素的能力较强。
[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，

本文编号：2011300