唐古特白刺二氢黄酮醇4-还原酶基因（DFR）的克隆与功能分析

发布时间：2018-04-14 01:36

本文选题：唐古特白刺 + 花青素　；参考：《内蒙古大学》2017年硕士论文

【摘要】：植物在生长发育的过程中会遭受各种各样的非生物胁迫,如UV-B、干旱、盐胁迫等。植物能通过积累多种小分子次生代谢产物,帮助其抵抗各种胁迫,花青素就是其中重要的一类。花青素具有良好的活性氧自由基(ROS)清除能力,能减轻非生物胁迫带来的氧化损伤。二氢黄酮醇4-还原酶(dihydroflavonol 4-reductase,DFR)是花青素合成途径下游的关键酶,决定植物积累花青素的种类,这对植物通过积累花青素来抵抗逆境胁迫具有十分重要的意义。唐古特白刺生长于自然环境恶劣的干旱和半干旱地区,对严峻条件下的各种非生物胁迫具有极强的耐受力和抵抗力。本研究利用RT-PCR和RACE技术,克隆唐古特白刺二氢黄酮醇4-还原酶(NtDFR)基因cDNA全长序列,该基因全长1392bp,其开放阅读框(ORF,Open Reading Frame)长度为1020bp,编码339个氨基酸。将扩增得到的ORF片段与pPZP221载体连接重组,得到pPZP221-NtDFR植物表达载体。农杆菌浸染拟南芥并筛选至T3代纯合子,得到了 6个转基因拟南芥株系,并进行PCR验证,结果均有条带,无假阳性。选取L2、L3、L5三个株系的转基因拟南芥用于后续实验。对野生型和转基因型拟南芥进行Oh、2h、4h和6h的UV-B胁迫,Od、10d和15d的干旱胁迫,以及OmM、75mM和150mM的NaCl胁迫。野生型和转基因型拟南芥的花青素含量,结果表明3种胁迫条件下转基因型拟南芥中的花青素含量高于野生型,证实了NtDF 的过表达能诱导转基因型拟南芥中花青素的大量积累。同时对拟南芥的其它一些生理生化指标经行了测量,包括鲜重、叶绿素含量、SOD、CAT活性和MDA、GSH含量。结果表明花青素的积累能改善拟南芥在胁迫条件下的生长状况,调节拟南芥的抗氧化系统,使其抗逆性增强。证实了NtDF 的过表达和花青素的积累对拟南芥在应对非生物胁迫的过程中发挥了积极的作用。
[Abstract]:Plants are subjected to various abiotic stresses, such as UV-B, drought, salt stress and so on.Anthocyanins are one of the most important secondary metabolites that can be accumulated by plants to help them resist all kinds of stresses.Anthocyanin has good scavenging ability of reactive oxygen free radical (Ros) and can reduce oxidative damage caused by abiotic stress.Dihydroflavonol 4-reductase (DFR) is a key enzyme downstream of anthocyanin synthesis pathway, which determines the type of anthocyanin accumulation in plants, which is of great significance for plants to resist stress by accumulation of anthocyanins.Tangut Spurs, which grows in arid and semi-arid areas with harsh natural environment, has strong tolerance and resistance to various abiotic stresses under severe conditions.In this study, RT-PCR and RACE techniques were used to clone the full-length cDNA sequence of the dihydroflavonol 4-reductase (NtDFR) gene from Tangut Spurs. The gene was 1392 BP in length and its open reading frame was 1020 BP, encoding 339 amino acids.The amplified ORF fragment was ligated with the pPZP221 vector to obtain the pPZP221-NtDFR plant expression vector.Six transgenic Arabidopsis strains were obtained by Agrobacterium tumefaciens soaking and screening to T3 generation homozygote. The results were verified by PCR, and there were no false positive bands.Transgenic Arabidopsis thaliana was selected from L _ 2 and L _ 3 and L _ 5 for further experiment.Wild-type and transgenic Arabidopsis thaliana were subjected to drought stress of 10 d and 15 d at UV-B stress of 2 h and 6 h, and NaCl stress of OmMN 75 mm and 150mM at 4 h and 6 h.The anthocyanin content of wild and transgenic Arabidopsis thaliana was higher than that of wild-type Arabidopsis under three stress conditions. It was proved that overexpression of NtDF could induce a large amount of anthocyanin accumulation in transgenic Arabidopsis.Meanwhile, some other physiological and biochemical indexes of Arabidopsis thaliana were measured, including fresh weight, chlorophyll content, activity of SODX cat and content of MDA-GSH in Arabidopsis thaliana.The results showed that anthocyanin accumulation could improve the growth of Arabidopsis thaliana under stress, regulate the antioxidant system of Arabidopsis thaliana and enhance its resistance to stress.It is confirmed that overexpression of NtDF and accumulation of anthocyanins play an active role in the response to abiotic stress in Arabidopsis thaliana.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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