本文選題：唐古特白刺 + 花青素�。� 參考：《內(nèi)蒙古大學(xué)》2017年碩士論文

【摘要】：植物在生長(zhǎng)發(fā)育的過(guò)程中會(huì)遭受各種各樣的非生物脅迫,如UV-B、干旱、鹽脅迫等。植物能通過(guò)積累多種小分子次生代謝產(chǎn)物,幫助其抵抗各種脅迫,花青素就是其中重要的一類。花青素具有良好的活性氧自由基(ROS)清除能力,能減輕非生物脅迫帶來(lái)的氧化損傷。二氫黃酮醇4-還原酶(dihydroflavonol 4-reductase,DFR)是花青素合成途徑下游的關(guān)鍵酶,決定植物積累花青素的種類,這對(duì)植物通過(guò)積累花青素來(lái)抵抗逆境脅迫具有十分重要的意義。唐古特白刺生長(zhǎng)于自然環(huán)境惡劣的干旱和半干旱地區(qū),對(duì)嚴(yán)峻條件下的各種非生物脅迫具有極強(qiáng)的耐受力和抵抗力。本研究利用RT-PCR和RACE技術(shù),克隆唐古特白刺二氫黃酮醇4-還原酶(NtDFR)基因cDNA全長(zhǎng)序列,該基因全長(zhǎng)1392bp,其開放閱讀框(ORF,Open Reading Frame)長(zhǎng)度為1020bp,編碼339個(gè)氨基酸。將擴(kuò)增得到的ORF片段與pPZP221載體連接重組,得到pPZP221-NtDFR植物表達(dá)載體。農(nóng)桿菌浸染擬南芥并篩選至T3代純合子,得到了 6個(gè)轉(zhuǎn)基因擬南芥株系,并進(jìn)行PCR驗(yàn)證,結(jié)果均有條帶,無(wú)假陽(yáng)性。選取L2、L3、L5三個(gè)株系的轉(zhuǎn)基因擬南芥用于后續(xù)實(shí)驗(yàn)。對(duì)野生型和轉(zhuǎn)基因型擬南芥進(jìn)行Oh、2h、4h和6h的UV-B脅迫,Od、10d和15d的干旱脅迫,以及OmM、75mM和150mM的NaCl脅迫。野生型和轉(zhuǎn)基因型擬南芥的花青素含量,結(jié)果表明3種脅迫條件下轉(zhuǎn)基因型擬南芥中的花青素含量高于野生型,證實(shí)了NtDF 的過(guò)表達(dá)能誘導(dǎo)轉(zhuǎn)基因型擬南芥中花青素的大量積累。同時(shí)對(duì)擬南芥的其它一些生理生化指標(biāo)經(jīng)行了測(cè)量,包括鮮重、葉綠素含量、SOD、CAT活性和MDA、GSH含量。結(jié)果表明花青素的積累能改善擬南芥在脅迫條件下的生長(zhǎng)狀況,調(diào)節(jié)擬南芥的抗氧化系統(tǒng),使其抗逆性增強(qiáng)。證實(shí)了NtDF 的過(guò)表達(dá)和花青素的積累對(duì)擬南芥在應(yīng)對(duì)非生物脅迫的過(guò)程中發(fā)揮了積極的作用。
[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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

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