唐古特白刺类黄酮-3-O-葡萄糖基转移酶基因(NtUFGT)的克隆与功能分析

doi:10.11686/cyxb2019334

摘要/Abstract

摘要： 花青素是植物体内重要的黄酮类次生代谢产物,其强大的抗氧化能力对植物抵抗由各种非生物胁迫带来的氧化损伤发挥着重要的作用。本研究基于转录组数据,从唐古特白刺cDNA中克隆得到一个类黄酮3-O-葡萄糖基转移酶基因,将其命名为NtUFGT。该基因开放阅读框长度为1407 bp,编码468个氨基酸,预测该基因编码的蛋白质相对分子质量为51.37 kDa。多重序列比对分析结果显示,其编码蛋白属于UDP-glycosyltransferases蛋白家族。实时荧光定量PCR(qRT-PCR)分析了该基因在唐古特白刺中的表达模式,发现该基因的表达具有组织特异性,由高到低依次为花>果实>茎>叶>根;同时该基因能被聚乙二醇(PEG)和脱落酸(ABA)等非生物胁迫强烈诱导表达。构建该基因的真核表达载体pPZP221:35S:NtUFGT,使用花序浸染法转化拟南芥并筛选至T₃代,RT-PCR验证表明,NtUFGT基因在转基因拟南芥3个株系中均明显表达。测定干旱胁迫条件下野生型(WT)和转基因拟南芥(OE株系)生长状况和抗逆相关生理生化指标,结果发现转基因拟南芥生长状况明显优于野生型,根长更长,鲜重和叶绿素含量更高,同时OE株系积累了更多的花青素和总黄酮。与表型一致,相较于WT,OE株系具有更高的抗氧化酶活性[超氧化物歧化酶(SOD);过氧化物酶(POD);过氧化氢酶(CAT)],积累了更多的还原性谷胱甘肽(GSH)和脯氨酸,同时其丙二醛(MDA)、过氧化氢(H₂O₂)含量显著低于WT;基因定量分析结果显示,OE株系拟南芥中抗逆相关基因AtCAT1、AtPOD1、AtRD29A及脯氨酸合成基因AtP5CS的表达量明显高于WT。以上结果说明,NtUFGT能有效提高转基因拟南芥中花青素和总黄酮含量,赋予植物更强的活性氧清除能力和渗透调节能力,从而增强了植物对干旱胁迫的耐受性。

关键词: 类黄酮-3-O-葡萄糖基转移酶, 唐古特白刺, 干旱胁迫, 花青素, 活性氧

Abstract: Anthocyanidins are important secondary metabolites of flavonoids. Their significant antioxidant effects can assist plants to alleviate oxidative damage caused by various abiotic stressors. Based on transcriptome data, a flavonoid 3-O-glucosyltransferase gene was cloned from the cDNA of Nitraria tangutorum, and was named NtUFGT. The open reading frame of NtUFGT has 1407 bp, and encodes a protein with 468 amino acids. The relative molecular mass of the protein encoded by this gene is predicted to be 51.37 kDa. Multiple sequence alignment analysis showed that the NtUFGT protein belongs to the UDP-glycosyltransferase protein family. Quantitative real-time PCR (qRT-PCR) was carried out to analyze gene expression patterns. NtUFGT had tissue-specific expression patterns. From high to low expression: flower>fruit>stem>leaf>root. Also, it was rapidly up-regulated by polyethylene glycol and abscisic acid treatments. The plasmid of pPZP221:35S:NtUFGT was constructed and introduced into a wild-type (WT) Arabidopsis thaliana by a floral-dip method. qRT-PCR was carried out to confirm that the NtUFGT gene was expressed in three T₃ generations. The growth status and stress-resistant physiological and biochemical indexes of wild-type and transgenic A. thaliana (OE lines) under drought stress were measured. The OE lines showed a stronger phenotype with longer root length, heavier fresh weight and higher chlorophyll content than the WT. OE lines also accumulated more flavonoids and anthocyanin. Consistent with phenotype, OE lines had increased levels of antioxidant enzyme (superoxide dismutase, peroxidase, and catalase) activity, and accumulated more glutathione, proline and less malondialdehyde and H₂O₂. The results of qRT-PCR analysis showed that the expression levels of the stress-related genes (AtCAT1, AtPOD1, AtRD29A) and the proline synthesis gene AtP5CS in OE lines were significantly higher than those in the WT. Our observations indicate that the NtUFGT can facilitate stronger active oxygen scavenging capacity and osmotic adjustment ability by effective promotion of the synthesis of anthocyanidins and flavonoids in transgenic Arabidopsis, thus enhancing the tolerance of plants to drought stress.

Key words: NtUFGT, Nitraria tangutorum, drought stress, anthocyanins, ROS

高子奇, 王佳, 汤宇晨, 王迎春. 唐古特白刺类黄酮-3-O-葡萄糖基转移酶基因(NtUFGT)的克隆与功能分析[J]. 草业学报, 2020, 29(5): 159-170.

GAO Zi-qi, WANG Jia, TANG Yu-chen, WANG Ying-chun. Cloning and functional analysis of the gene NtUFGT in Nitraria tangutorum[J]. Acta Prataculturae Sinica, 2020, 29(5): 159-170.

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