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七彩红竹中类黄酮-3-O-葡萄糖基转移酶基因的克隆及功能分析(PDF)

《广西植物》[ISSN:1000-3142/CN:45-1134/Q]

期数:
2015年02期
页码:
244-249
栏目:
遗传与育种
出版日期:
2015-04-20

文章信息/Info

Title:
Cloning and functional analysis of a flavonoid-3-O- glucosyltransferas gene from Indosasa hispida
文章编号:
1000-3142(2015)02-0244-06
作者:
王 毅12 王晨晨13 周 旭13 毕 玮12 杨宇明12 王 娟12*
1. 云南省林业学科院 国家林业局云南珍稀濒特森林植物繁育和保护重点实验室, 昆明 650201; 2. 云南林业学科院, 云南省森林植物培育与开发利用重点实验室, 昆明 650201; 3. 西南林业大学, 昆明650224
Author(s):
WANG Yi12 WANG Chen-Chen13 ZHOU Xu13 BI Wei12 YANG Yu-Ming12 WANG Juan12*
1. The Key Laboratory of Rare and Endangered Forest Plan ts of State Forestry Administration, Yunnan Academy of Forestry, Kunming 650204, China; 2. The Key Laboratory of Foresty Plant Cultivation and Utilization, Yunnan Academy of Forestry, Kunming 650204, China; 3. Southwest Forestry University, Kunming 650204, China
关键词:
七彩红竹 3GT cDNA 克隆 基因表达分析
Keywords:
Indosasa hispida Mc Clure cv. Rainbow flavonoid-3-O-glucosyltransferas gene cDNA clone gene expression analysis
分类号:
Q945
DOI:
10.11931/guihaia.gxzw201401011
文献标识码:
A
摘要:
类黄酮-3-O-葡萄糖基转移酶(Flavonoid-3-O-glucosyltransferase,3GT)是花青苷(Anthocyanins)生物合成途径中的关键酶,它主要负责将不稳定的花色素转变为稳定的花色素苷,虽然目前已经从其他植物中克隆获得类黄酮-3-O-葡萄糖基转移酶,但是对竹子中的类黄酮-3-O-葡萄糖基转移酶并不清楚。该文以产生花青素的七彩红竹(Indosasa hispida McClure cv. Rainbow)为材料,首先通过3GT的同源比对后设计3GT基因特异引物,获得3GT基因片段; 然后运用RT-PCR及RACE技术从七彩红竹茎中克隆得到完整的3GT基因(Ih3GT)。结果表明:Ih3GT基因的cDNA全长序列为1 730 bp,含有1个1 425 bp的开放阅读框(ORF),编码474个氨基酸; 系统进化分析显示,七彩红竹3GT与其他禾本科植物的3GT聚类到同一个分支; 该基因推断的蛋白与水稻(Oryza sativa)3GT蛋白的相似性为69%,与二穗短柄草(Brachypodium distachyon)3GT的相似性为67%; 经氨基酸序列比对,推断七彩红竹3GT含有糖基转移酶基因家族特有的结构域PSPG-box; 半定量PCR的结果显示,七彩红竹3GT基因在微红的幼茎中大量表达,而在其他组织中并不表达,说明Ih3GT具有组织表达特异性。该结果为今后深入研究七彩红竹花色苷的形成机理、鉴定Ih3GT酶活性以及利用Ih3GT基因培育竹子新品种奠定了基础。
Abstract:
Flavonoid -3-O- glucosyltransferase(3GT)is the key enzyme in anthocyanins biosynthesis,which transforms unstable anthocyanidins into stable anthocyanins. The function of Flavonoid -3-O- glucosyltransferase in bamboo is unclear,although many flavonoid -3-O- glucosyltransferases from other plant had been reported,so far. Indosasa hispida McClure cv. Rain bow is of few bamboo species which can produce anthocyanins in clum. Therefore,I. hispida McClure cv. Rain bow is very important material to reveal the function of Flavonoid -3-O- glucosyltransferase in bamboo and the mechanism of anthocyanins biosynthesis in bamboo. Therefore,it is first step to clone 3GT gene from I. hispida McClure cv. Rain bow. First,gene special primers of 3GT were obtained based on the homology analysis of reported flavonoid -3-O- glucosyltransferase,and the RNA was extracted from yound red clum which produce anthocyanins part in I. hispida McClure cv. Rain bow by Trizol method. And then gene fragment of I. hispida McClure cv. Rainbow was cloned with special primers of 3GT. According to the obtained fragment sequence,the primers were designed,which were used in rapid amplification of cDNA ends(RACE). Next,the 3’ end and 5’ end sequence of 3GT were obtained by RACE,the full length gene of Ih3GT was assembled by ATG software. Finally,the full length gene of Ih3GT was cloned from I. hispida McClure cv. Rainbow by reverse transcription-polymerase chain reaction(RT-PCR). ORF analysis program was used to confirm open reading frame,MEGA software was used to construct phylogenetic tree. the DNAman software was used in Homology analysis,and semi RT-PCR was applied in gene expression profile. The results showed that the cDNA sequence of Ih3GT consisted of 1 425 bp open reading frame(ORF)which encodes 474 amino acid, Ih3GT and 3GT from Poaceae were grouped in same clade,the deduced protein of 3GT from I. hispida McClure cv. Rain bow shared 69% identities with 3GT of Oryza sativa and shared 67% identities with 3GT of Brachypodium distachyon. Homology analysis showed that deduced Ih3GT protein had a glycosyltransferase signature domain PSPG-box. Expression profiling with semi RT-PCR analysis revealed that Ih3GT was expressed in young red culm and was not expressed in old culm,old leaf,young leaf and shoot. This implied that the expression of Ih3GT from I. hispida McClure cv. Rain bow showed obvious tissue specificity. This study will provide useful information to reveal mechanism of anthocyanins biosynthesis in I. hispida McClure cv. Rainbow in future. Ih3GT gene can be transformed into Eschera coli,and heterologous expression obtain Ih3GT protein and detect the enzyme activity of Ih3GT,and it can also be transformed into Arabidopsis thaliana or Oryza sativa,the function of Ih3GT will be confirmed by heterologous expression. The obtained Ih3GT also can been implied in bamboo breeding or other horticultural plants by genetic engineering.

参考文献/References

Aizza LCB,Dornelas MC. 2011. A genomic approach to study anthocyanin synthesis and flower pigmentation in passionflowers[J]. J Nucleic Acids,DOI:10.4061/2011/371517
Katsumoto Y,Fukuchi-Mizutani M,Fukui Y,et al. 2007. Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin[J]. Plant Cell Physiol,48(11):1 589-1 600
Nishihara M,Nakatsuka T. 2011. Genetic engineering of flavonoid pigments to modify flower color in floricultural plants[J]. Biotechnol Lett,33(3):433-441
Singh SR,Singh R,Kalia S,et al. 2013. Limitations,progress and prospects of application of biotechnological tools in improvement of bamboo—— a plant with extraordinary qualities[J]. Physiol Mol Biol Plants,19(1):21-41
Springob K,Nakajima J,Yamazaki M,et al. 2003. Recent advances in the biosynthesis and accumulation of anthocyanins[J]. Nat Prod Rep,20(3):288-303
Sui X,Gao X,Ao M,et al. 2011. cDNA cloning and characterization of UDP-glucose:anthocyanidin 3-O-glucosyltransferase in Freesia hybrida[J]. Plant Cell Rep,30(7):1 209-1 218
Tang YP(唐亚萍),Yuan H(原慧),Qin JB(覃建兵). 2012. Identification and characterization of a flavonoid-3-O-glucosyltransferas gene from Saussurea involucrata(天山雪莲 UDP 葡萄糖-类黄酮-3-O-葡萄糖基转移酶基因的克隆及功能分析)[J]. Chin J Biotechnol(生物工程学报),28(6):705-714
Vogt T,Jones P. 2000. Glycosyltransferases in plant natural product synthesis:characterization of a supergene family[J]. Trends Plant Sci,5(9):380-386
Wang J(王娟),Sun H(孙浩),Peng GS(彭桂莎),et al. 2012. Phylogenetic study on anthocyanin produced mutant of Indosasa hispida based on rDNA ITS sequences(浦竹仔红色秆变异个体的 rDNA ITS 序列及系统发育研究)[J]. J West Chin For Sci(西部林业科学),41(1):1-6
Yoshida K,Toyama Y,Kameda K,et al. 2000,Contribution of each caffeoyl residue of the pigment molecule of gentiodelphin to blue color development[J]. Phytochemistry,54(1):85-92
Zhang XM(张新明). 1999. Function and development tendency of ornimental bamboo in gardening(观赏竹在园林绿化中的功用及其发展方向)[J]. J Bamboo Res(竹子研究汇刊),18(4): 24-26
Zhao ZC,Hu GB,Hu FC,et al. 2012 The UDP glucose:flavonoid-3-O-glucosyltransferase(UFGT)gene regulates anthocyanin biosynthesis in litchi(Litchi chinesis Sonn. )during fruit coloration[J]. Mol Biol Rep,39(6):6 409-6 415
Zhou QM(赵启明),Li F(李范),Li P(李萍). 2012. Research advances on core enzymes of anthocyanidin biosynthesis(花青素生物合成关键酶的研究进展)[J]. Biotechnol Bull(生物技术通报),12:25-32

备注/Memo

备注/Memo:
收稿日期: 2014-06-23修回日期: 2014-08-21
基金项目: 云南省应用基础研究重点项目(2013FA054); 云南省中青年学术技术带头人后备人才培养项目(2010CI016); 云南省科技计划项目(20141A013)。
作者简介: 王毅(1981- ),男,四川广安人,博士,助理研究员,主要从事植物学和分子生物学研究,(E-mail)22825818@qq.com。 *通讯作者: 王娟,博士,教授,硕士生导师,主要从事生物多样性保护生态学和竹类植物的微观研究,(E-mail)schima@163.com。
更新日期/Last Update: 2015-04-20