欢迎访问作物学报,今天是

作物学报 ›› 2015, Vol. 41 ›› Issue (03): 479-486.doi: 10.3724/SP.J.1006.2015.00479

• 研究简报 • 上一篇    下一篇

飞机草类黄酮3’-羟化酶基因(CoF3’H)的克隆及其在烟草中的表达

何海旺,潘华清,张铙丹,何龙飞*   

  1. 广西大学农学院,广西南宁 530004
  • 收稿日期:2014-08-12 修回日期:2014-12-19 出版日期:2015-03-12 网络出版日期:2014-12-29
  • 通讯作者: 何龙飞, E-mail: lfhe@gxu.edu.cn, Tel: 0771-3235212-801
  • 基金资助:

    本研究由广西自然科学基金项目(桂科自0832047)资助。

Chromolaena odorata Flavonoid 3’-hydroxylase Gene Cloning and Its Expression in Tobacco

HE Hai-Wang,PAN Hua-Qing,ZHANG Nao-Dan,HE Long-Fei*   

  1. Agricultural College of Guangxi University, Nanning 530004, China
  • Received:2014-08-12 Revised:2014-12-19 Published:2015-03-12 Published online:2014-12-29
  • Contact: 何龙飞, E-mail: lfhe@gxu.edu.cn, Tel: 0771-3235212-801

摘要:

利用RT-PCR和RACE技术从飞机草中克隆得到1628 bp的类黄酮3’-羟化酶cDNA的全长序列,命名为CoF3H,GenBank登录号为HQ268505.1。CoF3’H基因的编码区长度为1524 bp,编码507个氨基酸。氨基酸序列含P450蛋白结构域和半胱氨酸亚铁血红素结合域保守区(F××G×R×C×G),利用DNAMAN软件比对显示,CoF3’H与其他植物的F3’H蛋白的同源性很高。通过农杆菌介导成功地将CoF3H基因导入烟草,T2代的转CoF3H基因烟草植株的黄酮含量显著高于野生型。说明CoF3¢H在黄酮的生物合成中起重要作用,为后续研究飞机草的化感作用及综合利用提供基础。

关键词: 飞机草, 类黄酮3’-羟化酶, 克隆, 表达

Abstract:

A flavonoid 3’-hydroxylase gene cDNA sequence was cloned by RACE and RT-PCR techniques from Chromolaena odorata. The obtained full-length cDNA was named as CoF3H with GenBank accession number HQ268505.1. It is 1628 bp in length, containing a 1524 bp open reading frame, encoding 507 amino acid residues. The amino acid sequence of CoF3H contains cytochrome P450 domain and cysteineheme binding region (F××G×R×C×G). Homology analysis by DNAMAN software showed that the deduced CoF3H protein was highly homologous to F3’H proteins from different species. CoF3H gene was transferred into tobacco by agrobacterium-mediated genetic transformation. There was higher content of flavonvoids in T2 transgenic tobacco than in the wild type. This result shows that CoF3H plays an important role in flavonoid biosynthesis, and provides a basis for researches on allelopathy and comprehensive utilization of C. odorata in the future.

Key words: Chromolaena odorata, Flavonoid 3’-hydroxylase, Clone, Expression

[1]VonSengerI, Barker N, Zachariades C. Preliminary phylogeography of Chromolaena odorata: finding the origin of a South African weed. In: Proceedings 2002 Fifth International Workshop on Biological Control and Management of Chromolaena odorata, Durban, South Africa, 2002: 90–99



[2]Witkowski E T F, Wilson M. Changes in density, biomass, seed production and soil seed banks of the non-native invasive plant, Chromolaena odorata, along a 15 year chronosequence. Plant Ecol, 2001, 152: 13–27



[3]Ye W H, Mu H P, Cao H L, Ge X J. Genetic structure of the invasive Chromolaena odorata in China. Weed Res, 2004, 44: 129–135



[4]Biswas K. Some foreign weeds and their distribution in India and Burma. Indian forest, 1934, 60: 861–865



[5]Hall J B, Kumar R, Enti A A. The obnoxious weed Eupatorium odoratum (Compositae) in Ghana. Ghana J Agric Sci, 1972, 5(1): 75–78



[6]刘金海, 黄必志, 罗富成. 飞机草的危害及防治措施简介. 草业科学, 2006, 23(10): 73–77



Liu J H, Huang B Z, Luo F C. The damage and control measures of Eupatorium odoratum. Pratac Sci, 2006, 23(10): 73–77 (in Chinese with English abstract)



[7]吴邦兴. 滇南飞机草群落的初步研究. 云南植物研究, 1982, 4: 177–184



Wu B X. The primary studies on Eupatorium odoratum community in Southern Yunnan. Acta Bot Yunnanica, 1982, 4: l77–184 (in Chinese with English abstract)



[8]Patane K. A, Setter S, Graham M. Effect of foliar herbicides on the germination and viability of Siam weed (Chromolaena odorata) seeds located on plants at the time of application. Plant Prot Quart, 2009, 24(4): 138–140



[9]侯杰, 佟玲, 崔国新, 许志茹, 李玉花. 植物类黄酮3’-羟化酶(F3’H)基因的研究进展. 植物生理学报, 2011, 47: 641–647



Hou J, Tong L, Cui G X, Xu Z R, Li Y H. Research advances of plant flavonoid 3’-hydroxylase (F3’H) gene. Plant Physiol J, 2011, 47: 641–647 (in Chinese with English abstract)



[10]Brugliera F, Barri-Rewell G, Holton T A, Mason J G. Isolation and characterization of a flavonoid 3’-hydroxylase cDNA clone corresponding to the Ht1 locus of petunia hybrid. Plant J, 1999, 19: 441–451



[11]Schoenbohm C, Martens S, Eder C, Forkmann G, Weisshaar B. Identification of the Arabidopsis thaliana flavonoid 3’-hydroxylase gene and functional expression of the encoded p450 enzyme. Biol Chem, 2005, 381: 749–753



[12]Seitz C, Eder C, Deiml B, Kellner S, Martens S, Forkmann G. Cloning, functional identification and sequence analysis of flavonoid 3’-hydroxylase and flavonoid 3’,5’-hydroxylase cDNAs reveals independent evolution of flavonoid 3’,5’-hydroxylase in the Asteraceae family. Plant Mol Biol, 2006, 61: 365–381



[13]Xu B B, Li J N, Zhang X K, Wang R, Xie L L, Chai Y R. Clone and molecular characterization of a functional flavonoid 3’-hydroxylase gene from Brassica napus. J Plant Physiol, 2007, 164: 350–363



[14]许志茹, 崔国新, 李春雷, 孙燕, 李玉花. 芜菁的类黄酮3’-羟化酶基因克隆和UV-A诱导表达特性. 植物生理学通讯, 2008, 44: 931–935



Xu Z R, Cui G X, Li C L, Sun Y, Li Y H. Cloning and UV-Ainduced expression trait of flavonoid 3’-hydroxylase gene in turnip (Brassica rapa L.). Plant Physiol Commun, 2008, 44: 931–935 (in Chinese with English abstract)



[15]陶吉寒, 招雪晴, 苑兆和, 尹燕雷, 冯立娟. 石榴花类黄酮3’-羟化酶基因的克隆及序列分析. 河南农业科学, 2013, 42(4): 121–124



Tao J H, Zhao X Q, Yuan Z H, Yin Y L, Feng L J. Cloning and sequence analysis of flavonoid 3’-hydroxylase gene in pomegranate flower. J Henan Agric Sci, 2013, 42(4): 121–124 (in Chinese with English abstract)



[16]Castellarin S D, Gaspero G D, Marconi R, Nonis A, Peterlunger E, Paillard S, Adam-Blondon A F, Testolin R. Colour variation in red grapevines (Vitis vinifera L.): genomic organization, expression of flavonoid 3’-hydroxylase, flavonoid 3’,5’-hydroxylase genes and related metabolite profiling of red cyaniding-/blue delphinidin-based anthocyanins in berry skin. BMC Genomic, 2006, 7: 12



[17]Toda K, Yand D, Yamanaka N, Watanabe S, Harada K, Takahashi R. A single-base deletion in soybean flavonoid 3’-hydroxylase gene if associated with gray pubescence color. Plant Mol Biol, 2002, 50: 187–196



[18]Jeong S T, Yamamoto N G, Hashizume K, Esaka M. Expression of the flavonoid 3’-hydroxylase and flavonoid 3’,5’-hydorxylase genes and flavonoid composition in grape (Vitis vinifera). Plant Sci, 2006, 170: 61–69



[19]Holton T A, Brugliera F, Lester D R, Tanaka Y, Hyland C D, Menting J G T, Lu C Y, Farcy E, Stevenson T W, Cornish E C. Cloning and expression of cytochrome P450 genes controlling flower colour. Nature, 1993, 366: 276–279



[20]张松焕, 郭惠明, 裴熙祥, 李春奇, 程红梅. 紫茎泽兰类黄酮3’-羟化酶在烟草中的表达. 中国农业科学, 2009, 44: 4182–4186



Zhang S H, Guo H M, Pei X X, Li C Q, Cheng H M. Expression of flavonoid 3’-hydrolyase from Eupatorium adenophorum in tobacco. Sci Agric Sin, 2009, 44: 4182–4186 (in Chinese with English abstract)



[21]何海旺, 许春燕, 李文, 何龙飞, 李创珍, 韦善清. 玻璃粉吸附核酸及其在植物核酸提取中应用的研究. 生物技术进展, 2012, 2: 359–365



He H W, Xu C Y, Li W, He L F, Li C Z, Wei S Q. The research of glass powder absorbing nucleic acid and its application of nucleic acid extraction from plant tissue. Curr Biotechnol, 2012, 2: 359–365 (in Chinese with English abstract)



[22]Holsters M, de Waele D, DepickerA, Messens E, van Montagu M, Schell J. Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet, 1978, 163: 181–187



[23]Horsch R B, Fry J E, Hoffmann N L, Eicholtz D, Rogers S G, Fraley R T. A simple and general method for transferring genes into plants. Science, 1985, 227: 1229–1231



[24]李增富, 吴荣锋. 葛根总黄酮的提取研究. 应用化工, 2008, 38: 1353–1358



Li Z F, Wu R F. Study on extracting total puerar in flavonoids. Appl Chem Ind, 2008, 38: 1353–1358 (in Chinese with English abstract)



[25]梁国兴, 赵缓, 王彩芳. 入侵物种飞机草(Eupatorium odoratum)化学成份及生物活性研究进展. 北京师范大学学报(自然科学版), 2011, 47: 611–617



Liang G X, Zhao Y, Wang C F. Chemical constituents and their bioactivity of the invasive species Eupatorium odoratum L. J Beijing Norm Univ (Nat Sci), 2011, 47: 611–617 (in Chinese with English abstract)



[26]陈向明, 马云飞. 山核桃外果皮黄酮提取液对小麦和绿豆幼苗的化感效应. 西北植物学报, 2010, 30 : 645–651



Chen X M, Ma Y F. Allelopathy of flavonoid extract from cary a cathayensisexocarp on wheat and mung bean seedlings. Acta Bot Boreal-occident Sin, 2010, 30: 645–651 (in Chinese with English abstract)



[27]郭亚姣, 郑书馨,李洁, 江莎. 黄顶菊叶中类黄酮对白菜种子萌发的影响. 内蒙古农业大学学报, 2011, 32(4): 105–109



Guo Y J, Zheng S X, Li J, Jing S. The effects of flavonoids extracted from flaveriabidentis leaves on seed germination of brassica pekinensi. J Inner Mongolia Agric Univ, 2011, 32(4): 105–109 (in Chinese with English abstract)



[28]雷磊. 飞机草中化感物质的提取分离与鉴定. 吉林农业大学硕士学位论文, 吉林长春, 2011. p 31



Lei L. Extraction separation and identification of allelochemicals in Chromolaena odoratum. MS Thesis of Jilin Agricultural University, Changchun, China, 2011. p 31 (in Chinese with English abstract)



[29]Phan T T, Wang L, See P, Grayer R J, Chan S Y, Lee S T. Phenolic compounds of Chromolaena odorata protect cultured skin cells from oxidative damage: implication for cutaneous wound healing. Biol Pharm Bull, 2001, 21: 1373–1379



[30]Suksamram A, Chotipong A, Suavansri T, Boongird S, Timsuksai P, Vimuttipong S, Chuaynugul A. Antimycobacterial activity and cytotoxicity of flavonoids from the flowers of Chromolaena odorata. Arch Pharm Res, 2004, 27: 507–511



[31]Owoyele B V, Oguntoye S O, Dare K, Ogunbiyi B A, Aruboula E A, Soladoye A O. Analegsic anti-inflammatory and antipyretic activities from flavonoid fractions of Chromolaena odorata. J Med Plants Res, 2008, 2: 219–225



[32]张松焕, 郭惠明, 裴熙祥, 李春奇, 程红梅. 紫茎泽兰类黄酮3’-羟化酶在烟草中的表达. 中国农业科学, 2009, 42: 4182–4186



Zhang S H, Guo H M, Pei X X, Li C Q, Cheng H M. Expression of Flavonoid 3’-hydrolyase from Eupatorium adenophorum in tobacco. Sci Agric Sin, 2009, 42: 4182–4186 (in Chinese with English abstract)



[33] Nakatsula T, Nishihara M, Mishiba K, Yamamura S. Heterologous expression of two gentian cytochrome P450 genes can modulate the intensity of ?ower pigmentation in transgenic tobacco plants. Mol Breed, 2006, 17: 91–99



[34]乔小燕, 马春雷, 陈亮. 植物类黄酮生物合成途径及重要基因的调控. 天然产物研究与开发, 2009, 21: 354–360



Qiao X Y, Ma C L, Chen L. Plant flavonoid biosynthesis pathway and regulation of its important genes. Nat Prod Res Dev, 2009, 21: 354–360 (in Chinese with English abstract)

[1] 陈雪燕, 何华川, 李政嘉, 董新盼, 李藕琪, 刘小云, 李丹萍, 陈志伟, 刘国霞, 吕胜源, 吴印莹, 赵振东, 曹新有, 万何平. 水培盐碱复合胁迫下‘济麦60’苗期根系有机酸分泌动态变化及其转录调控机制[J]. 作物学报, 2026, 52(6): 1859-1875.
[2] 左同鸿, 张贺翠, 曾静, 朱利泉. 甘蓝自交不亲和相关基因BoPUB3L的克隆与表达分析[J]. 作物学报, 2026, 52(6): 1698-1710.
[3] 王文辕, 燕雪嘉, 刘玉霖, 孙晓彤, 李亚楠, 唐鑫华, 石瑛. 耐弱光马铃薯品种筛选及转录因子编码基因StPIF3的克隆与功能分析[J]. 作物学报, 2026, 52(6): 1631-1645.
[4] 胡赵, 钱润, 谢丰璞, 应素平. 水稻SPX基因家族鉴定及响应磷处理的表达分析[J]. 作物学报, 2026, 52(6): 1902-1912.
[5] 孙淑凤, 许振南, 黄嘉鑫, 翁建峰, 李新海. 玉米MAPK家族全基因组鉴定及其对拟轮枝镰孢菌感染的响应[J]. 作物学报, 2026, 52(5): 1291-1308.
[6] 张曦, 王广恩, 李邵琦, 刘祎, 李俊兰, 钱玉源. 基于转录组测序解析陆海杂交姊妹系马克隆值差异的形成机制[J]. 作物学报, 2026, 52(5): 1442-1458.
[7] 韩亚鑫, 何冠华, 张小琼, 张登峰, 李永祥, 刘旭洋, 王天宇, 黎裕, 邹华文, 李春辉. 基于RNA-Seq和BSA-Seq联合分析挖掘玉米侧根密度基因资源[J]. 作物学报, 2026, 52(5): 1341-1352.
[8] 田立涛, 丁宁, 王树林, 齐恩芳, 张荣, 王蕊蕊, 马利雯, 李建武, 杨江伟. 马铃薯Argonaute基因家族鉴定及晚疫病诱导表达分析[J]. 作物学报, 2026, 52(4): 1116-1126.
[9] 汪玲, 胡好, 宋家凤, 程洁蓝, 陈颖, 郑婷婷, 吕钊彦, 朱晓彪, 侯华兰. 马铃薯UDP-糖基转移酶基因StUGT52的克隆及功能验证[J]. 作物学报, 2026, 52(3): 665-676.
[10] 杨宗桃, 杨婷, 王禹童, 艾静, 李燕烨, 刘家勇, 邓军, 赵勇, 张跃彬. 甘蔗CLC基因家族鉴定与表达分析[J]. 作物学报, 2026, 52(3): 722-734.
[11] 孟成, 王哲. 玉米ZmPFK基因家族全基因组鉴定及响应胁迫表达分析[J]. 作物学报, 2026, 52(3): 764-779.
[12] 杨飚, 杜帅康, 张继旺, 石瑛, 张丽莉. 马铃薯III类POD基因家族的全基因组鉴定及其表达谱分析[J]. 作物学报, 2026, 52(2): 405-420.
[13] 余开航, 周洪斌, 罗亮扎, 王玫郦, 姜瑞梅, 董陈文华, 李仕金, 毛孝强, 陈升位. 大麦亮氨酸富集重复型类受体激酶基因HvLRR-RLK-510的克隆和表达分析[J]. 作物学报, 2026, 52(2): 421-432.
[14] 詹戈锐, 余文, 李锋, 武明珠, 徐馨, 罗朝鹏, 巫升鑫, 杨军, 张智强, 王中. 烟草NtWRKY6基因响应ABA表达及其调控多酚合成的功能研究[J]. 作物学报, 2026, 52(2): 446-458.
[15] 张力岚, 杨军, 王让剑. 基于WGCNA发掘茶树糖苷类香气前体含量性状相关的候选基因[J]. 作物学报, 2026, 52(2): 494-513.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!