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2025年8月14日 星期四
农业生物技术学报  2019, Vol. 27 Issue (4): 636-644    DOI: 10.3969/j.issn.1674-7968.2019.04.007
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
茶树CsANS基因的克隆及在转基因烟草中的功能分析
齐勇1, 2, 赵德刚1, 2, 吕立堂1, 3, *
1 贵州大学 生命科学学院/农业生物工程研究院,贵州省农业生物工程重点实验室/山地植物资源保护与种质创新省部共建教育部重点实验室,贵阳 550025;
2 贵州大学 贵州省山地生态与农业生物工程2011协同创新中心,贵阳 550025;
3 贵州大学 茶学院,贵阳 550025
Cloning of CsANS Gene from Tea Plant (Camellia sinensis) and Its Functional Analysis in Transgenic Tobacco (Nicotiana tabacum)
QI Yong1, 2, ZHAO De-Gang1, 2, LV Li-Tang1, 3, *
1 The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education)/Guizhou Key Lab of Agro-Bioengineering, Institute of Agro-Bioengineering and College of Life Sciences, Guizhou University, Guiyang 550025, China;
2 The 2011 Collaboration Innovation Center for Mountain Ecology and Agro-Bioengineering in Guizhou Province, Guizhou University, Guiyang 550025, China;
3 Tea Academy, Guizhou University, Guiyang 550025, China
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摘要 花青素合成酶(anthocyanidin synthase, ANS)是合成植物花青素末端的重要催化酶,能够使无色花青素催化为有色花青素。本研究以茶树全长转录组测序数据为依据,利用反转录PCR(reverse transcription-polymerase chain reaction, RT-PCR)克隆了编码茶树(Camellia sinensis)ANS基因的cDNA序列(GenBank No.AY830416),编码区全长1 068 bp,编码355个氨基酸。构建载体pSH-CsANS,利用农杆菌(Agrobacterium tumefaciens)侵染烟草(Nicotiana tabacun)进行遗传转化,组织培养获得35株转基因植株。选取3个经PCR验证的阳性烟草株系TP-1、TP-2和TP-4进行基因表达以及花青素和原花青素含量分析。qRT-PCR分析显示,转基因烟草植株中原花青素生物合成途径基因查儿酮异构酶(chalcone isomerase, CHI)、黄烷酮-3-羟化酶((2S)-flavanone 3-hydroaylase, F3H)和二羟黄酮醇还原酶(dihydroflavonol4-reductase, DFR)表达上调,黄酮醇合成酶(flavonol synthase, FLS)基因表达下调。结果表明,超量表达茶树CsANS基因能够促进烟草花青素的合成,花青素含量比野生型提高45%左右。而且,超量表达CsANS基因能增加原花青素合成前提物质黄烷-3-醇含量,使转基因植株中原花青素含量比野生型升高34%左右。本研究结果为进一步对该基因的表达规律和功能分析提供理论基础。
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齐勇
赵德刚
吕立堂
关键词 茶树花青素合成酶(CsANS)烟草原花青素黄烷-3-醇    
Abstract:Anthocyanin synthase (ANS) is a key enzyme at the end of plant anthocyanin biosynthetic pathway,which catalyzes leucoanthocyanins into anthocyanins.In this study, the ANS gene of Camellia sinensis was cloned by reverse transcription-polymerase chain reaction (RT-PCR) based on tea full-length transcriptome sequencing data (GenBank No. AY830416).The full-length cDNA coding region was 1 068 bp and encoded 355 amino acids. The plant expression vector pSH-CsANS was constructed, and Agrobacterium tumefaciens was used to infect tobacco (Nicotiana tabacun) leaves for genetic transformation. Thirty-five transgenic plants were obtained by tissue culture. And after resistance selection and PCR identification, three PCR-positive tobacco lines TP-1, TP-2 and TP-4 were selected for gene expression and anthocyanin and proanthocyanidin content analysis. Quantitative real-time PCR (qRT-PCR) analysis showed that over-expression CsANS gene resulted in up-regulation of endogenous flavonoid biosynthesis pathway genes chalcone isomerase(CHI), (2S)-flavanone 3-hydroaylase (F3H) and dihydroflavonol4-reductase (DFR) and down-regulation of flavonol synthase (FLS) gene in tobacco. The results showed that over-expression of CsANS gene promoted the synthesis of tobacco anthocyanins, and the anthocyanin content was increased by about 45% compared with wild type. Moreover, over-expression of CsANS gene could increase the content of flavan-3-ol, a prerequisite material for the synthesis of proanthocyanidins, and increase the content of proanthocyanidins in transgenic plants by about 34% compared with wild type. This research has provided a foundation for future study on gene expression and functional analysis.
Key wordsTea anthocyanin synthase (CsANS)    Tobacco    Procyanidins    Flavan-3-ol
收稿日期: 2018-11-04     
ZTFLH:  S-3  
  Q812  
基金资助:国家自然科学基金(No. 31160149)、国家转基因生物新品种培育重大专项(No. 2014ZX 08010-003-2016ZX08010-003)和利用转基因油菜生产超长链多不饱和脂肪酸(黔科合LH字[2014]7680)
通讯作者: ltlv@gzu.edu.cn   
引用本文:   
齐勇, 赵德刚, 吕立堂. 茶树CsANS基因的克隆及在转基因烟草中的功能分析[J]. 农业生物技术学报, 2019, 27(4): 636-644.
QI Yong, ZHAO De-Gang, LV Li-Tang. Cloning of CsANS Gene from Tea Plant (Camellia sinensis) and Its Functional Analysis in Transgenic Tobacco (Nicotiana tabacum). 农业生物技术学报, 2019, 27(4): 636-644.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.04.007     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I4/636
 
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