Cloning and Bioinformatical Analysis of Anthocyanin Synthase Gene and Its Promoter in Camellia sinensis

JIN Qifang; CHEN Zhidan; SUN Weijiang; LIN Fuming; XUE Zhihui; HUANG Yan; TANG Xiuhua

doi:10.13305/j.cnki.jts.2016.02.014

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Journal of Tea Science ›› 2016, Vol. 36 ›› Issue (2) : 219-228. DOI: 10.13305/j.cnki.jts.2016.02.014

Cloning and Bioinformatical Analysis of Anthocyanin Synthase Gene and Its Promoter in Camellia sinensis

JIN Qifang^1,3, CHEN Zhidan^2,3, SUN Weijiang^1,2,3,*, LIN Fuming^2,3, XUE Zhihui^2,3, HUANG Yan^2,3, TANG Xiuhua^1,3

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Abstract

The full length cDNA sequence of anthocyanin synthase gene（CsANS）was cloned from ‘Wuyi qizhong C18’ (Camellia sinensis) using rapid amplification of cDNA ends（RACE）technology. The promoter of CsANS was isolated using Genome Walking technology. The gene expression of ANS under different shading treatments was analyzed by the real-time PCR. The results showed that the full length cDNA of CsANS was 1 000 bp, and it contained a 957 bp open reading frame (ORF), which encoding a protein of 320 amino acid, including two conservative functional domains, DIOX-N and 20G-Fell-Oxy. A sequence containing 1 010 bp was isolated and found to be the promoter of CsANS, which contained many cis-acting elements related to anthocyanin biosynthetic pathway, such as the core promoter element TATA-box and light responsive element (including ACE, GT1-motif and Sp1), circadian (cis-acting regulatory element involved in circadian control). The real-time PCR analysis revealed that the gene was highly expressed under the sunshine (CK), while lowly expressed under the 75% shading. This study reveals that the expression of CsANS was regulated by light intensity.

Key words

Camellia sinensis / bioinformatics analysis / CsANS gene / promoter

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JIN Qifang, CHEN Zhidan, SUN Weijiang, LIN Fuming, XUE Zhihui, HUANG Yan, TANG Xiuhua. Cloning and Bioinformatical Analysis of Anthocyanin Synthase Gene and Its Promoter in Camellia sinensis[J]. Journal of Tea Science. 2016, 36(2): 219-228 https://doi.org/10.13305/j.cnki.jts.2016.02.014

References

[1] 金琦芳, 孙威江, 陈志丹. 光照对紫色芽叶茶花青素合成的调控机理[J]. 生物技术通报, 2015, 31(6): 20-27.
[2] 刘富知, 黄建安, 付冬和. 茶树上红紫色芽叶部分生化特性研究[J]. 湖南农业学报, 2000, 26(1): 55-57.
[3] 萧力争, 苏晓倩, 李勤. 紫芽品种茶树芽叶多酚类物质组成特征[J]. 湖南农业大学学报: 自然科学版, 2008, 34(1) : 77-79.
[4] Sheehan MJ, Farmer PR, Brutnell TP.Structure and expression of maize phytochrome family homeologs[J]. Genetics, 2004, 167(88): 1395-1405.
[5] 胡可, 韩科厅, 戴思兰. 环境因子调控植物花青素苷合成及呈色的机理[J]. 植物学报, 2010, 45(3): 307-317.
[6] 林玉玲. 龙眼体胚发生过程中SOD基因家族的克隆及表达调控研究[D]. 福州: 福建农林大学, 2011: 31-37.
[7] Lin Y L, Lai Z X.Superoxide dismutase multigene family in longan somatic embryos: a comparison of CuZn-SOD, Fe-SOD, and Mn-SOD gene structure, splicing, phylogeny, and expression[J]. Molecular Breeding, 2013, 32(3): 595-615.
[8] 孙君, 陈桂信, 叶乃兴, 等. 茉莉花香气相关基因JsDXS及其启动子的克隆与表达分析[J]. 园艺学报, 2014, 41(6): 1236-1244.
[9] Lescot M, Dehais P, Moreau Y, et al.PlantCARE: A database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J]. Nucleic Acids Res, 2002, 30(1): 325-327.
[10] Higo K, Ugawa Y, Iwamoto M, et al.Plant cis-acting regulatory DNA elements (PLACE) atabase[J]. Nucleic Acids Research, 1999, 27(1): 297-300.
[11] 刘志钦, 马小玲, 严雁, 等. 拟南芥AtLURP1基因启动子在转基因烟草和水稻中的功能分析[J]. 热带作物学报, 2013, 34(2): 308-313.
[12] Shahmuradov I A, Solovyev V, Gammerman A J.Plant promoter prediction with confidence estimation[J]. Nucleic Acids Res, 2005, 33(3): 1069-1076.
[13] Nakajima J, Tanaka Y, Yamazaki M, et al.Reaction mechanism from leucoanthocyanidin to anthocyanidin 3-glucoside, a key reaction for coloring in anthocyanin biosynthesis[J]. Journal Biology Chemistry, 2001, 276(1): 25797-25803.
[14] 田佶, 沈红香, 张杰,等. 苹果属观赏海棠McANS基因克隆与不同叶色品种间表达差异分析[J]. 园艺学报, 2010, 37(6): 939-948.
[15] Menssen A, Hohmann S, MartinW, et al. The En/Spm transposable element of zea mays contains splice sites at the temini generating a novel intron frodSpm element in the A2 gene[J]. EMBO Journal, 1990, 9(2): 3051-3057.
[16] Tanaka Y, Fukui Y, Mizutani M F, et al.Molecular cloning and characterization of rosa hybrid dihydroflavonol 4-reductase gene[J]. Plant Cell Physiology, 1995, 36(6): 1023-1031.
[17] Rosati C, Cadic A, Duron M, et al.Molecular characterization of the anthocyanidin synthase gene in forsythiax intermedia reveals organ-specific expression during flower development[J]. Plant Science, 1999, 149(3): 73-79.
[18] David W.Regulation of flower pigmentation and growth: multiple signaling pathways control anthocyanin synthesis in expanding petals[J]. Physiol Plant, 2000, 110(28): 152-157.
[19] Mori K, Sugaya S, Gemma H.Decreased anthocyanin biosynthesis in grape berries grown under elevated night temperature condition[J]. Sci Hortic, 2005, 105(11): 319-330.
[20] Mori K, Goto-Yamamoto N, Kitayama M, et al.Loss of anthocyanins in red-winegrape under high temperature[J]. J Exp Bot, 2007, 58(68): 1935-1945.
[21] Hong G, Wang J, Zhang Y, et al.Biosynthesis of catechin components is differentially regulated in dark-treated tea (Camellia sinensis L.)[J]. Plant Physiol Biochem, 2014, 78(4): 49-52.
[22] 刘畅, 缪军, 霍雨猛, 等. 洋葱ANS基因启动子的克隆与瞬时表达分析[J]. 山东农业科学, 2013, 5(1): 13-17.
[23] 尚啸. 三色堇F3'H的克隆分析及ANS、DFR启动子的克隆[D]. 海口: 海南大学, 2014: 13-16.
[24] 董薇, 高峰. 紫心甘薯花青素合成酶基因ANS启动子的克隆及其与转录因子的相互作用研究[R]. 广东省植物学会, 2012: 8-10.
[25] Papi M, Sabatini S, Altamura MM.Inactivation of the phloem-specific dof zinc finger gene DAG1 affects response to light and integrity of the testa of arabidopsis seeds[J]. Plant Physiol, 2002, 128(2): 411-417.
[26] Ward JM, Cufr CA, Denzel MA.The dof transcription factor OBP3 modulates phytochrome and cryptochrome signaling in Arabidopsis[J]. Plant Cell, 2005, 17(2): 475-485.