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作物学报 ›› 2016, Vol. 42 ›› Issue (06): 850-859.doi: 10.3724/SP.J.1006.2016.00850

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

紫色不结球白菜花色苷合酶基因BrcANS的克隆与表达分析

许玉超1,侯喜林1,徐玮玮1,沈露露2,张仕林1,刘世拓1,胡春梅1,*   

  1. 1 南京农业大学作物遗传与种质创新国家重点实验室，江苏南京 210095；2 安徽省合肥市肥西县农业委员会，安徽合肥230001
  • 收稿日期:2015-11-11 修回日期:2016-03-14 出版日期:2016-06-12 网络出版日期:2016-03-21
  • 通讯作者: 胡春梅, E-mail: jjjhcm@njau.edu.cn
  • 基金资助:

    本研究由江苏省农业科技自主创新项目[CX(15)1015]和江苏省科技支撑计划项目(BE2013429)资助。

Cloning and Expression Analysis of Anthocyanidin Synthase Gene BrcANS from Purple Non-heading Chinese Cabbage

XU Yu-Chao1,HOU Xi-Lin1,XU Wei-Wei1,SHEN Lu-Lu2,ZHANG Shi-Lin1,LIU Shi-Tuo1,HU Chun-Mei1,*   

  1. 1 State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China; 2 Agriculture Committee of Feixi County, Hefei 230001, China
  • Received:2015-11-11 Revised:2016-03-14 Published:2016-06-12 Published online:2016-03-21
  • Contact: 胡春梅, E-mail: jjjhcm@njau.edu.cn
  • Supported by:

    The work was supported by the Independent Innovation Fund for Agricultural Science and Technology of Jiangsu Province [CX (15)1015], the Science-technology Support Program of Jiangsu Province (BE2013429).

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摘要：

以不结球白菜紫色品系NJZX1-3和其绿色突变体NJZX1-0及其后代F2的2个株系NJZX2-1和NJZX2-2为材料，研究花色苷合酶基因在紫色不结球白菜叶片花色苷合成途径中的作用。利用同源克隆的方法，分别在NJZX1-3及NJZX1-0中克隆到花色苷合酶基因；经序列比对发现，花色苷合酶基因的核苷酸和氨基酸序列在2种材料和大白菜中完全一致，长度为1077 bp，编码358个残基，第211~307肽段具有2OG-Fe (II)双加氧酶家族基因的结构域，被命名为BrcANS。BrcANS蛋白与同科芥菜的同源性高达99%，进化关系亦与其最相近。在全部4种材料鲜叶中，总花色苷的含量(TAC)与叶片紫色程度是一致的，其中，NJZX1-3叶片中总花色苷含量最高，达到80.15±5.74 mg 100 g–1 FW；BrcANS表达量为NJZX1-0 < NJZX2-1 < NJZX2-2< NJZX1-3，与其总花色苷含量呈正相关。BrcANS的mRNA在NJZX1-3和NJZX1-0两种材料的不同组织中特异性表达：在叶片中高度表达，而在其他组织中表达较弱；另外，在两种材料间的表达亦存在显著差异，在NJZX1-3叶片中的表达丰度显著高于NJZX1-0。随着叶龄的增大，紫色不接球白菜叶片紫色变浅，BrcANS的表达量下降，但在NJZX1-3和NJZX1-0间的表达差异亦明显减小。以上结果表明，BrcANS基因是紫色不结球白菜中花色苷合成的关键基因之一，其mRNA表达量与叶片紫色直接相关，可能在其转录水平上调控叶片中紫色的形成。

关键词: 不结球白菜, 花色苷合酶, 同源克隆, 序列分析, 总花色苷含量, 基因表达

Abstract:

Purple non-heading Chinese cabbage cultivar NJZX1-3, its green leaf mutant line NJZX1-0, and their progeny F2: NJZX2-1 and NJZX2-2 were used to study the function of anthocyanidin synthase gene in the anthocyanin biosynthesis of non-heading Chinese cabbage leaf. Homology-based cloning was used and anthocyanidin synthase gene was respectively cloned from two cultivars (NJZX1-3 and NJZX1-0). The gene nucleotides and amino acids sequences found in the two materials and Chinese cabbage were exactly the same, with a length of 1077 bp and encoding a peptide with 358 residues. Furthermore, a 2OG-Fe (II) dioxygenase super family domain was found in the amino acid sequence from the 211th to the 307th amino acids and the gene was named as BrcANS. The homology between BrcANS protein and BjANS protein was up to 99%, in accordance with the close relationship between them. Their total anthocyanin content (TAC) was consistent with the degree of purple in fresh leaves of the four materials, of which total anthocyanin content in cultivar NJZX1-3 leaves was the highest, up to 80.15±5.74 mg 100 g–1 FW. Simultaneously, the expression level of BrcANS (NJZX1-0 < NJZX2-1 < NJZX2-2< NJZX1-3) was positively correlated with the increasing trend of TAC. The mRNA of BrcANS exhibited tissue-specific expression in both materials, showing high level in leaves and lower level in other organs. In addition, the expression of two materials was significantly different, indicating that the expression of BrcANS in cultivar NJZX1-3 leaves was obviously higher than that in mutant line NJZX1-0. With the increasing of leaf age, the leaf color became shallow and the expression of BrcANS reduced. Meanwhile, the difference of expression between NJZX1-3 and NJZX1-0 decreased significantly. These results indicated that BrcANS gene is one of the key genes in the anthocyanin biosynthesis of non-heading Chinese cabbage leaf, and its expression level is directly related to the purple color of leaves, so the gene might regulate the formation of the purple color in leaves at transcriptional level.

Key words: Non-heading Chinese cabbage, Anthocyanidin synthase, Homology-based cloning, Sequence analysis, Total anthocyanin content /TAC, Gene expression

[1]侯喜林. 不结球白菜育种研究新进展. 南京农业大学学报, 2003, 26: 111–115
Hou X L. Advances in breeding of non-heading Chinese cabbage. J Nanjing Agric Univ, 2003, 26: 111–115 (in Chinese with English abstract)
[2]Grotewold E . The genetics and biochemistry of floral pigments. Annu Rev Plant Biol, 2006, 57: 761–780
[3]Strack D, Wray V. The anthocyanidins, in the flavonoids, advances in research since 1986 (Chapter 1), Chapman and Hall, London, 1994, pp 1–22
[4]Yoshikazu T, Nobuhiro S, Akemi O. Biosynthesis of plant pigments: anthocyanidins, betalains and carotenoids. Plant J, 2008, 54: 733–749
[5]Xie D Y, Sharma S B, Paiva N L, Ferreira D, Dixon R A. Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science, 2003, 299: 396–399
[6]Xie D Y, Dixon R A. Proanthocyanidin biosynthesis still more questions than answers? Phytochemistry, 2005, 66: 2127–2144
[7]Petroni K, Tonelli C. Recent advances on the regulation of anthocyanin synthesis in reproductive organs. Plant Science, 2011, 181: 219–229
[8]Wilmouth R C, Turnbull J J, Welford R W D, Clifton I J, Prescott A G, Schofield C J. Structure and mechanism of anthocyanidin synthase from Arabidopsis thaliana. Structure, 2002, 10: 93–103
[9]Yan M L, Liu X J, Guan C Y, Chen X B, Liu Z S. Cloning and expression analysis of an anthocyanidin synthase gene homolog from Brassica juncea. Mol Breed, 2011, 28: 313–322
[10]Samuelian S K, Camps C, Kappel C, Simova E P, Delrot S, Colova V M. Differential screening of overexpressed genes involved in flavonoid biosynthesis in North American native grapes: ‘Noble’ muscadinia var. and ‘Cynthiana’ aestivalis var. Plant Sci, 2009, 177: 211–221
[11]王冰, 王全逸, 印敬明, 陈敏, 杨清. 野生马铃薯ANS同源基因的克隆与表达分析. 植物生理学报, 2011, 47: 1103–1108
Wang B, Wang Q Y, Yin J M, Chen M, Yang Q. Molecular cloning and expression analysis of an ANS homologous gene from Solanum cardiphyllum. Plant Physiol J, 2011, 47: 1103–1108 (in Chinese with English abstract)
[12]缪军, 刘冰江, 杨妍妍, 霍雨猛, 张一卉, 霍凤梅, 修景润, 吴雄. 洋葱花青素合成酶基因的克隆和序列分析. 山东农业科学, 2010, (1): 1–5
Miao J, Liu B J, Yang Y Y, Huo Y M, Zhang Y H, Huo F M, Xiu J R, Wu X. Cloning and sequence analysis of anthocyanidin synthase gene in onion. Shandong Agric Sci, 2010, (1): 1–5 (in Chinese with English abstract)
[13]Holton T A, Cornish E C. Genetics and biochemistry of anthocyanidin biosynthesis. Plant Cell, 1995, 7: 1071–1083
[14]Walker A R, Davison P A, Bolognesi-Winfield A C, James C M, Srinivasan N, Blundell T L, Esch J J, Marks M D, Gray J C. The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiation and anthocyanidin biosynthesis in Arabidopsis, encodes a WD40 repeat protein. Plant Cell, 1999, 11: 1337–1349
[15]Payne C T, Zhang F, Lloyd A M. GL3 encodes a bHLH protein that regulates trichome development in Arabidopsis through interaction with GL1 and TTG1. Genetics, 2000, 156: 1349–1362
[16]Zhang F, Gonzalez A, Zhao M, Payne C T, Lloyd A. A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis. Development, 2003, 130: 4859–4869
[17]Baudry A, Caboche M, Lepiniec L. TT8 controls its own expression in a feedback regulation involving TTG1 and homologous MYB and bHLH factors, allowing a strong and cell-specific accumulation of flavonoids in Arabidopsis thaliana. Plant J, 2006, 46: 768–779
[18]Gonzalez A, Zhao M, Leavitt J M, Lloyd A M. Regulation of the anthocyanidin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J, 2008, 53: 814–827
[19]Lv S W, Zhang C W, Tang J, Li Y X, Wang Z, Hou X L. Genome-wide Analysis and identification of TIR-NBS-LRR genes in Chinese cabbage (Brassica rapa ssp. pekinensis) reveal expression patterns to TuMV infection. Physiol Mol Plant Pathol, 2015, 90: 89–97
[20]谭国飞, 王枫, 贾晓玲, 李岩, 熊爱生. 芹菜甘露醇脱氢酶基因的分离与表达分析. 园艺学报, 2013, 40: 2189–2198
Tan G F, Wang F, Jia X L, Li Y, Xiong A S. Isolation and expression of mannitol dehydrogenase gene in celery. Acta Hort Sin, 2013, 40: 2189–2198 (in Chinese with English abstract)
[21]Guo N, Wu J, Zheng S N, Cheng F, Liu B, Liang J L, Cui Y, Wang X W. Anthocyanin profile characterization and quantitative trait locus mapping in zicaitai (Brassica rapa L. ssp. chinensis var. purpurea). Mol Breed, 2015, 35: 113
[22]Lee J, Durst R W, Wrolstad R E. Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: collaborative study. J AOAC Int, 2005, 88: 1269–1278
[23]Podsedek A. Natural antioxidants and antioxidant capacity of Brassica vegetables: a review. LWT-Food Sci Technol, 2007, 40: 1–11
[24]张淑江, 马越, 徐学玲, 钱伟, 章时蕃, 李菲, 张慧, 孙日飞. 芸薹属5种紫红色蔬菜花青素苷含量及组分分析. 园艺学报, 2014, 41: 1451–1460
Zhang S J, Ma Y, Xu X L, Qian W, Zhang S F, Li F, Zhang H, Sun R F. Components and amounts of anthocyanidins in several Brassica vegetables. Acta Hort Sin, 2014, 41: 1451–1460 (in Chinese with English abstract)
[25]田佶, 沈红香, 张杰, 姚允聪, 宋婷婷, 耿慧. 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. 园艺学报, 2010, 37: 939–948
Tian J, Shen H X, Zhang J, Yao Y C, Song T T, Geng H. Cloning of McANS Gene in Malus crabapple and expression analysis in different cultivars. Acta Hort Sin, 2010, 37: 939–948 (in Chinese with English abstract)
[26]Cheng L Q, Xu Y J, Grotewold E, Jin Z P, Wu F Y, Fu C X, Zhao D X. Characterization of anthocyanidin synthase (ANS) gene and anthocyanidin in rare medicinal plant-Saussurea medusa. Plant Cell Tiss Organ Cult, 2007, 89: 63–73
[27]Xie D Y, Jackson L A, Cooper J D, Ferreira D, Paiva N L. Molecular and biochemical analysis of two cDNA clones encoding Dihydroflavonol-4-Reductase from Medicago truncatula. Plant Physiol, 2004, 134: 979–994
[28]许志茹, 李春雷, 崔国新, 孙燕, 李玉花. 芜菁花青素合成酶基因的克隆、序列分析及表达. 生物技术通讯, 2009, 20: 66–68
Xu Z R, Li C L, Cui G X, Sun Y, Li Y H. Cloning, sequence analysis and expression of anthocyanidin synthase dene in turnip. Lett Biotechnol, 2009, 20: 66–68 (in Chinese with English abstract)
[29]蒋明, 陈孝赏, 李金枝. 紫菜薹花青素合成酶基因BcANS的克隆、表达与序列分析. 浙江大学学报(农业与生命科学版), 2011, 37: 393–398
Jiang M, Chen X S, Li J Z. Cloning, expression and sequence analysis of anthocyanidin synthase gene BcANS in Brassica campestris var. purpurea. J Zhejiang Univ (Agric•& Life Sci), 2011, 37: 393–398 (in Chinese with English abstract)
[30]Lepiniec L, Debeaujon I, Routaboul J M, Baudry A, Pourcel L, Nesi N, Caboche M. Genetics and biochemistry of seed flavonoids. Annu Rev Plant Biol, 2006, 57: 405–430
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