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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

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