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作物学报 ›› 2018, Vol. 44 ›› Issue (03): 376-384.doi: 10.3724/SP.J.1006.2018.00376

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米胚乳母本印记基因ZmVIL1的克隆及印记特性分析

刘朝显*, 王久光*, 梅秀鹏, 余婷婷, 王国强, 周练, 蔡一林*()   

  1. 西南大学玉米研究所 / 南方山地农业教育部工程研究中心, 重庆400715
  • 收稿日期:2017-07-11 接受日期:2017-11-21 出版日期:2018-03-12 网络出版日期:2017-12-04
  • 通讯作者: 刘朝显,王久光,蔡一林
  • 作者简介:

    第一作者联系方式: 王瑞, E-mail: 18612261636@163.com; 张秀艳, E-mail: xyzhang0818@163.com

  • 基金资助:
    本研究由中国博士后科学基金项目(2014M552303), 中央高校基本科研业务费(XDJK2015B009)和杂交玉米新品种“中科玉9699”和“西大白糯1号”的技术集成与示范项目(cstc2015jcsf-nycgzhA80006)资助

Cloning and Imprinting Characterization Analyses of Paternally Expressed Gene ZmVIL1 in Maize Endosperm

Chao-Xian LIU**, Jiu-Guang WANG**, Xiu-Peng MEI, Ting-Ting YU, Guo-Qiang WANG, Lian ZHOU, Yi-Lin CAI*()   

  1. Maize Research Institute, Southwest University / Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, China
  • Received:2017-07-11 Accepted:2017-11-21 Published:2018-03-12 Published online:2017-12-04
  • Contact: Chao-Xian LIU,Jiu-Guang WANG,Yi-Lin CAI
  • Supported by:
    This study was supported by China Postdoctoral Science Foundation (2014M552303), Fundamental Research Funds for the Central Universities (XDJK2015B009), and the Technology Integration and Demonstration of Zhongkeyu 9699, and Xidabainuo 1 (cstc2015jcsf-nycgzhA80006).

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摘要：

印记基因在玉米籽粒发育中具有重要作用。玉米ZmVIL1是母本印记基因。本研究通过RACE扩增, 获得了ZmVIL1基因的cDNA全长, 约2.2 kb, 编码598个氨基酸。根据对B73和Mo17正反交授粉后14 d胚乳和胚中ZmVIL1等位基因的表达分析, 该基因仅在玉米胚乳中表现母本印记, 而在胚中则无印记现象。在郑58和昌7-2、黄C和178、PH6WC和PH4CV正反交14 d胚乳中ZmVIL1也表现印记, 因此该基因为基因特异性的二元印记。ZmVIL1在B73、Mo17正反交授粉后12~28 d胚乳中处于持续印记状态。组织表达模式研究表明ZmVIL1在营养生长和生殖生长阶段均有表达; ZmVIL1在玉米授粉后10 d籽粒、雌穗、雄穗、花丝中表达量较高, 其次是根、胚珠及授粉后25 d的胚中, 推测ZmVIL1在玉米籽粒及花发育过程中均具有重要功能。

关键词: ZmVIL1, 基因克隆, 基因印记, 印记模式

Abstract:

Imprint genes play important roles in maize seed development. ZmVIL1 is a paternal-preferentially expressed gene. In this study, the full length of ZmVIL1 cDNA was cloned by RACE, which is about 2.2 kb and encodes a protein consisting of 598 amino acids. The analyses of allele expression of ZmVIL1 in endosperm and embryo at 14 days after pollination (DAP) of hybrids from reciprocal crosses between B73 and Mo17 indicated that ZmVIL1 was maternally imprinted in endosperm, while not in embryo. The allele expression was extensively evaluated in 14 DAP endosperm of hybrids form reciprocal crosses of Zheng 58 and Chang 7-2, Huang C and 178, PH6WC and PH4CV, and in 10-28 DAP endosperm of hybrids form reciprocal crosses of B73 and Mo17, suggesting that ZmVIL1 was gene-specific and binary imprinting and consistently imprinted in endosperm from 12 to 28 DAP. Expression pattern analyses demonstrated ZmVIL1 was constitutively expressed from vegetative to reproductive stages. ZmVIL1 was strongly expressed in seed at 10 DAP, immature tassels, immature ears and silk, and secondly expressed in ovule, embryo at 25 DAP and root, suggesting that ZmVIL1 plays important roles in maize seed and flower development.

Key words: ZmVIL1, gene cloning, gene imprinting, imprinting pattern

表1

ZmVIL1 5° RACE、3° RACE、CDS扩增及印记特性分析引物序列"

引物名称
Primer name		 正向引物序列
Forward primer sequence (5°-3°)		 反向引物序列
Reverse primer sequence (5°-3°)		 内切酶
Restriction enzyme
ZmVIL1-5° race GCCCATCGCATTCCAACTGCCTTACTAC
ZmVIL1-5° race-nest-1 GCGTTTAGGTCGGGGACAGAAGATGAG
ZmVIL1-5° race-nest-2 GGTCAGGAGAACTCAACCACTCGTCAGC
ZmVIL1-3° race CTCGGCAAGTAAGCAAGGTGAATAGCGT
ZmVIL1-3° race-nest-1 GCAACTGGATGGGGATTATGAGCACTG
ZmVIL1-3° race-nest-2 AGGTAGTAAGGCAGTTGGAATGCGATGG
ZmVIL1-CDS ATGCCCAAAACACCTCCTGT CTCAGCGAACCACAACAACAAA
ZmVIL1-imprinting ATTCAAAGAGCTGACGAGTGGT TCATAATCCCCATCCAGTTGCC Hph I

图1

ZmVIL1基因5° RACE和3° RACE A、B、C: ZmVIL1第1轮、第2轮和第3轮RACE产物电泳。L1、L3、L5: ZmVIL1 cDNA 5° RACE产物电泳; L2、L4、L6: ZmVIL1 cDNA 3° RACE产物电泳; Marker: D2000 plus。"

图2

ZmVIL1与拟南芥VIN3、VIL1蛋白序列比对 淡绿色和橙色方框内分别为保守的锌指蛋白和纤连蛋白结构域。"

图3

B73和Mo17中ZmVIL1 CDS序列单核苷酸多态性位点 箭头所示位置为B73和Mo17 ZmVIL1 CDS在第1380、第1450和第1530碱基位置的单核苷酸多态性位点。"

图4

ZmVIL1在B73、Mo17正反交授粉后14 d胚乳及胚中的印记特性分析 A: 授粉后14 d胚乳中ZmVIL1的印记特性分析; B: 授粉后14 d胚中ZmVIL1的印记特性分析; Marker: D2000 plus。"

图5

ZmVIL1在黄C和178、郑58和昌7-2及PH4CV和PH6WC正交反授粉后14 d胚乳中印记特性分析 H: 黄C; C: 昌7-2; Z: 郑58; 4C: PH4CV; 6W: PH6WC; Marker: D2000 plus。"

图6

ZmVIL1在B73、Mo17正反交授粉后10~28 d胚乳中印记特性分析 A: ZmVIL1在B73×Mo17授粉后10~28 d胚乳中的印记特性分析; B: ZmVIL1在Mo17×B73授粉后10~28 d胚乳中的印记特性分析; Marker: D2000 plus。"

图7

ZmVIL1在玉米不同组织中的表达 Root: 根; SAM (shoot apical meristem): 顶端分生组织; Leaf: 叶; Ligule: 叶舌; Sheath: 叶鞘; Immature ear: 雌穗; Immature tassel: 雄穗; Anther: 花药; Pollen: 花粉; Silk: 花丝; Ovule: 胚珠; Seed_5 DAP: 授粉后5 d籽粒; Seed_10 DAP: 授粉后10 d籽粒; Embryo_25 DAP: 授粉后25 d胚; Endosperm_25 DAP: 授粉后25 d胚乳。"

图8

ZmVIL1与拟南芥、水稻中VIN3蛋白家族分子进化 关系分析"

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