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紫心甘薯转录因子bHLH的基因克隆及功能研究

惠亚可 胡敏伦 郭晋雅 高峰

惠亚可, 胡敏伦, 郭晋雅, 高峰. 紫心甘薯转录因子bHLH的基因克隆及功能研究[J]. 华南师范大学学报(自然科学版), 2020, 52(4): 63-70. doi: 10.6054/j.jscnun.2020062
引用本文: 惠亚可, 胡敏伦, 郭晋雅, 高峰. 紫心甘薯转录因子bHLH的基因克隆及功能研究[J]. 华南师范大学学报(自然科学版), 2020, 52(4): 63-70. doi: 10.6054/j.jscnun.2020062
HUI Yake, HU Minlun, GUO Jinya, GAO Feng. The Cloning and Functional Analysis of the Transcription Factor bHLH in Purple-Fleshed Sweet Potato[J]. Journal of South China normal University (Natural Science Edition), 2020, 52(4): 63-70. doi: 10.6054/j.jscnun.2020062
Citation: HUI Yake, HU Minlun, GUO Jinya, GAO Feng. The Cloning and Functional Analysis of the Transcription Factor bHLH in Purple-Fleshed Sweet Potato[J]. Journal of South China normal University (Natural Science Edition), 2020, 52(4): 63-70. doi: 10.6054/j.jscnun.2020062

紫心甘薯转录因子bHLH的基因克隆及功能研究

doi: 10.6054/j.jscnun.2020062
基金项目: 

国家自然科学基金项目 31171601

详细信息
    通讯作者:

    高峰,教授,Email:593197289@qq.com

  • 中图分类号: S432.45;Q782

The Cloning and Functional Analysis of the Transcription Factor bHLH in Purple-Fleshed Sweet Potato

  • 摘要: 通过对紫心甘薯中bHLH类转录因子的基因进行分子克隆,对其结构、表达模式及功能进行研究,明确了其结构特征和生物学功能.通过采用RACE克隆方法,获得了编码bHLH基因且长度分别为2 516 bp和2 304 bp的cDNA全序列.基于DNA序列的分子进化树分析结果表明:两者分别属于植物bHLH1bHLH2基因家族的成员,分别将其命名为IbbHLH1(GenBank登录号:KC708871)和IbbHLH2 (GenBank登录号:JF508437);IbbHLH1和IbbHLH2蛋白均定位于细胞核; 在3个甘薯品种(系)的块根中,IbbHLH2基因与花色素苷合成途径中的酶基因(CHSCHIF3HDFRANS和3GT)的表达量变化趋势相一致,初步推测转录因子IbbHLH2可能参与了紫心甘薯花色素苷合成途径一系列酶基因的表达与调控.
  • 图  1  “山川紫”IbbHLH1的全长cDNA序列及其编码的氨基酸序列

    Figure  1.  The full-length cDNA and deduced amino acid sequences of IbbHLH1 from "Yamakawamurasaki"

    图  2  “山川紫”IbbHLH2的全长cDNA序列及其编码的氨基酸序列

    Figure  2.  The full-length cDNA and deduced amino acid sequences of IbbHLH2 from "Yamakawamurasaki"

    图  3  IbbHLH1IbbHLH2基因NJ系统进化树的构建

    注:各节点处数字表示bootstrap值,重复1 000次

    Figure  3.  The phylogenetic tree of IbbHLH1 and IbbHLH2 generated with the neighbor joining method

    图  4  洋葱表皮细胞中IbbHLH1-EGFP和IbbHLH2-EGFP融合蛋白的亚细胞定位

    Figure  4.  The subcellular localization of IbbHLH1-EGFP and IbbHLH2-EGFP fusional protein in onion epidermis cells

    图  5  不同品种(系)甘薯块根中各基因的表达量

    Figure  5.  The expression of gene in the root of different sweet potato cultivars(strains)

    表  1  RACE引物使用表

    Table  1.   The RACE primer use table

    引物名称 引物序列
    IbbHLH1F-core 5’- CGTTCAACACCTAAAAACCTCGTATC -3’
    IbbHLH1R-core 5’- GAGGACATCAGATAACTGTGGCACTAAC -3’
    IbbHLH2F-core 5’-GTGGTTCTACCTCATGTGCATCTCC-3’
    IbbHLH2R-core 5’-CGTACTCAATGGTGTCGCCGA-3’
    UPM long:5’-CTAATACGACTCACTATAGGGCAAGCAGTGG
    TATCAACGCAGAGT-3’short: 5’- CTAATACGACTCACTATAGGGC-3’
    NUPM 5’-AAGCAGTGGTATCAACGCAGAGT-3’
    IbbHLH1F-1 5’- CGTTCAACACCTAAAAACCTCGTATC -3’
    IbbHLH1F-2 5’- GAGATGGTTGAGCATTCCCCGC -3’
    IbbHLH2F-1 5’-TAGAGCAATTCTCGCCAAGTGTG -3’
    IbbHLH2F-2 5’-CCGAAACAATATCGAGCATCC-3’
    IbbHLH1R-1 5’- CAGTGACTCCAAGCTCGATAACGCCTCCCAAA -3’
    IbbHLH1R-2 5’- TCCATCTGCCTGGGGAGCATTGCATAGCC -3’
    IbbHLH2R-1 5’-CGTACTCAATGGTGTCGCCGA-3’
    IbbHLH2R-2 5’-GTTCCCAGTTCAACCACGCC-3’
    下载: 导出CSV

    表  2  甘薯半定量RT- PCR引物使用表

    Table  2.   The semi-quantitative RT-PCR primers for sweet potato

    引物名称 引物序列
    IbbHLH1RTF 5'-GAAAGGCATCAAAATTAACGTCACC-3
    IbbHLH1RTR 5'-CTGTAAACAATTCCCTCAACTGGTA-3
    IbbHLH2RTF 5'-TAGGGAGCTTGGACTAGAAATCACG-3
    IbbHLH2RTR 5'-TGGCTACCTTGATGTTGTCTCTTGG-3
    CHSRTF 5'-GCAGACCTTGGCTCCCGATAG-3'
    CHSRTR 5'-TTAAGTTGGGACGCTATGGAGG-3'
    CHIRTF 5'-GTTAAGTGGAACGGGAAAAG -3'
    CHIRTR 5'- GAGACGACCGTTTGTGGAAT -3'
    F3HRTF 5'-GGTGGAGGGCAGTAACGGAAAAGTA-3'
    F3HRTR 5'-CGCCGGATTCTGAAACGTCGCTATA-3'
    DFRRTF 5'-TGAACTACCGAAAGCGGACAC-3'
    DFRRTR 5'-GGTGGGAATGTTGGGGTGAT-3'
    ANSRTF 5'-CCTCACCTTCATCCTCCACAACA-3'
    ANSRTR 5'-AACCCATAATAATACCAAACGGACT-3'
    UF3GTRTF 5'-TTCGGGAAGATCGTGCCGTGGG-3'
    UF3GTRTR 5'-CCAATTTCCCATACACTCTCCACA-3'
    G14RTF 5'-ATGTCGGACAAGTGCGGAAACTGCG-3'
    G14RTF 5'-TTAGTGGCCACAGGTGCGGTCGGTA-3'
    下载: 导出CSV

    表  3  IbbHLH1进化保守的氨基酸序列

    Table  3.   The evolutionarily conserved amino acid sequence of IbbHLH1

    位点 名称
    433 谷氨酸
    434 精氨酸
    436 精氨酸
    441 天冬酰胺
    444 苯丙氨酸
    447 亮氨酸
    456 赖氨酸
    459 赖氨酸
    462 异亮氨酸
    463 亮氨酸
    466 苏氨酸
    467 异亮氨酸
    469 酪氨酸
    473 亮氨酸
    下载: 导出CSV

    表  4  IbbHLH2进化保守的氨基酸序列

    Table  4.   The evolutionarily conserved amino acid sequence IbbHLH2

    位点 名称
    477 谷氨酸
    478 精氨酸
    480 精氨酸
    484 亮氨酸
    485 天冬酰胺
    488 苯丙氨酸
    491 亮氨酸
    492 赖氨酸
    500 赖氨酸
    503 赖氨酸
    506 异亮氨酸
    507 亮氨酸
    510 苏氨酸
    511 异亮氨酸
    513 酪氨酸
    514 缬氨酸
    517 亮氨酸
    下载: 导出CSV
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  • 收稿日期:  2019-09-10
  • 刊出日期:  2020-08-25

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