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作物学报 ›› 2018, Vol. 44 ›› Issue (03): 397-404.doi: 10.3724/SP.J.1006.2018.00397

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝转录因子BoLH27的克隆与转基因甘蓝的表型分析

梁云飞1,*, 张林成1,*, 蒲全明2, 雷镇泽1, 施松梅1, 姜宇鹏1, 任雪松1, 高启国1,*()   

  1. 1西南大学园艺园林学院 / 南方山地园艺学教育部重点实验室, 重庆 400716
    2南充市农业科学院蔬菜研究所, 四川南充 637000
  • 收稿日期:2017-06-06 接受日期:2017-11-21 出版日期:2018-03-12 网络出版日期:2017-12-11
  • 通讯作者: 梁云飞,张林成,高启国
  • 作者简介:

    18847123096@163.com

  • 基金资助:
    本研究由国家重点基础研究发展计划项目(973计划)(2012CB113900)和国家自然科学基金项目(30900986)资助

Cloning of BoLH27 Gene from Cabbage and Phenotype Analysis of Transgenic Cabbage

Yun-Fei LIANG1,**, Lin-Cheng ZHANG1,**, Quan-Ming PU2, Zhen-Ze LEI1, Song-Mei SHI1, Yu-Peng JIANG1, Xue-Song REN1, Qi-Guo GAO1,*()   

  1. 1 College of Horticulture and Landscape Architecture, Southwest University / Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education, Chongqing 400716, China
    2 Institute of Vegetables, Nanchong Academy of Agricultural Sciences, Nanchong 637000, Sichuan, China
  • Received:2017-06-06 Accepted:2017-11-21 Published:2018-03-12 Published online:2017-12-11
  • Contact: Yun-Fei LIANG,Lin-Cheng ZHANG,Qi-Guo GAO
  • Supported by:
    This study was supported by the National Program on Key Basic Research Project (973 program)(2012CB113900) and the National Natural Science Foundation of China (30900986).

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摘要：

bHLH转录因子对植物生长发育、形态调控有重要作用, 为了研究BoLH27基因在甘蓝叶片发育及形态建成中的调控功能, 本文以甘蓝品种519为材料, 克隆出转录因子BoLH27基因。序列分析表明, 该基因含有一个795 bp的开放阅读框, 编码264个氨基酸, 具有一个保守的典型bHLH结构域。以农杆菌介导的遗传转化方法将正义BoLH27基因导入甘蓝品种519中以增强表达, 通过PCR筛选出9株T0代转基因单株, 结合T2代单株的qRT-PCR分析表明, 筛选的转基因植株内BoLH27基因的表达积累量明显高于野生型。试验基地隔离网内种植的转基因甘蓝表型明显, 主要表现为莲座期茎叶间距拉长、叶柄伸长以及植株茎和叶柄显示紫色, 植株叶片平展, 无向上向内卷曲趋势, 表明BoLH27基因可能对甘蓝叶片发育有重要的调控作用。

关键词: 甘蓝, BoLH27, 基因克隆, 转基因, 表型

Abstract:

bHLH transcription factor plays an important role in plant growth, development and morphological control. In order to explore BoLH27 gene regulatory function for leaf development and morphologic formation, the BoLH27 gene was cloned from cabbage (Brassica oleracea L.) variety 519. Sequence analysis indicated that the length of BoLH27 gene was 795 bp, which encoded 264 amino acids. The BoLH27 protein contained conservative structure domains of the bHLH family. The sense BoLH27 gene was transformed into cabbage variety 519 by Agrobacterium mediated method, PCR analysis exhibited that BoLH27 was genetically transformed into nine individual plants, qRT-PCR analysis revealed that BoLH27 had higher expression level in T2 transgenic cabbage than in wild type. The phenotype at rosette stage of transgenic cabbage grown in the test base was obvious, showing elongated the stems and leaves, elongated petiole, purple stems and petiole, and flat leaves without upward inward curling trend. It suggested that BoLH27 may play important roles in the control of leaves development.

Key words: Brassica oleracea, BoLH27, gene cloning, transgene, phenotype

表1

试验使用的引物"

引物名称及序列
Primer name and sequence (5°→3°)		 退火温度
Annealing temperature (°C)
klBoLH27S: ATGGAAGACCTCGAAGATGAGTACAAG
klBoLH27AS: GGTTTTTGGTACAATGAAACAAACTAGAAG		 63
pcBoLH27S: GGATCCATGGAAGACCTCGAAGATGAGTACAAG
pcBoLH27AS: GGATCCGGTTTTTGGTACAATGAAACAAACTAGAAG
NOS: CCCGATCTAGTAACATAGATGACAC		 62
jcBoLH27S: GGATCCATGGAAGACCTCGAAGATGAGTACAAG
jcBoLH27AS: GGATCCGGTTTTTGGTACAATGAAACAAACTAGAAG		 56.8
qBoLH27S: AGATTAGAAGCAGAGATCCAAGAGC
qBoLH27AS: GAAGTATTGTAATCCATCTGCCTGAAC		 58

图1

甘蓝BoLH27 cDNA及其推导的氨基酸序列黑色阴影示bHLH结构域; 实线框示N-糖基化位点; 虚线框示cAMP磷酸位点; 下画线示蛋白激酶C磷酸化位点; 波浪线示酪蛋白II磷酸化位点; 椭圆框示N-豆蔻酰化位点。"

图2

BoLH27及其同源序列bHLH结构域比对分析星号表示bHLH结构域保守的氨基酸位点; 三角表示参与二聚体形成的位点。"

图3

BoLH27及其同源氨基酸序列进化分析"

图4

pC35S-BoLH27转基因植株的获得 A: 抗性芽的生长; B、C: 抗性苗生根; D: 抗性植株幼苗。"

图5

转基因植株PCR检测 M: DL2000; 1~10: 转基因甘蓝; +: 阳性对照; -: 非转基因甘蓝。"

图6

BoLH27在野生型和转基因甘蓝中的表达分析 WT: 野生型株系; S13, S14, S33: 超表达株系。"

图7

野生型株系和BoLH27超表达株系表型 A: 10片叶幼苗期; B: 24片叶莲座期侧视图; C: 24片叶莲座期俯视图; WT: 野生型; BoLH27: 超表达株系; partial: 超表达株系局部放大。"

表2

超表达株系平均叶柄长度和平均叶间距"

超表达株系
Overexpressed lines		 平均叶柄长度
Average petiole length (cm)		 平均叶间距
Average leaf
spacing (cm)
1 16.8 1.7
2 15.2 1.8
3 14.5 1.6
[1] Ludwig S R, Habera L F, Dellaporta S L, Wessler S R.Lc, a member of the maize R gene family responsible for tissue- specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc Natl Acad Sci USA, 1989, 86: 7092-7096
[2] Ling H Q, Bauer P, Bereczky Z, Keller B, Ganal M.The tomato FER gene encoding a bHLH protein controls iron-uptake responses in roots.Proc Natl Acad Sci USA, 2002, 99: 13938-13943
[3] Kiribuchi K, Jikumaru Y, Kaku H, Minami E, Hasegawa M, Kodama O.Involvement of the basic helix-loop-helix transcription factor RERJ1 in wounding and drought stress responses in rice plants.Biosci Biotechnol Biochem, 2005, 69: 1042-1044
[4] Carreteropaulet L, Galstyan A, Roigvillanova I, Martínezgarcía J F, Bilbaocastro J R, Robertson D L.Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae.Plant Physiol, 2010, 153: 1398-1412
[5] Pires N, Dolan L.Origin and diversification of basic-helix- loop-helix proteins in plants.Mol Biol Evol, 2010, 27: 862-874
[6] Grove C A, De M F, Barrasa M I, Newburger D E, Alkema M J, Bulyk M L.A multiparameter network reveals extensive divergence between C. elegans bHLH transcription factors. Cell, 2009, 138: 314-327
[7] Toledo-Ortiz G, Huq E, Quail P H.The Arabidopsis basic-helix-loop-helix transcription factor family. Plant Cell, 2003, 15: 1749-1770
[8] Li X, Duan X, Jiang H, Sun Y, Tang Y, Yuan Z.Genome-wide analysis of basic-helix-loop-helix transcription factor family in rice and Arabidopsis. Plant Physiol, 2006, 141: 1167-1184
[9] Atchley W R, Terhalle W, Dress A.Positional dependence, cliques, and predictive motifs in the bHLH protein domain.J Mol Evol, 1999, 48: 501-516
[10] Heim M A, Jakoby M, Werber M, Martin C, Weisshaar B, Bailey P C.The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity.Mol Biol Evol, 2003, 20: 735-747
[11] Yin J, Chang X, Kasuga T, Mai B, Reid M S, Jiang C Z.A basic helix-loop-helix transcription factor, PhFBH4, regulates flower senescence by modulating ethylene biosynthesis pathway in petunia.Hortic Res, 2015, 2: 15059-15068
[12] Ko S S, Li M J, Sun-Ben K M, Ho Y C, Lin Y J, Chuang M H. The bHLH142 transcription factor coordinates with TDR1 to modulate the expression of EAT1 and regulate pollen development in rice.Plant Cell, 2014, 26: 2486-2504
[13] Takahashi Y, Ebisu Y, Kinoshita T, Doi M, Okuma E, Murata Y. bHLH transcription factors that facilitate K+ uptake during stomatal opening are repressed by abscisic acid through phosphorylation. Sci Signal, 2013, 6: ra48
[14] Shen Q, Lu X, Yan T, Fu X, Lv Z, Zhang F.The jasmonate-responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua. New Phytol, 2016, 210: 1269-1281
[15] Kurbidaeva A, Ezhova T, Novokreshchenova M.Arabidopsis thaliana ICE2 gene: phylogeny, structural evolution and functional diversification from ICE1. Plant Sci, 2014, 229: 10-22
[16] Makkena S, Lamb R S.The bHLH transcription factor SPATULA is a key regulator of organ size in Arabidopsis thaliana. Plant Signal Behav, 2013, 8: e24140
[17] An R, Liu X, Wang R, Wu H, Liang S, Shao J, Qi Y, An L, Yu F.The over-expression of two transcription factors, ABS5/bHLH30 and ABS7/MYB101, leads to upwardly curly leaves.PLoS One, 2014, 9: e107637
[18] Nath U, Crawford B C, Carpenter R, Coen E.Genetic control of surface curvature.Science, 2003, 299: 1404-1407
[19] Qin G, Gu H, Zhao Y, Ma Z, Shi G, Yang Y.An indole-3-acetic acid carboxyl methyltransferase regulates Arabidopsis leaf development.Plant Cell, 2005, 17: 2693-2704
[20] Sarvepalli K, Nath U.Hyper-activation of the TCP4 transcription factor in Arabidopsis thaliana accelerates multiple aspects of plant maturation. Plant J, 2011, 67: 595-607
[21] Efroni I, Blum E, Goldshmidt A, Eshed Y.A protracted and dynamic maturation schedule underlies Arabidopsis leaf development. Plant Cell, 2008, 20: 2293-2306
[22] 莫晓婷. 玉米逆境相关转录因子的克隆与初步分析. 中国农业科学院硕士学位论文. 北京, 2013
Mo X T.Cloning and Functional Characterization of Stress-related Transcription Factors in Maize. MS Thesis of Chinese Academy of Agricultural Sciences, Beijing,China, 2013 (in Chinese with English abstract)
[23] 何绍敏, 李春雨, 兰彩耘, 邹敏, 任雪松, 司军, 李成琼, 宋洪元. 转MLPK反义基因对甘蓝自交不亲和性的影响. 园艺学报, 2015, 42: 252-262
He S M, Li C Y, Lan C Y, Zou M, Ren X S, Si J, Li C Q, Song H Y.Effect of antisense RNA of the MLPK gene on self-incompatibility in cabbage.Acta Hortic Sin, 2015, 42: 252-262 (in Chinese with English abstract)
[24] Heim M A, Jakoby M, Werber M, Martin C, Weisshaar B, Bailey P C.The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity.Mol Biol Evol, 2003, 20: 735-747
[25] Grotewold E, Sainz M B, Tagliani L, Hernandez J M, Bowen B, Chandler V L.Identification of the residues in the Myb domain of maize C1 that specify the interaction with the bHLH cofactor R.Proc Natl Acad Sci USA, 2000, 97: 13579-13584
[26] Bernhardt C, Lee M M, Gonzalez A, Zhang F, Lloyd A, Schiefelbein J.The bHLH genes GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the Arabidopsis root. Development, 2003, 130: 6431-6439
[27] Zhang L Y, Bai M Y, Wu J, Zhu J Y, Wang H, Zhang Z.Antagonistic HLH/bHLH transcription factors mediate brassinosteroid regulation of cell elongation and plant development in rice and Arabidopsis. Plant Cell, 2009, 21: 3767-3780
[28] Meng Y, Li H, Wang Q, Liu B, Lin C.Blue light-dependent interaction between cryptochrome 2 and CIB1 regulates transcription and leaf senescence in soybean.Plant Cell, 2013, 25: 4405-4420
[29] Husbands A, Bell E M, Shuai B, Smith H M S, Springer P S. LATERAL ORGAN BOUNDARIES defines a new family of DNA-binding transcription factors and can interact with specific bHLH proteins.Nucl Acids Res, 2007, 35: 6663-6671
[30] Shuai B, Reynagapeña C G, Springer P S.The lateral organ boundaries gene defines a novel, plant-specific gene family.Plant Physiol, 2002, 129: 747-761
[31] Aguilar-Martínez J A, Sinha N. Analysis of the role of Arabidopsis class I TCP genes AtTCP7, AtTCP8, AtTCP22, and AtTCP23 in leaf development. Front Plant Sci, 2013, 8:e24140
[32] Guo Z, Fujioka S, Blancaflor E B, Miao S, Gou X, Li J.TCP1 modulates brassinosteroid biosynthesis by regulating the expression of the key biosynthetic gene DWARF4 in Arabidopsis thaliana. Plant Cell, 2010, 22: 1161-1173
[33] Palatnik J F, Allen E, Wu X, Schommer C, Schwab R, Carrington J C.Control of leaf morphogenesis by microRNAs.Nature, 2003, 425: 257-263
[34] Chandler V L, Radicella J P, Robbins T P, Chen J, Turks D.Two regulatory genes of the maize anthocyanin pathway are homologous: isolation of B utilizing R genomic sequences.Plant Cell, 1989, 1: 1175-1183
[35] de Vetten N, Quattrocchio F, Mol J, Koes R. The an11 locus controlling flower pigmentation in petunia encodes a novel WD-repeat protein conserved in yeast, plants, and animals.Genes Dev, 1997, 11: 1422-1434
[36] 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 Mol Biol, 2006, 46: 768-779
[37] 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
[38] Liu X F, Yin X R, Allan A C, Wang K L, Shi Y N, Huang Y J.The role of MrbHLH1, and MrMYB1, in regulating anthocyanin biosynthetic genes in tobacco and Chinese bayberry (Myrica rubra) during anthocyanin biosynthesis. Plant Cell Tissue Org, 2013, 115: 285-298
[39] Chiu L W, Zhou X, Burke S, Wu X, Prior R L, Li L.The purple cauliflower arises from activation of a MYB transcription factor.Plant Physiol, 2010, 154: 1470-1480
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