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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (03): 397-404.doi: 10.3724/SP.J.1006.2018.00397

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2018-03-12 Published:2017-12-11
  • Contact: Yun-Fei LIANG,Lin-Cheng ZHANG,Qi-Guo GAO E-mail:gaoqg031@swu.edu.cn
  • 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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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

Table 1

Primers used in this study"

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

Fig. 1

cDNA sequence of Brassica oleracea L. BoLH27 and its deduced amino acid sequence The black shadow shows bHLH domain; the full line box shows N-glycosylation site; the dotted line box shows cAMP phosphorylation site; the underline shows cGMP-dependent protein kinase phosphorylation site; the wavy line shows casein kinase II phosphorylation site; the oval box shows N-glycosylation site."

Fig. 2

bHLH domain alignment among BoLH27 and other homologous proteins Stars indicate the conserved amino acids of bHLH domain; triangle indicates amino acid involved in dimer formation."

Fig. 3

Phylogenetic relationship of amino acid sequences between Brassica oleracea L. BoLH27 and its homologous proteins"

Fig. 4

The process of obtaining pC35S-BoLH27 transgenic cabbage plants A: the growth of resistant bud; B, C: the rooting of resistant seedling; D: the seedling of transgenic cabbage."

Fig. 5

PCR analysis of transgenic plants M: DL2000; 1-10: transgenic cabbage; +: positive plamid; -: non-transgenic cabbage."

Fig. 6

Relative expression of BoLH27 in wild-type and transgenic cabbage lines WT: non-transgenic lines; S13, S14, S33: transgenic lines."

Fig. 7

Phenotypes of non-transgenic lines and BoLH27 overexpressed lines A: seedling stage with ten leaves; B: side view of rosette stage with twenty-four leaves; C: top view of rosette stage with twenty-four leaves; WT: non-transgenic lines; BoLH27: overexpressed lines; partial: partial enlarged drawing of overexpressed lines."

Table 2

Average petiole length and average leaf spacing in overexpressed lines"

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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