白桦BpMYB21基因的克隆及其表达模式分析

孙璐; 杨杰; 王思瑶; 李欣; 李影; 徐志强; 詹亚光; 尹静

doi:10.3969/j.issn.1000-2006.201708017

白桦BpMYB21基因的克隆及其表达模式分析

孙璐,杨杰,王思瑶,李欣,李影,徐志强,詹亚光,尹静

南京林业大学学报（自然科学版） ›› 2018, Vol. 42 ›› Issue (04) : 119-126.

PDF(11653137 KB)

国家林草科技领军期刊
中国精品科技期刊
中国高校百佳科技期刊
江苏省新闻出版政府奖期刊奖
RCCSE林学权威期刊（A+）
CSCD核心期刊
Scopus数据库收录期刊
中文核心期刊
SCD核心期刊

作者加群：102861116

微信公众号：南京林业大学学报

高级检索

PDF(11653137 KB)

南京林业大学学报（自然科学版） ›› 2018, Vol. 42 ›› Issue (04) : 119-126. DOI: 10.3969/j.issn.1000-2006.201708017

研究论文

白桦BpMYB21基因的克隆及其表达模式分析

孙璐¹,杨杰¹,王思瑶¹,李欣¹,李影¹,徐志强¹,詹亚光^1,2,尹静^1,2*

作者信息 +

Cloning and expression pattern of BpMYB21 from Betula platyphylla Suk.

SUN Lu¹, YANG Jie¹, WANG Siyao¹, LI Xin¹, LI Ying¹, XU Zhiqiang¹, ZHAN Yaguang^1,2, YIN Jing^1,2*

Author information +

文章历史 +

摘要

【目的】研究了白桦(Betula platyphylla Suk.)MYB基因功能,分析其在茉莉酸(MeJA)和水杨酸(SA)诱导下的表达特性。【方法】以白桦为试材,通过RT-PCR等方法克隆得到1条白桦MYB转录因子家族基因序列,运用生物信息学方法,分析其蛋白质性质和亲缘关系,通过实时荧光定量PCR分析MeJA和SA诱导处理下该基因的表达特性及组织特异性。【结果】获得了1条新的白桦MYB转录因子家族基因,命名为BpMYB21,包含完整的开放阅读框,长度为1 014 bp,编码337个氨基酸。BpMYB 21蛋白与其他植物MYB氨基酸同源性为51%～64%,其中与胡桃(Juglans regia)MYB30-Like的亲缘关系最近。BpMYB21 在白桦根、茎、叶中均有表达,且在叶中的表达量高于茎和根,但差异不显著; 与对照相比,100 μmol/L MeJA和50 mg/L SA分别处理白桦苗6 h时,BpMYB21在茎和叶中的表达较高,处理12～48 h逐渐降低。【结论】BpMYB21为MYB转录因子家族新成员,与胡桃MYB基因亲缘关系较近。该基因响应激素诱导,推测其可能在白桦生长发育及在次生代谢过程发挥重要作用。

Abstract

Abstract: 【Objective】In this study, we explored the biological functions of the MYB gene in triterpenoid metabolism and examine MYB expression patterns induced by MeJA and SA treatment and expression specificity in different birch(Betula platyphylla Suk.)tissues.【Method】A new MYB transcription factor gene from birch was obtained by RT-PCR, and its protein characteristics and phylogenetic relationships were analyzed using bioinformatics methods. The expression pattern induced by MeJA and SA treatment was determined using real-time fluorescence quantitative PCR.【Result】A new gene of the MYB family transcription factor, named BpMYB21, was cloned from birch. The gene contains a complete 1 014 bp open reading frame(ORF), encoding a 337 amino acid protein. The BpMYB21 protein has an amino acid homology of 51%-64% with other plants and is most closely related to the walnut(Juglans regia)MYB30-Like protein. BpMYB21 was expressed in the roots, stems and leaves of birch and the expression level in the leaves was higher than those in the stems and roots; however, the difference was not significant. In birch culture plants treated with 100 μmol/L MeJA and 50 mg/L SA for 6 h, the expression of BpMYB21 was the highest in the stems and leaves and expression levels gradually decreased with time from 12 to 48 h. 【Conclusion】 BpMYB21 is a new member of the MYB transcription factor family, which is closely related to walnut(Juglans regia). This gene is responsive to hormone induction and may play an important role in the growth and development and secondary metabolism of birch.

引用本文

EndNote

Ris (Procite)

Bibtex

导出引用

孙璐,杨杰,王思瑶,李欣,李影,徐志强,詹亚光,尹静. 白桦BpMYB21基因的克隆及其表达模式分析[J]. 南京林业大学学报（自然科学版）. 2018, 42(04): 119-126 https://doi.org/10.3969/j.issn.1000-2006.201708017

SUN Lu, YANG Jie, WANG Siyao, LI Xin, LI Ying, XU Zhiqiang, ZHAN Yaguang, YIN Jing. Cloning and expression pattern of BpMYB21 from Betula platyphylla Suk.[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2018, 42(04): 119-126 https://doi.org/10.3969/j.issn.1000-2006.201708017

中图分类号： Q78

参考文献

[1] 尹静,詹亚光,肖佳雷. 白桦三萜的合成和调控[J]. 植物生理学通讯,2009,45(5):520-526. DOI:10.13592/j.cnki.ppj.2009.05.019. YIN J, ZHAN Y G, XIAO J L.Synthesis and regulation of triterpene from Betula platyphylla Suk.[J].Plant Physiology Communications,2009,45(5):520-526.
[2] 叶银英, 何道伟, 叶文才,等. 23-羟基白桦酯酸体外抗恶性肿瘤作用的研究[J].东南大学学报(医学版),2001,20(3):141-146. YE Y Y,HE D W,YE W C,et al. The anticancer effect of 23-hydroxyl betulinic acid in vitro[J]. Journal of Nanjing Railway Medical College,2001,20(3):141-146.
[3] 李薇,李岩,金雄杰.白桦三萜类物质的抗肿瘤作用及其对免疫功能的增强效应[J].中国免疫学杂志,2000,16(9):485-490.DOI:10.3321/j.issn:1000-484X.2000.09.008. LI W,LI Y,JIN X J. Antitumor activity and immunoregulatory effect of triterpenes isolated from Betula platyphylla[J]. Chinese Journal of Immunology,2000,16(9):485-490.
[4] 李岩,金雄杰,谢湘林,等.白桦三萜类物质抗黑色素瘤B16、S180肉瘤作用及其机制的实验研究[J].中国药理学通报,2000,16(3):279-281.DOI:10.3321/j.issn:1001-1078.2000.03.012. LI Y,JIN X J,XIE X L,et al.Inhibitory effects of TFM on L-thyroxine-inducedheart hypertrophy in rats[J].Chinese Pharmacological Bulletin,2000,16(3):279-281.
[5] FUJIOKA T,KASHIWADA Y,KILKUSKIE R E,et al.Anti-AIDS agents.11.Betulinic acid and platanic acid as anti-HIV principles from Syzigium claviflorum, and the anti-HIV activity of structurally related triterpenoids[J]. J Nat Prod(Lloydia),1994,57(2):243-247.DOI:10.1021/np50104a008.
[6] FULDA S,JEREMIAS I,STEINER H H,et al. Betulinc acid:a new cytotoxio-agent against malignant brain-tumor cells Cancer[J]. View issue TOC,1999,82(3):435-441. DOI:10.1002/(SIII)1097-0215(19990730)
[7] FALAMAS A,PINZARU C S,DEHELEAN C A,et al. Betulin and its natural resource as potential anticancer drug candidate seen by FT-Raman and FT-IR spectroscopy[J]. Raman Spectrosc, 2011,42(1): 97-107. DOI:10.1002/jrs.2658.
[8] 李春晓,尹静,詹亚光,等. 水分、氮肥及 MeJA处理对白桦三萜积累特性的影响[J].西北植物学报,2012,32(1):155-161. LI C X,YIN J,ZHAN Y G,et al. Effects of water,nitrogen and methyl jasmonate treatment on triterpenes accumulation in birch(Betula platyphylla Suk.)[J]. Acta Botanica Boreali-Occidentalia Sinica,2012,32(1):155-161.
[9] 牛义岭,姜秀明,许向阳.植物转录因子MYB基因家族的研究进展[J].分子植物育种,2016,14(8):2050-2059. DOI:10.13271/j.mpb.014.002050. NIU Y L,JIANG X M,XU X Y. Reaserch advances on transcription factor MYB gene family in plant[J].Molecular Plant Breeding,2016,14(8):2050-2059.
[10] TIAN C Q,HUANG H,SU S S,et al. Regulation of Jasmonate-mediated stamen development and seed production by a bHLH-MYB complex in Arabidopsis[J]. Plant Cell,2015,27(6):1620-1633. DOI:10.1105/tpc.15.00116.
[11] SHENG N L,WEN Y W,JIN L G,et al. MYB75 Phosphorylation by MPK4 is required for light-induced anthocyanin accumulation in Arabidopsis[J]. Plant Cell, 2016, 28(11):2866-2883. DOI:10.1105/tpc.16.0013.
[12] 邢文,金晓玲.调控植物类黄酮生物合成的MYB转录因子研究进展[J].分子植物育种,2015,13(3):689-696. DOI:10.13271/j.mpb.013.000689. XING W,JIN X L. Recent advances of MYB transcription factors involved in the regulation of flavonoid biosynthesis [J].Molecular Plant Breeding,2015,13(3):689-696.
[13] 雒晓鹏,朱冬寅,黄云吉,等.茉莉酸甲酯对芽期苦荞黄酮积累及相关基因表达的影响[J].基因组学与应用生物学,2015,34(5):1040-1046. DOI:10.13417/j.gab.034.001040. LUO X P,ZHU D Y,HUANG Y J,et al. Effects of methyl jasmonate accumulation of flavonoids and related gene expression of buckwheat sprouts[J]. Genomics and Applied Biology,2015,34(5):1040-1046.
[14] 马杰,胡文忠,毕阳,等.茉莉酸甲酯处理对鲜切莴苣和甘蓝苯丙烷代谢的影响[J].食品工业科技,2013,34(7):333-339. DOI:10.13386/j.issn1002-0306.2013.07.086. MA J,HU W Z,BI Y,et al. Effect of MeJA treatments on benzene propane metabolism in tissues of fresh-cut lettuce and cabbage [J]. Science and Technology of Food Industry,2013,34(7):333-339.
[15] 常志凯,朱珠,董恒,等.茉莉酸甲酯结合高温胁迫对白桦悬浮细胞三萜合成的影响[J].中草药,2016,47(13):2333-2340. DOI: 10.7501/j.issn.0253-2670.2016.13.022. CHANG Z K,ZHU Z,DONG H,et al. Effect of MeJA combined with high temperature stress in treatment for accumulation of triterpenoids in birch suspension cells[J]. Chinese Traditional and Herbal Drugs,2016,47(13):2333-2340.
[16] 李光哲,李玲美,姚展鹏,等.乙烯和水杨酸信号转导在拟南芥对聚乙二醇胁迫应答中的相互作用[J].沈阳师范大学学报(自然科学版),2017,35(2):214-219. DOI:10.3969/j.issn.1673-5862.2017.02.018. LI G Z,LI L M,YAO Z P,et al. Interaction between ethylene and salicylic acid-signaling in Arabidopsis response to polyethylene glycol stress[J].Journal of Shenyang Normal University(Natural Science Edition),2017,35(2):214-219.
[17] 王艳,尹静,马泓思,等.钙离子在介导 SA 诱导白桦悬浮细胞三萜合成途径中的作用[J]. 北京林业大学学报,2014,36(2):51-58. DOI:10.13332/j.cnki.jbfu.2014.02.013. WANG Y, YIN J, MA H S, et al. Role of calcium ion in mediating the triterpenoid synthesis induced by SA in suspension cells of Betula platyphylla[J].Journal of Beijing Forestry University,2014,36(2):51-58.
[18] 王艳.钙离子在SA和MeJA诱导白桦三萜合成中的作用及机制[D].哈尔滨: 东北林业大学,2014. WANG Y. The role and mechanism of Ca²⁺ in SA and Me JA induce the synthesis of birch triterpenoids[D].Harbin: Northeast Forestry University,2014.
[19] 李春晓. MeJA和SA对白桦幼树三萜合成调控及FPS基因克隆[D].哈尔滨: 东北林业大学,2012. LI C X. Study on triterpenoid biosynthesis regulation in birch saplings by Me JA and SA and the key enzyme gene FPS cloning[D]. Harbin: Northeast Forestry University,2012.
[20] PFAFFL M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Research,2001,29(9): 2002-2007.DOI: 10.1093/nar/29.9.e45.
[21] DUBOS C, STRACKE R, GROTEWOLD E, et al. MYB transcription factors in Arabidopsis[J]. Trends Plant Sci, 2010, 15(10): 573-581. DOI:10.1016/j.tplants.2010.06.005.
[22] STRACKE R, WERBER M, WEISSHAAR B. The R2R3-MYB gene family in Arabidopsis thaliana[J]. Curr Opin Plant Biol, 2001, 4(5): 447-456. DOI:10.1016/s1369-5266(00)00199-0.
[23] 李晓波,张俊武. 真核生物中锌指蛋白的结构与功能[J]. 中国生物化学与分子生物学报,2009,25(3):206-211. DOI:10.13865/j.cnki.cjbmb.2009.03.016. LI X B, ZHANG J W. Structure and function of zinc finger proteins in eucaryon[J]. Chinese Journal of Biochemistry and Molecular Biology,2009,25(3):206-211.
[24] BOYER L A, LATEK R R, PETERSON C L. The SANT domain: a unique histone-tail-binding module[J]. Nat Rev Mol Cell Biol, 2004, 5(2): 158-163. DOI:10.1038/nrm1314.
[25] 李欣,李影,曲子越,等. bHLH转录因子在茉莉酸信号诱导植物次生产物合成中的作用及分子机制[J]. 植物生理学报,2017,53(1): 1-8. DOI:10.13592/j.cnki.ppj.2016.0369. LI X,LI Y,QU Z Y,et al. bHLH transcription factors in the role of jasmonic acid signal induced plant secondary synthesis and molecular mechanism [J].Plant Physiology Journal,2017,53(1): 1-8.
[26] 马泓思,潘亚婕,王艳,等.Ca²⁺在介导MeJA诱导白桦悬浮培养三萜合成中的作用[J]. 植物研究,2015,35(1): 117-126. DOI:10.7525/j.issn.1673-5102.2015.01.018 MA H S,PAN Y J,WANG Y, et al. Effect of Ca²⁺ on mediating the Me JA-induced synthesis of triterpenoid in suspension cells of Betula platyphylla Suk. [J]. Bulletin of Botanical Research,2015, 35(1): 117-126.
[27] YIN J,LI C, ZHAN Y, et al. The response of physiological characteristics,expression of OSC genes,and accumulation of triterpenoids in Betula platyphylla Suk. to MeJA and SA treatment[J]. Plant Mol Biol Rep, 2016,34(2),427-439.DOI:10.1007/s11105-015-0931-5.
[28] ZHANG M,WANG S,YIN J,et al. Molecular cloning and promoter analysis of squalene synthase and squalene epoxidase genes from Betula platyphylla[J].Proto-plasma,2016,253(5): 1347-1363. DOI:10.1007/s00709-015-0893-3.
[29] CHATEL G,MONTIEL G,PRÉ M,et al. Cr MYC1,a Catharanthus roseus elicitor-and jasmonate-responsive bHLH transcription factor that binds the G-box element of the strictosidine synthase gene promoter[J]. J Exp Bo,2003,54(392): 2587-2588. DOI:10.1093/jxb/erg275.
[30] HUANG X S,WANG W,ZHANG Q,et al. A basic helix-loop-he-lix transcription factor,Ptrb HLH,of Poncirus trifoliata confers cold tolerance and modulates peroxidase-mediated scavenging of hydrogen peroxide[J]. Plant Physiol,2013,162(2): 1178-1194. DOI: 10.1104/pp.112.210740.
[31] XU W,DUBOS C,LEPINIEC L. Transcriptional control of flavo-noid biosynthesis by MYB-bHLH-WDR complexes[J]. Trends Plant Sci,2015,20(3): 176-185. DOI: 10.1111/nph.12620.
[32] AFRIN S,NURUZZAMAN M,ZHU J,et al. Combinatorial inter-actions of MYB and bHLH in flavonoid biosynthesis and their function in plants[J]. Plant Res,2015,3(2): 1123-1129. DOI: 10.1016/j.tplants.2014.12.001.
[33] BULGAKOV V P,VEREMEICHIK G N,GRIGORCHUK V P,et al. The rolB gene activates secondary metabolism in Arabidopsis calli via selective activation of genes encoding MYB and b HLH transcription factors[J]. Plant Physiol Bioch,2016,102: 70-79. DOI: 10.1016/j.plantly.2016.02.015.
[34] WANG F, ZHU H,CHEN D,et al. A grape bHLH transcription factor gene,VvbHLH1,increases the accumulation of flavonoids and enhances salt and drought tolerance in transgenic Arabidopsis thaliana[J]. Plant Cell Tiss Organ Cult,2016,125(2): 387-398. DOI:10.1007/s11240-416-0953-1.
[35] LICAUSI F,GIORGI FM,ZENONI S,et al. Genomic and transcriptomic analysis of the AP2/ERF superfam-ily in Vitis vinifera[J]. BMC Genomics,2010,11(1): 719. DOI:10.1186/1471-2164-11-719.
[36] BAUDRY A,HEIM M A,DUBREUCQ B,et al. TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesiss in Arabidopsis thaliana[J]. Plant J,2004,39(3): 366-380. DOI:10.1111/j.1365-313X.2004.02138.x.
[37] LI H,FLACHOWSKY H,FISCHER T C,et al. Maize Lc transcription factor enhances biosynthesis of anthocyanins,dis-tinct proanthocyanidins and phenylpropanoids in apple(Malus domestica Borkh.)[J]. Planta,2007,226(5): 1243-1254. DOI:10.1007/s00425-007-0573-4.
[38] FERNÁNDEZ-CALVO P, CHINI A,FERNÁNDEZ-BARBERO G,et al. The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jas-monate responses[J]. Plant Cell,2011,23(2): 701-715. DOI:10.1105/tpc.110.080788.
[39] BOTER M,GOLZ J F,GIMÉNEZ-iBAÑEZ S,et al. FILAMENTOUS FLOWER is a direct target of JAZ3 and modulates responses to jasmonate[J]. Plant Cell, 2015,27(11): 3160-3174. DOI:10.1105/tpc.15.00220.
[40] 赵恒伟,葛锋,孙颖,等.植物萜类物质生物合成的相关转录因子及其应用前景[J].中草药,2012,43(12):2512-2519. ZHAO H W,GE F,SUN Y,et al. Transcription factors involved in plant terpenoid biosynthesis and their application prospect[J]. Chinese Traditional and Herbal Drugs,2012,43(12):2512-2519.
[41] 刘翔,左开井,张飞,等.MYB 类转录因子在植物腺毛发育中的作用研究进展[J]. 上海交通大学学报(农业科学版),2010,28(2):188-194. DOI:10.3969/j.issn.1671-9964.2010.02.018. LIU X,ZUO K J,ZHANG F,et al. Progress on the roles of MYB transcription factors on plant trichome development[J]. Journal of Shanghai Jiaotong University(Agricul Tural Science),2010,28(2):188-194.
[42] 李欣.白桦BpMYC4和BpbHLH9基因的克隆、表达特性及功能研究[D]. 哈尔滨: 东北林业大学,2017. LI X. Molecular cloning, expression characteristics,and function of BpMYC4 and BpbHLH9 genes from birch(Betula platyphylla Suk.)[D].Harbin: Northeast Forestry University,2017.
[43] 谭何新,肖玲,周正,等.青蒿素生物合成分子机制及调控研究进展[J].中国中药杂志,2017,42(1):10-19. DOI:10.19540/j.cnki.cjcmm.20161222.019. TAN H X,XIAO L,ZHOU Z,et al. Molecular mechanism of artemisinin biosynthesis and regulation in Artemisia annua[J].China Journal of Chinese Materia Medica,2017,42(1):10-19.
[44] 葛楠,赵凯,赵立斐,等. 红豆杉细胞培养合成紫杉醇的代谢调控研究进展[J]. 黑龙江医药,2008,21(1):16-19. DOI:10.14035 /j.cnki.hljyy.2008.01.001. GE N,ZHAO K,ZHAO L F,et al. Research progress in metabolic regulation of taxol production by Taxus cell culture[J].Heilongjiang Medicine Journal,2008,21(1):16-19.
[45 ]OHNO S,HOSOKAWA M,HOSHINO A, et al. A bHLH transcription factor,DvIVS, is involved in regulation of anthocyanin synthesis in dahlia(Dahlia variabilis)[J]. J Exp Bot,2011, 62(14): 5105-5116. DOI: 10.1093/jxb/err216.
[46] CARRETERO-PAULET L,GALSTYAN A,ROIG-VILLANOVA I,et al. Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae[J]. J Plant Physiol,2010,153(3): 1398-1412. DOI:10.1104/pp.110.153593.
[47] FELLER A,MACHEMER K,BRAUN E L,et al. Evolutionary and comparative analysis of MYB and bHLH plant transcription factors[J]. Plant J, 2011,66(1): 94-116. DOI: 10.1111/j.1365-313X.2010.04459.x.
[48] CHICO J M,FERNÁNDEZ-BARBERO G,CHINI A, et al. Repression of jasmonate-dependent of MYC transcription factors and their JAZ repressors in Arabidopsis[J]. Plant Cell,2014,26(5): 1967-1980. DOI:10.1105/tpc.114.125047.