作物栽培·遗传育种

转录因子NtMYC2b参与烟草烟碱含量杂种优势形成的机制研究

  • 吴宇瑶 ,
  • 谢瑞 ,
  • 杨友成 ,
  • 刘坤华 ,
  • 聂琼
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  • 1.贵州大学 农学院,贵州 贵阳 550025;2.贵州大学 贵州省烟草品种研究重点实验室,贵州 贵阳 550025;3.遵义市农业科学研究院,贵州 遵义563000;4.遵义市烟草公司,贵州遵义 563000)
吴宇瑶(1993-),女,贵州黎平人,在读硕士研究生,研究方向:烟草遗传育种。E-mail:15761630170@163.com

收稿日期: 2019-05-22

  网络出版日期: 2019-11-15

基金资助

贵州省烟草公司项目(黔烟科201302);贵州省烟草公司重大专项(黔烟科201602);贵州省烟草公司遵义市公司项目(遵烟计[2016]07号);贵州省烟草品质遗传改良与生物转化科技创新人才团队项目(黔科合人才团队[2015]4001)

 Transcription Factor NtMYC2b Is Involved in the Formation of Heterosis of Nicotine Content Trait

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  •  ( 1.College of Agriculture,Guizhou University,Guiyang 550025,China; 2.Guizhou Provincial Key Laboratory of Tobacco Variety Research,Guizhou University,Guiyang 550025,China; 3.Zunyi Academy of Agricultural Science,Zunyi 563000,China; 4. Zunyi Tobacco Company,Zunyi 563000,China)

Received date: 2019-05-22

  Online published: 2019-11-15

摘要

为探讨bHLH转录因子家族成员MYC2b基因与烟叶中烟碱含量及烟碱含量杂种优势表现的相关性,利用RT-qPCR分析NtMYC2b基因与烟碱合成途径关键酶基因的表达,测定烟碱含量和烟碱杂种优势表现。结果表明,移栽后80 d,NtMYC2b在烟草亲本GDH88和巴斯玛根部的表达量较移栽后66 d上调,其根和叶中烟碱含量增加,杂交种根中NtMYC2b、鸟氨酸脱羧酶基因(ODC)、精氨酸脱羧酶基因(ADC)、N-甲基转移酶基因(PMT)、N-甲基腐胺氧化酶基因(MPO)的表达量相对于亲本平均表达量均明显上调,且除MPO外,NtMYC2bODC、ADC、PMT、QPT(喹啉酸磷酸核糖转移酶基因)在强优势组合VA116×巴斯玛中的相对表达量均明显高于弱优势组合VA116×GDH88,根和叶中烟碱含量及其杂种优势表现也具有相同的趋势,表明NtMYC2b参与了烟碱的合成,还可能参与了烟碱含量杂种优势的形成。相关性分析表明,在亲本根部,NtMYC2b表达量与PMT表达量呈显著正相关;在杂交种根部,NtMYC2b表达量与ADC、MPO表达量呈显著正相关;在杂交种叶片中,NtMYC2b表达量与烟碱含量杂种优势呈显著正相关,PMT表达量与ODC、ADC表达量均呈显著正相关。关键酶基因启动子序列分析表明,ADC中含有1个MYC结合位点和1个G-box元件;PMT中含有7个MYC结合位点和1个G-box元件。推测NtMYC2b可能通过与ADC、PMT的顺式元件MYC结合位点或G-box元件特异性结合调控ADC、PMT的表达,进而促进烟碱的合成,且NtMYC2b还参与了烟碱含量杂种优势的形成。

本文引用格式

吴宇瑶 , 谢瑞 , 杨友成 , 刘坤华 , 聂琼 . 转录因子NtMYC2b参与烟草烟碱含量杂种优势形成的机制研究[J]. 河南农业科学, 2019 , 48(11) : 45 -53 . DOI: 10.15933/j.cnki.1004-3268.2019.11.007

Abstract

To investigate the correlation between transcription factor MYC2b and nicotine content and its heterosis in tobacco,RT-qPCR was used to analyze the expression of NtMYC2b and key enzyme genes in the nicotine synthesis pathway,and the nicotine content and its heterosis in roots and leaves of tobacco were determinated.The results showed that the expression of NtMYC2b in parent roots(GDH88 and Bas ma) was upregulated at 80 d compared with 66 d after transplanting,and their nicotine content in root and leaves was also increased.The expressions of NtMYC2b,ornithine decarboxylase gene(ODC),arginine de carboxylase gene(ADC) ,putrescine-N-methyltransferase gene (PMT) and N-methylputrescine oxidase gene(MPO) were significantly upregulated in root of hybrid relative to their parents.The relative expres sion levels of NtMYC2b,ODC,ADC,PMT and QPT(quinoline phosphoribosyltransferase gene) in the strong heterosis combination(VA116 × Basma) were significantly higher than those in the weak heterosis combination(VA116 × GDH88) ,meanwhile,the nicotine content and its heterosis also showed the same trend in roots and leaves of hybrid.It indicated that NtMYC2b was involved in the synthesis of nicotine and might also be involved in the formation of heterosis of nicotine content.Correlation analysis showed that the expression of NtMYC2b was significantly positively correlated with PMT in the parent roots,the same with ADC and MPO in hybrid roots.Moreover,between NtMYC2b expression and nicotinic content heterosis,between PMT expression and ODC and ADC expression,was also a significant positive correla tion in hybrid leaves.Sequence analysis showed that the promoter sequence of ADC contained 1 MYC binding site and 1 G-box element,the promoter sequence of PMT contained 7 MYC binding sites and 1 G box element.Therefore,it is speculated that NtMYC2b may regulate the expression of ADC and PMT by specifically binding with cis-element MYC binding site or G-box elementto promote the synthesis of nico tine.These findings suggest that NtMYC2b is involved in the synthesis of nicotine and the formation of het erosis in nicotine content.

参考文献

[1]ROEDER R G.The role of general initiation factors in transcription by RNA polymeraseⅡ[J].Trends in Biochemical Sciences,1996,21(9):327-335.
2]LEE T I,YOUNG R A.Transcription of eukaryotic proteincoding genes[J].Annual Review of Genetics,2000,34(1):77-137.
3]李罡,李文龙,许雪梅,等.MYC2转录因子参与植物发育调控的研究进展[J].植物生理学报,2019,55(2):125-132.
4]张麒,陈静,李俐,等.植物AP2/ERF转录因子家族的研究进展[J].生物技术通报,2018,34(8):1-7.
5]LIU L,WHITE M J,MACRAE T H.Transcription factors and their genes in higher plantsfunctional domains,evolution and regulation[J].European Journal of Biochemistry,1999,262(2):247-257.
6]LUTOVA L A,DODUEVA I E,LEBEDEVA M A,et al.Transcription factors in developmental genetics and the evolution of higher plants[J].Russian Journal of Genetics,2015,51(5):449-466.
7]RUSHTON P J,BOKOWIEC M T,HAN S,et al.Tobacco transcription factors:Novel insights into transcriptional regulation in the solanaceae[J].Plant Physiology,2008,147(1):280-295.
8]闫宁,张洪博.烟草重要基因篇:10.烟草转录因子基因[J].中国烟草科学,2015,36(4):117-120.
9]ZHANG H B,BOKOWIEC M T,RUSHTON P J,et al.Tobacco transcription factors NtMYC2a and NtMYC2b form nuclear complexes with the NtJAZ1 repressor and regulate multiple jasmonateinducible steps in nicotine biosynthesis[J].Molecular Plant,2012,5(1):73-84.
10]HAKKINEN S T,TILLEMAN S,WIATEK A,et al.Functional characterisation of genes involved in pyridine alkaloid biosynthesis intobacco[J].Phytochemistry,2007,68(22/24):2773-2785.
11]MARTON L J,PEGG A E.Polyamines as targets for therapeutic intervention[J].Annu Rev Pharmacol Toxicol,1995,35(1):55-91.
12]TIBURCIO A F,GALSTON A W.Arginine decarboxylase as the source of putrescine for tobacco alkaloids[J].Phytochemistry,1985,25(1):107-110.
13]SAUNDERS J W,BUSH L P.Nicotine biosynthetic enzyme activities in Nicotiana tabacum L.genotypes with different alkaloid levels[J].Plant Physiology,1979,64(2):236-240.14]FETH F,WAGNER R,WAGNER K G.Regulation in tobacco callus of enzyme activities of the nicotine pathway[J].Planta,1986,168(3):402-407.
15]SATO F.Inaugural article:Metabolic engineering of plant alkaloid biosynthesis[J].Proceedings of the National Academy of Sciences,2001,98(1):367-372.
16]CHINTAPAKORN Y,HAMILL J D.Antisensemediated downregulation of putrescine Nmethyltransferase activity in transgenic Nicotiana tabacum L.can lead to elevated levels of anatabine at the expense of nicotine[J].Plant Molecular Biology,2003,53(1/2):87-105.
17]黄新杰,李章海,王能如,等.中国主要烟区烟叶烟碱含量差异分析[J].湖南农业科学,2006(5):33-36.
18]张翼翾,张一宾.新烟碱类杀虫剂新用途的探索:对噻虫嗪作为多杀菌素增效剂的研究[J].世界农药,2013,35(4):23-26,33.
19]李晓敏,李静静,汤清波,等.烟碱对B型和Q型烟粉虱取食行为的影响:基于EPG和液体饲囊技术体系[J].中国农业科学,2013,46(10):2041-2049.
20]徐国华.烟碱的提取及在生物农药中的应用[D].青岛:青岛大学,2007.
21]刘增兵,姜景彬,杨欢欢,等.植物杂种优势的研究进展[J].分子植物育种,2019,17(12):4127-4134.
22]BUTELLI E,TITTA L,GIORGIO M,et al.Enrichment of tomato fruit with healthpromoting anthocyanins by expression of select transcription factors[J].Nature Biotechnology,2008,26(11):1301-1308.
23]CUTANDAPEREZ M C,AGEORGES A,GOMEZ C A,et al.Ectopic expression of VlmybA1 in grapevine activates a narrow set of genes involved in anthocyanin synthesis and transport[J].Plant Molecular Biology,2009,69(6):633-648.
24]杨堤贻,张子敬,王大会,等.转录因子KLF家族的结构、功能及调控机制研究进展[J].河南农业科学,2019,48(7):1-5.
25]李春丽,张前前,王磊,等.应用吸光度比值法识别硅藻和甲藻[J].中国海洋大学学报(自然科学版),2007,37(S2):161-164.
26]谢瑞.烟碱合成相关转录因子基因NtMYC2s的克隆、表达与SNP分析[D].贵阳:贵州大学,2018.
27]ZHANG H T,HEDHILI S,MONTIEL G,et al.The basic helixloophelix transcription factor CrMYC2 controls the jasmonateresponsive expression of the ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus[J].Plant Journal,2011,67(1):6171.
28]PIRES N,DOLAN L.Origin and diversification of basichelixloophelix proteins in plants[J].Molecular Biology & Evolution,2010,27(4):862874.
29]GASPERINI D,CHETELAT A,ACOSTA I F,et al.Multilayered organization of jasmonate signalling in the regulation of root growth[J].PLoS Genetics,2015,11(6):e1005300.
30]QI T,HUANG H,SONG S,et al.Regulation of jasmonatemediated stamen development and seed production by a bHLHMYB complex in Arabidopsis[J].The Plant Cell,2015,27(6):1620-1633.
31]LI T,XU Y X,ZHANG L C,et al.The jasmonateactivated transcription factor MdMYC2 regulates ethylene response factor and ethylene biosynthetic genes to promote ethylene biosynthesis during apple fruit ripening[J].The Plant Cell,2017,29(6):1316-1334.
32]GAO C H,QI S H,LIU K G,et al.MYC2,MYC3,and MYC4 function redundantly in seed storage protein accumulation in Arabidopsis[J].Plant Physiology and Biochemistry,2016,108:63-70.
33]HUANG C F,YU C P,WU Y H,et al.Elevated auxin biosynthesis and transport underlie high vein density in C4 leaves[J].Proceedings of the National Academy of Sciences of the United States of America,2017,114(33):E6884-E6891.
34]黄劭理.茎部环割处理对烤烟生长及烟碱和钾素运输与分配的影响[D].长沙:湖南农业大学,2015.
35]赵阳,秦智伟,周秀艳,等.植物杂种优势预测研究进展[J].北方园艺,2018(8):172-180.
36]SHULL G H.The composition of a field of maize[J].Journal of Heredity,1908,1(4):296-301.
37]BRUCE A B.The mendelian theory of heredity and the augmentation of vigor [J].Science,1910,32(827):627-628.
38]SCHNELL F W,COCKERHAM C C.Multiplicative vs.arbitrary gene action in heterosis[J].Genetics,1992,131(2):461-469.
39]KATOH A.Early steps in the biosynthesis of NAD in arabidopsis start with aspartate and occur in the plastid[J].Plant Physiology,2006,141(3):851-857.
40]IMANISHI S,HASHIZUME K,NAKAKITA M,et al.Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures[J].Plant Molecular Biology,1998,38(6):1101-1111.
41]HIBI N,HIGASHIGUCHI S,HASHIMOTO T,et al.Gene expression in tobacco lownicotine mutants [J].Plant Cell,1994,6(5):723-735.
42]KATOH A,SHOJI T,HASHIMOTO T.Molecular cloning of Nmethylputrescine oxidase from tobacco[J].Plant and Cell Physiology,2007,48(3):550-554.
43]TIAN M Z,NIE Q,LI Z H,et al.Transcriptomic analysis reveals overdominance playing a critical role in nicotine heterosis in Nicotiana tabacum L.[J].BMC Plant Biology,2018,18(1):48.
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