文心兰15个miRNAs及其候选靶标的表达特性
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摘要
为了解miRNAs及其候选靶标在文心兰不同组织部位及假鳞茎受软腐病病原菌侵染中的表达规律,基于文心兰转录组和miRNA数据库进行分析,从中筛选获得15个miRNAs,结合生物信息学法对其候选靶标进行预测和功能注释,并利用实时荧光定量技术检测miRNA及候选靶标在文心兰不同组织部位和假鳞茎受软腐病病原菌侵染的表达情况.结果表明,文心兰miRNA数据库的miRNA reads分析提示15个miRNAs不同成员可能在文心兰正常植株和感病不同阶段中差异表达. psRNATarget靶标预测显示,文心兰15个miRNAs有828个候选靶标,其中67个可能与抗病相关,包括丝氨酸/苏氨酸蛋白、F-box蛋白基因、锌指蛋白、RGA抗病蛋白等.基于文心兰miRNAs读取次数(Reads)和Unigene候选靶标RPKM值,发现miRNAs及其候选靶标在文心兰正常植株和假鳞茎不同感病时段表达均存在差异.实时荧光定量PCR分析显示,在文心兰不同组织部位中,miR159a、miR167b、miR168a、miR169a、miR171a和miR172a均在假鳞茎和叶中均大量表达,并与对应候选靶标的表达呈负调控关系,推测它们参与假鳞茎和叶的发育和形态建成.在软腐病病菌侵染文心兰假鳞茎中,miR159a、miR168a、miR169a、miR171a和miR172a在8 h处理时表达量高,并且与对应的候选靶标在侵染0-8 h表达呈负调控关系,推测上述miRNAs通过负调控候选靶标在中期响应软腐病病原菌侵染过程;而miR167b在处理0h高表达,并且与候选靶标在侵染0-8h表达呈负调控关系,推测其通过下调表达参与软腐病病原菌侵染过程的响应.上述研究表明,文心兰miRNAs通过介导候选靶标的裂解可能广泛参与文心兰不同组织的发育及软腐病病原菌侵染过程的响应.
This study aimed to investigate the response mode of miRNA and candidate target genes in the disease resistance process of Oncidium. The transcriptome data and miRNA database of Oncidium leaves infected by soft rot(normal, mild,and severe infection) pathogens were analyzed. Subsequently, 15 miRNAs were screened out, and their candidate targets and functional annotations were predicted by bioinformatics analysis. A real-time quantitative PCR analysis was applied to clarify the expression of miRNAs and their candidate targets in different tissues and pseudobulbs infected by soft rot. An miRNA read analysis based on the Oncidium miRNA database suggested that different miRNAs might have different functions in normal plants and in different stages of Oncidium susceptibility. Using the psRNATarget online software to predict the candidate targets of Oncidium miRNAs, we found 828 candidate targets for 15 miRNAs in Oncidium; among them, 67 targets,including serine/threonine-protein, F-box protein gene, zinc finger protein, resistance gene analog(RGA) protein, might be related to disease resistance. Based on the miRNA reads and the reads per kilobase per million mapped reads(RPKM) values of candidate targets, the miRNAs and their candidate targets were found to be differentially expressed in normal plants and in different susceptible periods of Oncidium. A real-time quantitative PCR analysis showed that miR159 a, miR167b, miR168a,miR169a, miR171a, and miR172a were abundantly expressed in the pseudobulbs and leaves of Oncidium and were negatively correlated with the expression of the corresponding candidate targets, suggesting that they are involved in the development and morphogenesis of pseudobulbs and leaves. In the Oncidium pseudobulbs infected by soft rot pathogen, the miRNAs miR159a,miR168a, miR169a, miR171a, and miR172a were highly expressed at 8 h and showed a negatively regulated relationship with the expression of the corresponding candidate targets at 0–8 h of infection. Thus, it was speculated that the above miRNAs respond to the infection process of the soft rot pathogens in the medium term through negatively regulated candidate targets.However, miR167b was highly expressed at 0 h of infection and showed negatively regulated relationship with candidate targets at 0–8 h of infection, suggesting that it is involved in the response of soft rot pathogen infected process by down-regulating its expression. The above research shows that miRNAs might be widely involved in the development of different tissues of Oncidium by mediating the cleavage of candidate targets and response of the infection process of soft rot pathogens.
This study aimed to investigate the response mode of miRNA and candidate target genes in the disease resistance process of Oncidium. The transcriptome data and miRNA database of Oncidium leaves infected by soft rot(normal, mild,and severe infection) pathogens were analyzed. Subsequently, 15 miRNAs were screened out, and their candidate targets and functional annotations were predicted by bioinformatics analysis. A real-time quantitative PCR analysis was applied to clarify the expression of miRNAs and their candidate targets in different tissues and pseudobulbs infected by soft rot. An miRNA read analysis based on the Oncidium miRNA database suggested that different miRNAs might have different functions in normal plants and in different stages of Oncidium susceptibility. Using the psRNATarget online software to predict the candidate targets of Oncidium miRNAs, we found 828 candidate targets for 15 miRNAs in Oncidium; among them, 67 targets,including serine/threonine-protein, F-box protein gene, zinc finger protein, resistance gene analog(RGA) protein, might be related to disease resistance. Based on the miRNA reads and the reads per kilobase per million mapped reads(RPKM) values of candidate targets, the miRNAs and their candidate targets were found to be differentially expressed in normal plants and in different susceptible periods of Oncidium. A real-time quantitative PCR analysis showed that miR159 a, miR167b, miR168a,miR169a, miR171a, and miR172a were abundantly expressed in the pseudobulbs and leaves of Oncidium and were negatively correlated with the expression of the corresponding candidate targets, suggesting that they are involved in the development and morphogenesis of pseudobulbs and leaves. In the Oncidium pseudobulbs infected by soft rot pathogen, the miRNAs miR159a,miR168a, miR169a, miR171a, and miR172a were highly expressed at 8 h and showed a negatively regulated relationship with the expression of the corresponding candidate targets at 0–8 h of infection. Thus, it was speculated that the above miRNAs respond to the infection process of the soft rot pathogens in the medium term through negatively regulated candidate targets.However, miR167b was highly expressed at 0 h of infection and showed negatively regulated relationship with candidate targets at 0–8 h of infection, suggesting that it is involved in the response of soft rot pathogen infected process by down-regulating its expression. The above research shows that miRNAs might be widely involved in the development of different tissues of Oncidium by mediating the cleavage of candidate targets and response of the infection process of soft rot pathogens.
引文
1 You SJ,Liau CH,Huang HE,Feng TY,Prasad V,Hsiao HH,Lu JC,Chan MT.Sweet pepper ferredoxin-like protein(pflp)gene as a novel selection marker for orchid transformation[J].Planta,2003,217(1):60-65
2叶炜,林玉玲,匡云波,江金兰,李永清,雷伏贵,赖忠雄.文心兰转化龙眼不同类型铁氧还蛋白基因提高抗病作用分析[J].福建农业学报,2015(9):850-855[Ye W,Lin YL,Kuang YB,Jiang JL,Li YQ,Lei FG,Lai ZX.Transformation of different types of ferredoxin gene from longan into Oncidium hybridum to improve disease resistance[J].JFujian Agric,2015(9):850-855]
3 Carrington JC,Ambros V.Role of microRNAs in plant and animal development[J].Science,2003,301(5631):336-338
4 Bar tel,Bonnie,Bar tel,David P.MicroRNAs:at the root of plant development?[J].Plant Physiol,2003,132(2):709-17
5 Rein har t BJ,Weinstein EG,R hoades M W,Bar tel B,Bar tel DP.MicroRNAs in plants[J].G D,2002,16(13):1616
6 Llave C,Carrington JC.Endogenous and silencing-associated small RNAs in plants[J].Plant Cell,2002,14(7):1605-1619
7 Park W,Li J,Song R,Messing J,Chen X.CARPEL FACTORY,a Dicer homolog,and HEN1,a novel protein,act in microRNA metabolism in Arabidopsis thaliana[J].Curr Biol,2002,12(17):1484-1495
8 Bartel DP.MicroRNAs:target recognition and regulatory functions[J].Cell,2009,136(2):215-233
9 Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297
10 Gao P,Bai X,Yang L,Lv D,Pan X,Li Y.Osa-miR393:a salinity and alkaline stress-related microRNA gene[J].Mol Biol Rep,2011,38(1):237
11éva V,Válóczi A,ákosá,Burgyán J,Havelda Z.Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1accumulation[J].Embo J,2010,29(20):3507-3519
12 Navarro L,Bari R,Seilaniantz A,Nemri A,Jones J DG.Roles of Plant Hormones in Plant Resistance and Susceptibility to Pathogens[M].Gen Dis,2008:1-10
13 Charkowski AO.Decaying signals:will understanding bacterial plant communications lead to control of soft rot[J].Curr Opin Biotechnol,2009,20(2):178-184
14 Sjahril R,Poh CD,Sher KR,Yamamura S,Nakamura I,Amemiya Y.Transgenic phalaenopsis plants with resistance to Erwinia carotovora produced by introducing wasabi defensin gene using Agrobacterium method[J].Plant Biotechnol,2006,23(2):191-194
15 Zhou JN,Lin BR,Shen HF,Pu XM,Chen ZN,Feng JJ.First report of a soft rot of Phalaenopsis aphrodita caused by Dickeya dieffenbachiae in China[J].Plant Dis,2012,96(5):760
16吴晓佩,谢礼洋,白玉,叶炜,林争春,张梓浩,陈裕坤,林玉玲,程春振,赖钟雄.文心兰铁氧还蛋白基因克隆定位及表达分析[J].西北植物学报,2017,37(1):48-58[Wu XP,Xie LY,Bai Y,Ye W,Lin ZC,Zhang ZH,Chen YK,Lin YL,Cheng CZ,Lai ZX.Cloning,subcellular localization and expression analysis of fer redoxin gene OnFd in Oncidium hybridum[J].J NW Bot Sci,2017,37(1):48-58]
17王培育,王丛巧,张舒婷,王雪晶,叶炜,赖钟雄,林玉玲.文心兰25条miRNA前体序列特性及其表达分析[J].西北植物学报,2018,38(9):1587-1597[Wang PY,Wang CQ,Zhang ST,Wang XJ,Ye W,Lai ZX,Lin YL.Sequences characteristics and expression patterns of 25 miRNAprecursors in Oncidium hybridum[J].J NW Bot Sci,2018,38(9):1587-1597]
18 Dai X,Zhao PX.psRNATarget:a plant small RNA target analysis server[J].Nucleic Acids Res,2011,39(Web Server issue):W155
19 Xue T,Liu Z,Dai X,Xiang F.Primary root growth in Arabidopsis thaliana is inhibited by the miR159 mediated repression of MYB33,MYB65 and MYB101[J].Plant Sci,2017,262:182
20赵丽娟,易小娅,曾幼玲.植物逆境相关C2H2型锌指蛋白的研究进展[J].分子植物育种,2016(3):578-585[Zhao LJ,Yi XY,Zeng YL.Advances in research on plant stress related C2H2 zinc finger protein[J].Mol plant,2016(3):578-585]
21蒋正宁,别同德,赵仁惠.受条锈菌诱导的小麦丝氨酸苏氨酸激酶基因TaS/TK的克隆与表达[J].江苏农业学报,2016,32(5):980-986[Jiang ZN,Bie TD,Zhao RH.Cloning and expression of the wheat serine threonine kinase gene TaS/TK induced by stripe rust[J].Jiangsu J Agric Sci,2016,32(5):980-986]
22廖维华,陈仲,叶梅霞,马焕弟,高凯,雷炳琪,安新民.毛白杨真菌胁迫下miRNA靶基因预测和生物信息分析[J].中国细胞生物学学报,2014(11):1506-1513[Liao WH,Chen Z,Ye MX,Ma HD,Gao K,Lei BQ,An XM.Prediction and bioinformatics analysis of miRNA target genes in populus tomentosa under fungus stress[J].Chin J Cell Biol,2014(11):1506-1513]
23王艳芳,赵彦宏,于佳丽,林德华.miRNA在植物病毒基因组中的靶基因预测及分析[J].植物保护,2015(6):132-135[Wang YF,Zhao YH,Yu JL,Lin DH.Target gene prediction and analysis of miRNA in plant virus genome[J].Plant Prot,2015(6):132-135]
24孙平.茶叶儿茶素合成相关m i R N A及靶基因的验证和表达分析[D].福州:福建农林大学,2017[Sun P.Stadies on identification and expression of miRNAs and target genes iovolved in the biosynthesis of catechins in Camellia sinensis[D].Fuzhou:Fujian Agriculture and Forestry University,2017]
25 Palatnik JF,Wollmann H,Schommer C,Schwab R,Boisbouvier J,Rodriguez R.Sequence and expression differences underlie functional specialization of Arabidopsis microRNAs miR159 and miR319[J].Dev Cell,2007,13(1):115-125
26 Palatnik JF,Allen E,Wu X,Schommer C,Schwab R,Carrington JC.Control of leaf morphogenesis by microRNAs[J].Nature,2003,425(6955):257
27 Du Q,Avci U,Li S,Gallegogiraldo L,Pattathil S,Qi L.Activation of miR165b represses AtHB15 expression and induces pith secondary wall development in Arabidopsis[J].Plant J,2015,83(3):388-400
28 Duclercq J,Assoumou Ndong YP,Guerineau F,Sangwan RS,Catterou M.Arabidopsis shoot organogenesis is enhanced by an amino acid change in the ATHB15 transcription factor[J].Plant Biol,2011,13(2):317
29 Glazińska P,Zienkiewicz A,Wojciechowski W,Kopcewicz J.The putative miR172 target gene InAPETALA2-like is involved in the photoperiodic flower induction of Ipomoea nil[J].J Plant Physiol,2009,166(16):1801-1813
30 Schommer C,Palatnik JF,Aggarwal P,Chételat A,Cubas P,Farmer EE.Control of jasmonate biosynthesis and senescence by miR319 targets[J].Plos Biol,2008,6(9):e230
31 Jagadeeswaran G,Zheng Y,Li YF,Shukl LI,Matts J,Hoyt P.Cloning and characterization of small RNAs from Medicago truncatula reveals four novel legume-specific microRNA families[J].New Phytol,2009,184(1):85-98
32 Navarro L,Jones JDG.A plant miRNA contributes to antibacterial resistance by repressing auxin signaling[J].Science,2006,312(5772):436-439
33宋锐.基于生物信息学方法预测玉米抗纹枯病相关m i R NA及功能分析[D].雅安:四川农业大学,2009[Song R.Prediction of Corn Resistance to Rhizoctonia solani based on bioinformatics method and its function analysis[D].Ya’an:Sichuan Agricultural University,2009]
2叶炜,林玉玲,匡云波,江金兰,李永清,雷伏贵,赖忠雄.文心兰转化龙眼不同类型铁氧还蛋白基因提高抗病作用分析[J].福建农业学报,2015(9):850-855[Ye W,Lin YL,Kuang YB,Jiang JL,Li YQ,Lei FG,Lai ZX.Transformation of different types of ferredoxin gene from longan into Oncidium hybridum to improve disease resistance[J].JFujian Agric,2015(9):850-855]
3 Carrington JC,Ambros V.Role of microRNAs in plant and animal development[J].Science,2003,301(5631):336-338
4 Bar tel,Bonnie,Bar tel,David P.MicroRNAs:at the root of plant development?[J].Plant Physiol,2003,132(2):709-17
5 Rein har t BJ,Weinstein EG,R hoades M W,Bar tel B,Bar tel DP.MicroRNAs in plants[J].G D,2002,16(13):1616
6 Llave C,Carrington JC.Endogenous and silencing-associated small RNAs in plants[J].Plant Cell,2002,14(7):1605-1619
7 Park W,Li J,Song R,Messing J,Chen X.CARPEL FACTORY,a Dicer homolog,and HEN1,a novel protein,act in microRNA metabolism in Arabidopsis thaliana[J].Curr Biol,2002,12(17):1484-1495
8 Bartel DP.MicroRNAs:target recognition and regulatory functions[J].Cell,2009,136(2):215-233
9 Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297
10 Gao P,Bai X,Yang L,Lv D,Pan X,Li Y.Osa-miR393:a salinity and alkaline stress-related microRNA gene[J].Mol Biol Rep,2011,38(1):237
11éva V,Válóczi A,ákosá,Burgyán J,Havelda Z.Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1accumulation[J].Embo J,2010,29(20):3507-3519
12 Navarro L,Bari R,Seilaniantz A,Nemri A,Jones J DG.Roles of Plant Hormones in Plant Resistance and Susceptibility to Pathogens[M].Gen Dis,2008:1-10
13 Charkowski AO.Decaying signals:will understanding bacterial plant communications lead to control of soft rot[J].Curr Opin Biotechnol,2009,20(2):178-184
14 Sjahril R,Poh CD,Sher KR,Yamamura S,Nakamura I,Amemiya Y.Transgenic phalaenopsis plants with resistance to Erwinia carotovora produced by introducing wasabi defensin gene using Agrobacterium method[J].Plant Biotechnol,2006,23(2):191-194
15 Zhou JN,Lin BR,Shen HF,Pu XM,Chen ZN,Feng JJ.First report of a soft rot of Phalaenopsis aphrodita caused by Dickeya dieffenbachiae in China[J].Plant Dis,2012,96(5):760
16吴晓佩,谢礼洋,白玉,叶炜,林争春,张梓浩,陈裕坤,林玉玲,程春振,赖钟雄.文心兰铁氧还蛋白基因克隆定位及表达分析[J].西北植物学报,2017,37(1):48-58[Wu XP,Xie LY,Bai Y,Ye W,Lin ZC,Zhang ZH,Chen YK,Lin YL,Cheng CZ,Lai ZX.Cloning,subcellular localization and expression analysis of fer redoxin gene OnFd in Oncidium hybridum[J].J NW Bot Sci,2017,37(1):48-58]
17王培育,王丛巧,张舒婷,王雪晶,叶炜,赖钟雄,林玉玲.文心兰25条miRNA前体序列特性及其表达分析[J].西北植物学报,2018,38(9):1587-1597[Wang PY,Wang CQ,Zhang ST,Wang XJ,Ye W,Lai ZX,Lin YL.Sequences characteristics and expression patterns of 25 miRNAprecursors in Oncidium hybridum[J].J NW Bot Sci,2018,38(9):1587-1597]
18 Dai X,Zhao PX.psRNATarget:a plant small RNA target analysis server[J].Nucleic Acids Res,2011,39(Web Server issue):W155
19 Xue T,Liu Z,Dai X,Xiang F.Primary root growth in Arabidopsis thaliana is inhibited by the miR159 mediated repression of MYB33,MYB65 and MYB101[J].Plant Sci,2017,262:182
20赵丽娟,易小娅,曾幼玲.植物逆境相关C2H2型锌指蛋白的研究进展[J].分子植物育种,2016(3):578-585[Zhao LJ,Yi XY,Zeng YL.Advances in research on plant stress related C2H2 zinc finger protein[J].Mol plant,2016(3):578-585]
21蒋正宁,别同德,赵仁惠.受条锈菌诱导的小麦丝氨酸苏氨酸激酶基因TaS/TK的克隆与表达[J].江苏农业学报,2016,32(5):980-986[Jiang ZN,Bie TD,Zhao RH.Cloning and expression of the wheat serine threonine kinase gene TaS/TK induced by stripe rust[J].Jiangsu J Agric Sci,2016,32(5):980-986]
22廖维华,陈仲,叶梅霞,马焕弟,高凯,雷炳琪,安新民.毛白杨真菌胁迫下miRNA靶基因预测和生物信息分析[J].中国细胞生物学学报,2014(11):1506-1513[Liao WH,Chen Z,Ye MX,Ma HD,Gao K,Lei BQ,An XM.Prediction and bioinformatics analysis of miRNA target genes in populus tomentosa under fungus stress[J].Chin J Cell Biol,2014(11):1506-1513]
23王艳芳,赵彦宏,于佳丽,林德华.miRNA在植物病毒基因组中的靶基因预测及分析[J].植物保护,2015(6):132-135[Wang YF,Zhao YH,Yu JL,Lin DH.Target gene prediction and analysis of miRNA in plant virus genome[J].Plant Prot,2015(6):132-135]
24孙平.茶叶儿茶素合成相关m i R N A及靶基因的验证和表达分析[D].福州:福建农林大学,2017[Sun P.Stadies on identification and expression of miRNAs and target genes iovolved in the biosynthesis of catechins in Camellia sinensis[D].Fuzhou:Fujian Agriculture and Forestry University,2017]
25 Palatnik JF,Wollmann H,Schommer C,Schwab R,Boisbouvier J,Rodriguez R.Sequence and expression differences underlie functional specialization of Arabidopsis microRNAs miR159 and miR319[J].Dev Cell,2007,13(1):115-125
26 Palatnik JF,Allen E,Wu X,Schommer C,Schwab R,Carrington JC.Control of leaf morphogenesis by microRNAs[J].Nature,2003,425(6955):257
27 Du Q,Avci U,Li S,Gallegogiraldo L,Pattathil S,Qi L.Activation of miR165b represses AtHB15 expression and induces pith secondary wall development in Arabidopsis[J].Plant J,2015,83(3):388-400
28 Duclercq J,Assoumou Ndong YP,Guerineau F,Sangwan RS,Catterou M.Arabidopsis shoot organogenesis is enhanced by an amino acid change in the ATHB15 transcription factor[J].Plant Biol,2011,13(2):317
29 Glazińska P,Zienkiewicz A,Wojciechowski W,Kopcewicz J.The putative miR172 target gene InAPETALA2-like is involved in the photoperiodic flower induction of Ipomoea nil[J].J Plant Physiol,2009,166(16):1801-1813
30 Schommer C,Palatnik JF,Aggarwal P,Chételat A,Cubas P,Farmer EE.Control of jasmonate biosynthesis and senescence by miR319 targets[J].Plos Biol,2008,6(9):e230
31 Jagadeeswaran G,Zheng Y,Li YF,Shukl LI,Matts J,Hoyt P.Cloning and characterization of small RNAs from Medicago truncatula reveals four novel legume-specific microRNA families[J].New Phytol,2009,184(1):85-98
32 Navarro L,Jones JDG.A plant miRNA contributes to antibacterial resistance by repressing auxin signaling[J].Science,2006,312(5772):436-439
33宋锐.基于生物信息学方法预测玉米抗纹枯病相关m i R NA及功能分析[D].雅安:四川农业大学,2009[Song R.Prediction of Corn Resistance to Rhizoctonia solani based on bioinformatics method and its function analysis[D].Ya’an:Sichuan Agricultural University,2009]