文心兰‘南茜’谷氧还蛋白基因克隆定位及表达
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摘要
为研究文心兰谷氧还蛋白超家族基因ROXY在花药发育中的功能,以文心兰‘南茜’(Oncidium Gower Ramsey)为试验材料,从‘南茜’的转录组网站选取一条与拟南芥At ROXY1/2相似度最高的基因序列(命名为OnROXY1),以该序列的cDNA为模版设计引物,用RT-PCR从‘南茜’花器官中扩增该基因,并对扩增产物进行测序和保守结构域分析.结果表明,OnROXY1基因编码区402 bp,编码133个氨基酸,与‘南茜’转录组网站中的预测序列只有一个碱基差异,但与该预测序列的氨基酸序列和保守结构域完全一致(GenBank登录号:MF696154).OnROXY1基因具有典型的谷氧还蛋白(Glutaredoxin,GRX)基因家族的结构域和CMCC活性位点,因此该基因属于GRX基因家族,并且属于亚类CC型的GRX基因.进化树分析表明,OnROXY1与水稻的Os ROXY1、Os ROXY2和拟南芥的At ROXY1、At ROXY2蛋白序列的一致性最高,进化距离最近.亚细胞定位结果显示,OnROXY1主要定位于细胞核中,少量定位在细胞质中.RT-PCR检测发现,在不同组织部位中,OnROXY1在假鳞茎中的表达量最高,唇瓣中的表达量最低;不同花期的定量结果显示,OnROXY1在脱落干枯期时的表达量最高,盛开期时的表达量最低.本研究推测谷氧还蛋白基因OnROXY1可能在文心兰假鳞茎与根的发育、消除植物体内过多的活性氧以及延缓文心兰花器官的衰老等方面具有重要作用.
In order to investigate the function of the glutathione superfamily ROXY gene during anther development in Oncidium(Orchidaceae), sequences in the glutaredoxin gene family from the Oncidium ‘Gower Ramsey' transcriptomic library were selected and aligned with AtROXY1 and AtROXY2 of Arabidopsis thaliana for genetic homology analysis. Next, OnROXY1(Gen Bank accession No. MF696154) was screened, and using RACE-PCR, several conserved amino acid sequence domains were revealed. The results showed that the complete cDNA sequence of OnROXY1 was 402 bp and encoded 133 amino acids, which was only one base different from the predicted sequence selected from the Oncidium ‘Gower Ramsey' transcriptomic library and completely consistent with the amino acid sequence and conserved domain of the predicted sequence. In addition, OnROXY1 belonged to the glutaredoxin(GRX) superfamily and had a c-type cytochrome in the CMCC superfamily domain. Moreover, protein sequence and phylogenetic tree analysis showed that OnROXY1 had the closest amino acid identity with OsROXY1, OsROXY2, AtROXY1, and AtROXY2. Subcellular localization using a GFP-fusion tag of OnROXY1 revealed nucleic and cytosolic localization. Differential expression profiles of OnROXY1 via quantitative real-time PCR produced a significantly greater amount of m RNA transcript in pseudobulb tissues and lesser in the labellum. Moreover, OnROXY1 exhibited the greatest gene expression in the withered and abscised period and lowest during the blooming periods. Here, the results showed the importance of OnROXY1, specifically in pseudobulbs and roots, via its function of scavenging excessive reactive oxygen species and delaying aging in the floral organs of Oncidium.
In order to investigate the function of the glutathione superfamily ROXY gene during anther development in Oncidium(Orchidaceae), sequences in the glutaredoxin gene family from the Oncidium ‘Gower Ramsey' transcriptomic library were selected and aligned with AtROXY1 and AtROXY2 of Arabidopsis thaliana for genetic homology analysis. Next, OnROXY1(Gen Bank accession No. MF696154) was screened, and using RACE-PCR, several conserved amino acid sequence domains were revealed. The results showed that the complete cDNA sequence of OnROXY1 was 402 bp and encoded 133 amino acids, which was only one base different from the predicted sequence selected from the Oncidium ‘Gower Ramsey' transcriptomic library and completely consistent with the amino acid sequence and conserved domain of the predicted sequence. In addition, OnROXY1 belonged to the glutaredoxin(GRX) superfamily and had a c-type cytochrome in the CMCC superfamily domain. Moreover, protein sequence and phylogenetic tree analysis showed that OnROXY1 had the closest amino acid identity with OsROXY1, OsROXY2, AtROXY1, and AtROXY2. Subcellular localization using a GFP-fusion tag of OnROXY1 revealed nucleic and cytosolic localization. Differential expression profiles of OnROXY1 via quantitative real-time PCR produced a significantly greater amount of m RNA transcript in pseudobulb tissues and lesser in the labellum. Moreover, OnROXY1 exhibited the greatest gene expression in the withered and abscised period and lowest during the blooming periods. Here, the results showed the importance of OnROXY1, specifically in pseudobulbs and roots, via its function of scavenging excessive reactive oxygen species and delaying aging in the floral organs of Oncidium.
引文
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2 陈永坤,赵启红,刘海珍,周海天,徐吉臣.美洲商陆谷氧还蛋白基因PaGRXC13的克隆与生物信息学分析[J].分子植物育种,2016,14(8):2019-2024[Chen YK,Zhao QH,Liu HZ,Zhou HT,Xu JC.Cloning and bioinformatics analysis of glutaredoxin gene PaGRXC13 in Phytolacca americana[J].Mol Plant Breed,2016,14(8):2019-2024
3 Pham K,Pal R,Qu Y,Liu X,Yu H,Shiao SL,Wang X,O’Brian Smith E,Cui X,Rodney GG,Cheng N.Nuclear glutaredoxin 3 is critical for protection against oxidative stress-induced cell death[J].Free Radical Biol Med,2015,85:197
4 Schieber M,Chandel N.ROS function in redox signaling and oxidative stress[J].Curr Biol CB,2014,24(10):R453
5 Reczek CR,Chandel NS.ROS-dependent signal transduction[J].Curr Opin Cell Biol,2015,33:8-13
6 Shelton MD,Chock PB,Mieyal JJ.Glutaredoxin:role in reversible protein s-glutathionylation and regulation of redox signal transduction and protein translocation[J].Antioxidants Redox Signaling,2005,7(3-4):348-366
7 Zhang H,Du Y,Zhang X,Lu J,Holmgren A.Glutaredoxin 2 reduces both thioredoxin 2 and thioredoxin 1 and protects cells from apoptosisinduced by auranofin and 4-hydroxynonenal[J].Antioxidants Redox Signaling,2014,21(5):669-681
8 Hong L,Tang D,Zhu K,Wang K,Li M,Cheng Z.Somatic and reproductive cell development in rice anther is regulated by a putative glutaredoxin[J].Plant Cell,2012,24(2):577-588
9 Guo Y,Huang C,Yan X,Song F,Zhou X.A tomato glutaredoxin gene SlGRX1 regulates plant responses to oxidative,drought and salt stresses[J].Planta,2010,232(6):1499-1509
10 Sharma R,Priya P,Jain M.Modified expression of an auxin-responsive rice CC-type glutaredoxin gene affects multiple abiotic stress responses[J].Planta,2013,238(5):871-884
11 苏慧慧,李涛,黎振兴,李植良,王永飞.番茄GRX基因家族的鉴定及表达分析[J].核农学报,2015,29(4):663-673[Su HH,Li T,Li ZX,Li ZL,Wang YF.Identification and expression analysis of tomato GRX gene family[J].J Nucl Agric Sci,2015,29(4):663-673]
12 Sundaram S,Wu S,Ma LQ,Rathinasabapathi B.Expression of a Pterisvittata glutaredoxin PvGRX5 in transgenic Arabidopsis thaliana increases plant arsenic tolerance and decreases arsenic accumulation in the leaves.[J].Plant Cell Environ,2009,32(7):851-858
13 Wu Q,Lin J,Liu JZ,Wang X,Lim W,Myungmin O.Ectopic expression of Arabidopsis glutaredoxin AtGRXs17,enhances thermotolerance in tomato[J].Plant Biotechnol J,2012,10(8):945
14 Ying H,Wu Q,Sprague SA,Park J,Oh M,Rajashekar CB,Hisashi K,Paul AN,Ninghui C,Kendal DH,Frank FW,Sunghun P.Tomato expressing Arabidopsis glutaredoxin gene AtGRXs17 confers tolerance to chilling stress via modulating cold responsive components[J].Horti Res,2015,2:15051
15 Wang Z,Xing S,Birkenbihl RP,Zachgo S.Conserved functions of Arabidopsis and rice CC-type glutaredoxins in flower development and pathogen response[J].Mol Plant,2009,2(2):323
16 Xing S,Rosso MG,Zachgo S.ROXY1,a member of the plant glutaredoxin family,is required for petal development in Arabidopsis thaliana[J].Development,2005,132(7):1555-1565
17 Xing S,Zachgo S.ROXY1 and ROXY2,two Arabidopsi s glutaredoxingenes,are required for anther development[J].Plant J,2008,53(5):790-801
18 Li S,Lauri A,Ziemann M,Busch A,Bhave M,Zachgo S.Nuclear activity of ROXY1,a glutaredoxin interacting with TGA factors,is required for petal development in Arabidopsis thaliana[J].Plant Cell,2009,21(2):429
19 Mark Z,Mrinal B,Sabine Z.Bioinformatic studies of the wheat glutaredoxin gene family and functional analysis of the ROXY1orthologues[J].Funct Plant Biol,2010,38(1):25
20 Rouhier N,Gelhaye E,Jacquot JP.Plant glutaredoxins:still mysterious reducing systems[J].Cell Mol Life Sci,2004,61(11):1266-1277
21 Murmu J,Bush MJ,Delong C,Li S,Xu M,Khan M,Malcolmson C,Fobert PR,Zachgo S,Hepworth SR.Arabidopsis basic leucinezipper transcription factors TGA9 and TGA10 interact with f loral glutaredoxins ROXY1 and ROXY2 and are redundantly required for anther development[J].Plant Physiol,2010,154(3):1492-1504
22 Lemaire SD.The glutaredoxin family in oxygenic photosynthetic organisms[J].Photosynth Res,2004,79(3):305
23 Xing S,Lauri A,Zachgo S.Redox regulation and flower development:anovel function for glutaredoxins[J].Plant Biol,2006,8(5):547
24 Ndamukong I,Abdallat AA,Thurow C,Fode B,Zander M,Weigel R,Gatz C.SA-inducible Arabidopsis glutaredoxin interacts with TGA factors and suppresses JA-responsive PDF1.2 transcription[J].Plant J,2007,50(1):128
25 Lemaire SD.The glutaredoxin family in oxygenic photosynthetic organisms[J].Photosynth Res,2004,79(3):305
26 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[Chen YK,Lin YL,Cheng CZ,Lai ZX.Cloning,subcellular localization and expression analysis of ferredoxin(OnFd)in Oncidium hybridum[J].J NW Bot Sci,2017,37(1):48-58]
27 Bagde P,Sharon M,Bagde P,Sharon M.In vitro regeneration of Oncidium Gower Ramsey by high frequency protocorm like bodies proliferation[J].Indian J Plant Physiol,1997,2(1):10-14
28 Ahmad Z,Hassan AA,Idris NA,Basiran MN.Effects of ion beam irradiation on Oncidiumlanceanum orchids[J].J Nucl Related Technol,2006,3(1-2):1-8
29 Singer RB,Koehler S.Notes on the pol l inat ion biology of Notylianemorosa(Orchidaceae:Oncidiinae):do pollinators necessarily promote cross-pollination?[J].J Plant Res Jpn,2003,116:19-25
30 易美秀.文心蘭授粉、結實與無菌播種之研究[D].台中:中兴大学,2000[Yi MX.Studies on pollination,capsule setting and asymbiotic seed germination of Oncidium[D].Taizhong:Chung Hsing University,2000]
31 罗远华,黄敏玲,林兵,钟淮钦,叶秀仙,吴建设.文心兰花粉活力与杂交结荚性研究[J].福建农业学报,2015(3):258-263[Luo YH,Huang ML,Lin B,Zhong HQ,Ye XX,Wu JJ.Studying on pollen viability and hybrid pod setting ability of Oncidium[J].J Fujian Agric,2015(3):258-263]
32 许玉妹,许哲夫,黄雅玲.花卉园艺作物改良.行政院农业委员会高雄区农业改良场百年纪念专刊[J],2002,3(2):38-46[Xu YM,Xu ZF,Huang Y.Flower horticultural crop improvement.Executive Committee Agricultural Committee Kaohsiung District Agricultural Improvement Field Centennial Special Issue[J],2002,3(2):38-46]
33 叶炜,尚伟,夏潮水.文心兰育种研究进展[J].三明农业科技,2008(3):17-19[Ye W,Shang W,Xia CS.Study on the research of Oncidium[J].Sanming Agric Sci Technol,2008(3):17-19]
34 谢礼洋,刘生财,白玉,柳燕,林觅,蔡顺亮,郑学立,谢晓清,冯新,程春振,陈欲坤,赖钟雄.苋菜甜菜红素合成相关基因AmMYB1的克隆及表达分析[J].西北植物学报,2016,36(6):1080-1090[Xie LY,Liu SC,Bai Y,Liu Y,Lin M,Cai SL,Zheng XL,Xie XQ,Feng X,Cheng CZ,Chen YK,Lai ZX.Cloning and expression analysis of betalainrelated transcription factor gene AmMYB1 in Amaranthus tricolor[J].JNW Bot Sci,2016,36(6):1080-1090]
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