无籽沙糖桔WUBE1基因的功能验证
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摘要
为了解泛素活化酶E1基因(UBE1)在无籽沙糖桔自交不亲和反应中的作用,通过根癌农杆菌介导法将来源于自交不亲和无籽沙糖桔(Citrus reticulata ‘Wuzishatangju’) WUBE1基因转化烟草(Nicotiana tabacum)。结果表明,外源基因WUBE1已导入烟草基因组中并得到表达。转WUBE1基因的自交授粉组合花粉管在生长过程中,部分花粉管出现停止生长的现象,到达花柱基部的花粉管数量少于异交授粉和野生型自交组合。转WUBE1基因烟草的花粉生活力、发芽率、自交和异交后每个果荚中的种子数与野生型烟草无显著差异。这表明单一的WUBE1基因不能调控无籽沙糖桔自交不亲和反应,很可能是通过Ub/26S途径参与了无籽沙糖桔自交不亲和反应。
In order to understand the role of UBE1 gene in self-incompatibility(SI) response of Citrus reticulata 'Wuzishatangju', a WUBE1 gene from the self-incompatible cultivar 'Wuzishatangju' was transferred into tobacco(Nicotiana tabacum) mediated by Agrobacterium tumefaciens. The results showed that the WUBE1 gene was integrated into genome and expressed in transgenic tobacco. Some pollen tubes of self-pollinated transgenic tobacco with WUBE1 became twisted and could not enter the embryo sac. The number of pollen tube entering into ovules in self-pollinated WUBE1 tobacco was fewer than that in wild type(WT) and cross-pollination(transgenic tobacco×WT). However, there was no significant difference in pollen viability, germination rate and seed number per pod between transgenic tobacco and WT. Therefore, it was suggested that only the WUBE1 could not regulate SI response of 'Wuzishatangju', and it was possible to form a complex in Ub/26 S pathway involved in the SI reaction.
In order to understand the role of UBE1 gene in self-incompatibility(SI) response of Citrus reticulata 'Wuzishatangju', a WUBE1 gene from the self-incompatible cultivar 'Wuzishatangju' was transferred into tobacco(Nicotiana tabacum) mediated by Agrobacterium tumefaciens. The results showed that the WUBE1 gene was integrated into genome and expressed in transgenic tobacco. Some pollen tubes of self-pollinated transgenic tobacco with WUBE1 became twisted and could not enter the embryo sac. The number of pollen tube entering into ovules in self-pollinated WUBE1 tobacco was fewer than that in wild type(WT) and cross-pollination(transgenic tobacco×WT). However, there was no significant difference in pollen viability, germination rate and seed number per pod between transgenic tobacco and WT. Therefore, it was suggested that only the WUBE1 could not regulate SI response of 'Wuzishatangju', and it was possible to form a complex in Ub/26 S pathway involved in the SI reaction.
引文
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[29]SHI G J,HOU X L.Measurement of self-incompatible by fluoroscope observation in non-heading Chinese cabbage[J].J Wuhan Bot Res,2004,22(3):197-200.doi:10.3969/j.issn.2095-0837.2004.03.003.史公军,侯喜林.白菜自交不亲和性的荧光测定[J].武汉植物学研究,2004,22(3):197-200.doi:10.3969/j.issn.2095-0837.2004.03.003.
[30]SMALLE J,VIERSTRA R D.The ubiquitin 26S proteasome proteolytic pathway[J].Annu Rev Plant Biol,2004,55:555-590.doi:10.1146/annurev.arplant.55.031903.141801.
[31]HUA Z H,VIERSTRA R D.The cullin-RING ubiquitin-protein ligases[J].Annu Rev Plant Biol,2011,62:299-334.doi:10.1146/annurevarplant-042809-112256.
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[33]INDRIOLO E,GORING D R.A conserved role for the ARC1 E3ligase in Brassicaceae self-incompatibility[J].Front Plant Sci,2014,5:181.doi:10.3389/fpls.2014.00181.
[2]MORREALE F E,WALDEN H.Types of ubiquitin ligases[J].Cell,2016,165(1):248-248.e1.doi:10.1016/j.cell.2016.03.003.
[3]ISONO E,NAGEL M K.Deubiquitylating enzymes and their emerging role in plant biology[J].Front Plant Sci,2014,5:56.doi:10.3389/fpls.2014.00056.
[4]VIERSTRA R D.The ubiquitin-26S proteasome system at the nexus of plant biology[J].Nat Rev Mol Cell Biol,2009,10(6):385-397.doi:10.1038/nrm2688.
[5]MOON J,PARRY G,ESTELLE M.The ubiquitin-proteasome pathway and plant development[J].Plant Cell,2004,16(12):3181-3195.doi:10.1105/tpc.104.161220.
[6]YU F F,WU Y R,XIE Q.Ubiquitin-proteasome system in ABAsignaling:From perception to action[J].Mol Plant,2016,9(1):21-33.doi:10.1016/j.molp.2015.09.015.
[7]KELLEY D R.E3 ubiquitin ligases:Key regulators of hormone signaling in plants[J].Mol Cell Proteom,2018,17(6):1047-1054.doi:10.1074/mcp.MR117.000476.
[8]CUI X,LU F L,LI Y,et al.Ubiquitin-specific proteases UBP12 and UBP13 act in circadian clock and photoperiodic flowering regulation in Arabidopsis[J].Plant Physiol,2013,162(2):897-906.doi:10.1104/pp.112.213009.
[9]FANG H M,MENG Q L,XU J W,et al.Knock-down of stress inducible OsSRFP1 encoding an E3 ubiquitin ligase with transcriptional activation activity confers abiotic stress tolerance through enhancing antioxidant protection in rice[J].Plant Mol Biol,2015,87(4/5):441-458.doi:10.1007/s11103-015-0294-1.
[10]BAHMANI R,KIM D,LEE B D,et al.Over-expression of tobacco UBC1 encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco(Nicotiana tabacum)[J].Plant Mol Biol,2017,94(4/5):433-451.doi:10.1007/s11103-017-0616-6.
[11]HE Q,McLELLAN H,BOEVINK P C,et al.U-box E3 ubiquitin ligase PUB17 acts in the nucleus to promote specific immune pathways triggered by Phytophthora infestans[J].J Exp Bot,2015,66(11):3189-3199.doi:10.1093/jxb/erv128.
[12]MILLYARD L,LEE J,ZHANG C J,et al.The ubiquitin conjugating enzyme,TaU4 regulates wheat defence against the phytopathogen Zymoseptoria tritici[J].Sci Rep,2016,6:35683.
[13]XU X S,PAIK I,ZHU L,et al.Phytochrome interacting factor1enhances the E3 ligase activity of constitutive photomorphogenic1 to synergistically repress photomorphogenesis in Arabidopsis[J].Plant Cell,2014,26(5):1992-2006.doi:10.1105/tpc.114.125591.
[14]CHANG L C,GUO C L,LIN Y S,et al.Pollen-specific SKP1-like proteins are components of functional SCF complexes and essential for lily pollen tube elongation[J].Plant Cell Physiol,2009,50(8):1558-1572.doi:10.1093/pcp/pcp100.
[15]DOUCET J,LEE H K,GORING D R.Pollen acceptance or rejection:A tale of two pathways[J].Trends Plant Sci,2016,21(12):1058-1067.doi:10.1016/j.tplants.2016.09.004.
[16]SUN P L,LI S,LU D H,et al.Pollen S-locus F-box proteins of Petunia involved in S-RNase-based self-incompatibility are themselves subject to ubiquitin-mediated degradation[J].Plant J,2015,83(2):213-223.doi:10.1111/tpj.12880.
[17]STONE S L,ANDERSON E M,MULLEN R T,et al.ARC1 is an E3ubiquitin ligase and promotes the ubiquitination of proteins during the rejection of self-incompatible Brassica pollen[J].Plant Cell,2003,15(4):885-898.doi:10.1105/tpc.009845.
[18]QIAO H,WANG H Y,ZHAO L,et al.The F-box protein Ah SLF-S2physically interacts with S-RNases that may be inhibited by the ubiquitin/26S proteasome pathway of protein degradation during compatible pollination in Antirrhinum[J].Plant Cell,2004,16(3):582-595.doi:10.1105/tpc.017673.
[19]ZHANG N,LIU J H,JIA C H,et al.The interaction of banana MADS-box protein MuMADS1 and ubiquitin-activating enzyme E-MuUBA in post-harvest banana fruit[J].Chin J Trop Crops,2014,35(2):307-312.doi:10.3969/j.issn.1000-2561.2014.02.016.张妮,刘菊华,贾彩红,等.香蕉Mu MADS1与泛素激活酶(MuUBA)在采后果实中的相互作用[J].热带作物学报,2014,35(2):307-312.doi:10.3969/j.issn.1000-2561.2014.02.016.
[20]JUE D W,SANG X L,SHU B,et al.Expression pattern analysis of an ubiquitin-activating enzyme gene CpUBA1 in Carica papaya[J].Guangdong Agric Sci,2017,44(11):20-25.doi:10.16768/j.issn.1004-874X.2017.11.004.决登伟,桑雪莲,舒波,等.番木瓜泛素活化酶基因CpUBA1的表达模式分析[J].广东农业科学,2017,44(11):20-25.doi:10.16768/j.issn.1004-874X.2017.11.004.
[21]LI X,ZHANG S S,MA J X,et al.TaUBA,a UBA domain-containing protein in wheat(Triticum aestivum L.),is a negative regulator of salt and drought stress response in transgenic Arabidopsis[J].Plant Cell Rep,2015,34(5):755-766.doi:10.1007/s00299-015-1739-3.
[22]YE Z X,ZENG T,XU J K,et al.Wuzishatangju,a new mandarin cultivar[J].J Fruit Sci,2006,23(1):149-150.doi:10.3969/j.issn.1009-9980.2006.01.037.叶自行,曾泰,许建楷,等.无子沙糖橘(十月橘)的选育[J].果树学报,2006,23(1):149-150.doi:10.3969/j.issn.1009-9980.2006.01.037.
[23]YE W J,QIN Y H,YE Z X,et al.Seedless mechanism of a new mandarin cultivar‘Wuzishatangju’(Citrus reticulata Blanco)[J].Plant Sci,2009,177(1):19-27.doi:10.1016/j.plantsci.2009.03.005.
[24]MIAO H X,QIN Y H,YE Z X,et al.Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata[J].Gene,2013,513(2):249-259.doi:10.1016/j.gene.2012.10.056.
[25]QIN Y H,HU G B.Experimental Guidance of Biotechnology for Horticultural Plants[M].2nd ed.Beijing:Chinese Agricultural Press,2016:51-53,82-84.秦永华,胡桂兵.园艺植物生物技术实验指导[M].第2版.北京:中国农业出版社,2016:51-53,82-84.
[26]MIAO H X,QIN Y H,DA SILVA J A T,et al.Cloning and expression analysis of S-RNase homologous gene in Citrus reticulata Blanco cv.Wuzishatangju[J].Plant Sci,2011,180(2):358-367.doi:10.1016/j.plantsci.2010.10.012.
[27]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods,2001,25(4):402-408.doi:10.1006/meth.2001.1262.
[28]TAO S T,ZHANG S L,CHEN D X,et al.Study on characteristics of in situ pollen germination and tube growth of Prunus mume[J].J Fruit Sci,2004,21(4):338-340.doi:10.3969/j.issn.1009-9980.2004.04.014.陶书田,张绍铃,陈迪新,等.果梅花粉原位萌发及花粉管生长特性的研究[J].果树学报,2004,21(4):338-340.doi:10.3969/j.issn.1009-9980.2004.04.014.
[29]SHI G J,HOU X L.Measurement of self-incompatible by fluoroscope observation in non-heading Chinese cabbage[J].J Wuhan Bot Res,2004,22(3):197-200.doi:10.3969/j.issn.2095-0837.2004.03.003.史公军,侯喜林.白菜自交不亲和性的荧光测定[J].武汉植物学研究,2004,22(3):197-200.doi:10.3969/j.issn.2095-0837.2004.03.003.
[30]SMALLE J,VIERSTRA R D.The ubiquitin 26S proteasome proteolytic pathway[J].Annu Rev Plant Biol,2004,55:555-590.doi:10.1146/annurev.arplant.55.031903.141801.
[31]HUA Z H,VIERSTRA R D.The cullin-RING ubiquitin-protein ligases[J].Annu Rev Plant Biol,2011,62:299-334.doi:10.1146/annurevarplant-042809-112256.
[32]KUBO K I,ENTANI T,TAKARA A,et al.Collaborative non-self recognition system in S-RNase-based self-incompatibility[J].Science,2010,330(6005):796-799.doi:10.1126/science.1195243.
[33]INDRIOLO E,GORING D R.A conserved role for the ARC1 E3ligase in Brassicaceae self-incompatibility[J].Front Plant Sci,2014,5:181.doi:10.3389/fpls.2014.00181.