流式细胞术测定苎麻基因组大小
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摘要
苎麻属于荨麻科苎麻属,是目前非常重要的纺织原料,有着广泛的开发前景。为了更好地研究苎麻基因组信息,拟通过流式细胞术测定苎麻基因组大小。本研究以水稻(Oryza sativa)日本晴为内标,湘苎3号为待测样品,分别分离水稻和苎麻叶片细胞的细胞核,PI染色,用流式细胞仪对水稻和苎麻样品的PI发射荧光强度进行测定。通过比较水稻和苎麻样品的荧光强度的倍数关系,计算得到苎麻基因组大小,并采用外标法和内标法同时进行测定和计算。以水稻日本晴为外标,苎麻3号为待测样品,测得苎麻基因组大小为332.5 Mb,而用内标法得到的苎麻基因组大小为300.5 Mb,相比之下内标法测定的变异系数更低,更为准确。苎麻基因组大小的测定为苎麻基因组文库的建立及其基因组相关研究提供了理论指导。
Boehmeria nivea(ramie), which belongs to Urticaceae in Boehmeria, is an important textile material at present with a broad development prospects. In order to better study the genomic information of ramie, the genomic size of ramie was determined by flow cytometry. One of the ramie variants Xiangzhu 3 was used as the material and Oryza sativa Japonica as reference, and the nucleus of the leaf cells of both species were isolated and stained with propidium iodide(PI), then the PI emission fluorescence intensity was measured by flow cytometry.Ramie's genome size was calculated according to the ratio of fluorescent peak values of Xiangzhu 3 to the rice reference subsequently. In addition, the external standard method and internal standard method are used for simultaneous determination and calculation. The estimated genome size of ramie was about 332.5 Mb by external standard method and about 330.5 Mb by internal standard method. The results showed that the internal method had smaller CV(coefficient of variation) value, which meant the result was more reliable. The determination of ramie's genome size laid an important foundation for the construction of its genomic library and its genome related studies.
Boehmeria nivea(ramie), which belongs to Urticaceae in Boehmeria, is an important textile material at present with a broad development prospects. In order to better study the genomic information of ramie, the genomic size of ramie was determined by flow cytometry. One of the ramie variants Xiangzhu 3 was used as the material and Oryza sativa Japonica as reference, and the nucleus of the leaf cells of both species were isolated and stained with propidium iodide(PI), then the PI emission fluorescence intensity was measured by flow cytometry.Ramie's genome size was calculated according to the ratio of fluorescent peak values of Xiangzhu 3 to the rice reference subsequently. In addition, the external standard method and internal standard method are used for simultaneous determination and calculation. The estimated genome size of ramie was about 332.5 Mb by external standard method and about 330.5 Mb by internal standard method. The results showed that the internal method had smaller CV(coefficient of variation) value, which meant the result was more reliable. The determination of ramie's genome size laid an important foundation for the construction of its genomic library and its genome related studies.
引文
Dolezel J.,and Bartos J.,2005,Plant DNA flow cytometry and estimation of nuclear genome size,Ann.Bot-London,95(1):99-110
Fujisawa M.,Baba T.,and Nagamura Y.,2005,The map-based sequence of the rice genome,Nature,436(7052):793-800
Gong X.,Liu Y.,Huang D.,Zeng G.,Liu S.,Tang H.,Zhou L.,Hu X.,Zhou Y.,and Tan X.,2016,Effects of exogenous calcium and spermidine on cadmium stress moderation and metal accumulation in Boehmeria nivea(L.)Gaudich,Environ.Sci.Pollut.R.,23(9):8699-8708
Jin L.,Zhang X.Y.,and Xue Q.Z.,2007,Comparison on the effects of different isolation buffer on the DNA resolution of the cell nucleus suspensions from rice,Zhejiang Nongye Xuebao(Acta Agriculturae Zhejiangensis),19(2):93-96(金亮,张宪银,薛庆中,2007,分离缓冲液对水稻细胞核悬液DNA分辨率效应的比较,浙江农业学报,19(2):1-96)
Johnston J.S.,Bennett M.D.,Rayburn A.L.,Galbraith D.W.,and Price H.J.,1999,Reference standards for determination of DNA content of plant nuclei,Am.J.Bot.,86(5):609-613
Lee J.,Kim A.R.,and Lee J.J.,2015,Ramie leaf extracts suppresses adipogenic differentiation in 3T3-L1 cells and pig preadipoc-ytes,Asian Austral.J.Anim.,29(9):1338-1344
Liu C.,Zeng L.,Zhu S.,Wu L.,Wang Y.,Tang S.,Wang H.Zheng X.,Zhao J.,Chen X.,Dai Q.,and Liu T.,2017,Draf genome analysis provides insights into the fiber yield crude protein biosynthesis,and vegetative growth of domesticated ramie(Boehmeria nivea L.Gaud),DNA Research25(2):173-181
Liu T.,Zhu S.,Tang Q.,and Tang S.,2014,Genome-wide transcriptomic profiling of ramie(Boehmeria nivea L.Gaud)in response to cadmium stress,Gene,558(1):131-137
Loureiro J.,Travnicek P.,Rauchova J.,Urfus T.,Vit P.,Stech M.Castro S.,and Suda J.,2010,The use of flow cytometry in the biosystematics,ecology and population biology of homoploid plants,Preslia,82(1):3-21
Luan B.,Jian J.,Chen P.,Chen J.H.,Gao Q.,Gao G.,Zhou J.H.Chen K.M.,Guang X.M.,Chen J.K.,Zhang Q.Q.,Wang XF.,Fang L.,Sun Z.M.,Bai M.Z.,Fang X.D.,Zhao S.C.Xiong H.P.,Yu C.M.,and Zhu A.G.,2018,Draft genome sequence of ramie,Boehmeria nivea(L.)gaudich,Mol.Ecol Resour.,18(3):639-645
Luan M.B.,Yang Z.M.,Zhu J.J.,Deng X.,Liu C.C.,Wang X.F.Xu Y.,Sun Z.M.,and Chen J.H.,2016,Identification,evaluation and application of the genomic-SSR loci in ramie Acta Soc.Bot.Pol.,85(3):3510
Mallón R.,Rodríguez-Oubina J.,and González M.L.,2010,In vitro propagation of the endangered plant Centaurea ultreiae:assessment of genetic stability by cytological studies,flow cytometry and RAPD analysis,Plant Cell Tiss.Org.,101(1):31-39
Otto F.,1990,Chapter 11 DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA,Method Cell Biol,33(33):105-110
Qin Y.H.,Xu X.,and Wang C.T.,2014,Measurement of DNAcontent and ploidy of rice cell nucleus suspensions by flow cytometry,Zhongnan Minzu Daxue Xuebao(Journal of South-Central University for Nationalities(Natural Science E-dition),33(1):28-30(覃永华,徐鑫,王春台,2014,水稻细胞核DNA含量和倍性的流式细胞术测定,中南民族大学学报(自然科学版),33(1):28-30)
Tang H.,Liu Y.,Gong X.,Zeng G.,Zheng B.,Wang D.,Sun Z.,Zhou L.,and Zeng X.,2015,Effects of selenium and silicon on enhancing antioxidative capacity in ramie(Boehmeria nivea(L.)Gaud.)under cadmium stress,Environ.Sci.Pollut.R.,22(13):9999-10008
Tiersch T.R.,Chandler R.W.,Wachtel S.S.,and Elias S.,1989,Reference standards for flow cytometry and application in comparative studies of nuclear DNA content,Cytometry,10(6):706-710
Vrána J.,Cápal P.,BednárováM.,and Dolenel J.,2014,Flow cytometry in plant research:a success story,Appl.Plant Cell Biol.,22:395-430
Yang B.,Zhou M.,Shu W.S.,Lan C.Y.,Ye Z.H.,and Qiu R.L.,2010,Constitutional tolerance to heavy metals of a fiber crop,ramie(Boehmeria nivea),and its potential usage,Environ.Pollut.,158(2):551-558
Yang B.,Zhou M.,Zhou L.L.,Xue N.D.,Zhang S.L.,and Lan C.Y.,2015,Variability of cadmium,lead,and zinc tolerance and accumulation among and between germplasms of the fiber crop Boehmeria nivea with different root-types,Environ.Sci.Pollut.R.,22(18):13960-13969
Zhu T.T.,Zhu A.G.,Yu Y.T.,Sun K.,Mao H.D.,and Chen Q.,2016,Research progress of ramie for feedstuff,Caoye Kexue(Pratacultural Science),33(2):338-347(朱涛涛,朱爱国,余永廷,孙凯,毛虎东,陈权,2016,苎麻饲料化的研究,草业科学,33(2):338-347)
Fujisawa M.,Baba T.,and Nagamura Y.,2005,The map-based sequence of the rice genome,Nature,436(7052):793-800
Gong X.,Liu Y.,Huang D.,Zeng G.,Liu S.,Tang H.,Zhou L.,Hu X.,Zhou Y.,and Tan X.,2016,Effects of exogenous calcium and spermidine on cadmium stress moderation and metal accumulation in Boehmeria nivea(L.)Gaudich,Environ.Sci.Pollut.R.,23(9):8699-8708
Jin L.,Zhang X.Y.,and Xue Q.Z.,2007,Comparison on the effects of different isolation buffer on the DNA resolution of the cell nucleus suspensions from rice,Zhejiang Nongye Xuebao(Acta Agriculturae Zhejiangensis),19(2):93-96(金亮,张宪银,薛庆中,2007,分离缓冲液对水稻细胞核悬液DNA分辨率效应的比较,浙江农业学报,19(2):1-96)
Johnston J.S.,Bennett M.D.,Rayburn A.L.,Galbraith D.W.,and Price H.J.,1999,Reference standards for determination of DNA content of plant nuclei,Am.J.Bot.,86(5):609-613
Lee J.,Kim A.R.,and Lee J.J.,2015,Ramie leaf extracts suppresses adipogenic differentiation in 3T3-L1 cells and pig preadipoc-ytes,Asian Austral.J.Anim.,29(9):1338-1344
Liu C.,Zeng L.,Zhu S.,Wu L.,Wang Y.,Tang S.,Wang H.Zheng X.,Zhao J.,Chen X.,Dai Q.,and Liu T.,2017,Draf genome analysis provides insights into the fiber yield crude protein biosynthesis,and vegetative growth of domesticated ramie(Boehmeria nivea L.Gaud),DNA Research25(2):173-181
Liu T.,Zhu S.,Tang Q.,and Tang S.,2014,Genome-wide transcriptomic profiling of ramie(Boehmeria nivea L.Gaud)in response to cadmium stress,Gene,558(1):131-137
Loureiro J.,Travnicek P.,Rauchova J.,Urfus T.,Vit P.,Stech M.Castro S.,and Suda J.,2010,The use of flow cytometry in the biosystematics,ecology and population biology of homoploid plants,Preslia,82(1):3-21
Luan B.,Jian J.,Chen P.,Chen J.H.,Gao Q.,Gao G.,Zhou J.H.Chen K.M.,Guang X.M.,Chen J.K.,Zhang Q.Q.,Wang XF.,Fang L.,Sun Z.M.,Bai M.Z.,Fang X.D.,Zhao S.C.Xiong H.P.,Yu C.M.,and Zhu A.G.,2018,Draft genome sequence of ramie,Boehmeria nivea(L.)gaudich,Mol.Ecol Resour.,18(3):639-645
Luan M.B.,Yang Z.M.,Zhu J.J.,Deng X.,Liu C.C.,Wang X.F.Xu Y.,Sun Z.M.,and Chen J.H.,2016,Identification,evaluation and application of the genomic-SSR loci in ramie Acta Soc.Bot.Pol.,85(3):3510
Mallón R.,Rodríguez-Oubina J.,and González M.L.,2010,In vitro propagation of the endangered plant Centaurea ultreiae:assessment of genetic stability by cytological studies,flow cytometry and RAPD analysis,Plant Cell Tiss.Org.,101(1):31-39
Otto F.,1990,Chapter 11 DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA,Method Cell Biol,33(33):105-110
Qin Y.H.,Xu X.,and Wang C.T.,2014,Measurement of DNAcontent and ploidy of rice cell nucleus suspensions by flow cytometry,Zhongnan Minzu Daxue Xuebao(Journal of South-Central University for Nationalities(Natural Science E-dition),33(1):28-30(覃永华,徐鑫,王春台,2014,水稻细胞核DNA含量和倍性的流式细胞术测定,中南民族大学学报(自然科学版),33(1):28-30)
Tang H.,Liu Y.,Gong X.,Zeng G.,Zheng B.,Wang D.,Sun Z.,Zhou L.,and Zeng X.,2015,Effects of selenium and silicon on enhancing antioxidative capacity in ramie(Boehmeria nivea(L.)Gaud.)under cadmium stress,Environ.Sci.Pollut.R.,22(13):9999-10008
Tiersch T.R.,Chandler R.W.,Wachtel S.S.,and Elias S.,1989,Reference standards for flow cytometry and application in comparative studies of nuclear DNA content,Cytometry,10(6):706-710
Vrána J.,Cápal P.,BednárováM.,and Dolenel J.,2014,Flow cytometry in plant research:a success story,Appl.Plant Cell Biol.,22:395-430
Yang B.,Zhou M.,Shu W.S.,Lan C.Y.,Ye Z.H.,and Qiu R.L.,2010,Constitutional tolerance to heavy metals of a fiber crop,ramie(Boehmeria nivea),and its potential usage,Environ.Pollut.,158(2):551-558
Yang B.,Zhou M.,Zhou L.L.,Xue N.D.,Zhang S.L.,and Lan C.Y.,2015,Variability of cadmium,lead,and zinc tolerance and accumulation among and between germplasms of the fiber crop Boehmeria nivea with different root-types,Environ.Sci.Pollut.R.,22(18):13960-13969
Zhu T.T.,Zhu A.G.,Yu Y.T.,Sun K.,Mao H.D.,and Chen Q.,2016,Research progress of ramie for feedstuff,Caoye Kexue(Pratacultural Science),33(2):338-347(朱涛涛,朱爱国,余永廷,孙凯,毛虎东,陈权,2016,苎麻饲料化的研究,草业科学,33(2):338-347)