F18大肠杆菌抗性型与敏感型苏太断奶仔猪十二指肠组织比较转录组分析
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摘要
旨在揭示培育品种—苏太猪(杜洛克×梅山猪)F18大肠杆菌抗性的调控通路和重要候选基因,同时进一步探究中外猪品种F18大肠杆菌抗性调控遗传基础的差异。本研究以课题组前期获得的苏太猪和梅山猪F18大肠杆菌抗性型与敏感型断奶仔猪全同胞个体为研究对象,通过转录组测序筛选苏太猪F18大肠杆菌抗性相关的调控通路以及重要候选基因,并在细胞水平,利用qPCR和Western blot分析F18大肠杆菌刺激小肠上皮细胞IPEC-J2后重要候选基因(蛋白)的表达变化,同时利用qPCR检测重要候选基因在苏太和梅山断奶仔猪F18大肠杆菌抗性型与敏感型个体十二指肠组织中的差异表达情况。结果显示:1)在苏太断奶仔猪F18大肠杆菌抗性型与敏感型个体中筛选出238个差异表达基因,主要参与Toll样受体信号通路(toll-like receptor signaling pathway)和糖脂类通路(glycosphingolipid biosynthesis-lacto and neolacto series),其中TLR5、IL-1β、FUT2为重要候选基因;2)不同血清型F18大肠杆菌(F18ac、F18ab)菌体分别刺激小肠上皮细胞IPEC-J2后,FUT2、IL-1β、TLR5基因mRNA表达水平显著上调(P<0.05),并且其蛋白表达水平也表现为明显的上调,由此表明,TLR5、IL-1β和FUT2在断奶仔猪F18大肠杆菌感染过程中发挥重要的调控作用;3)组织差异表达分析显示,TLR5、IL-1β和FUT2在苏太猪抗性型与敏感型个体十二指肠组织中表达差异均达到显著水平(P<0.05),而梅山猪中TLR5、IL-1β表达差异达到极显著水平(P<0.01),FUT2表达水平差异不显著(P>0.05)。结合课题组前期关于梅山猪及外来引进品种F18大肠杆菌抗性相关分子机制研究及报道,本研究进一步证明中外猪品种F18大肠杆菌抗性调控的遗传基础确实存在差异,Toll样受体信号通路及CD14、TLR5等基因可能在中国地方品种—梅山猪抵抗F18大肠杆菌感染过程中发挥着免疫调控作用,而鞘糖脂合成通路及FUT2等基因可能在外来猪品种F18大肠杆菌受体形成过程中起关键作用。
To reveal the regulatory pathways and important candidate genes related to E.coli F18 resistance in Sutai pig(Duroc× Meishan pigs),and further explore the differences of hereditarybasis in regulating E.coli F18 resistance between Chinese domestic and foreign pig breeds,Sutai and Meishan populations previously established with resistance and sensitivity to E.coli F18 were selected as experiment objects.Using transcriptome sequencing,we screened out the regulatory pathways and candidate genes related to E.coli F18 resistance in Sutai pig,then analyzed the expressions of candidate genes(proteins)in different serotype of E.coli F18(F18 ac and F18 ab)-stimulated pig intestinal epithelial cell(IPEC-J2)by qPCR and Western blot.Additionally,the differential expressions of candidate genes in duodenal tissues between E.coli F18-resistant and E.coli F18-sensitive weaned piglets were detected by qPCR.The results showed that:1)there were 238 differentially expressed genes in duodenum between Sutai E.coli F18-resistant and E.coli F18-sensitive weaned piglets,which were mainly involved in toll-like receptor signaling pathway and glycosphingolipid biosynthesis-lacto and neolacto series pathway,including TLR5,IL-1βand FUT2 genes.2)The mRNA expression levels of IL-1β,TLR5,FUT2 were all significantly up-regulated after F18 ac-,F18 ab-stimulated IPEC-J2,respectively(P<0.05),and the corresponding proteins expression were also obviously increased,which indicated that IL-1β,TLR5 and FUT2 might play important regulatory roles in the process of Sutai piglets responsing to E.coli F18 infection.3)Tissue expressions analysis showed that the expression levels of TLR5,IL-1β,FUT2 genes in duodenum tissues between Sutai E.coli F18-resistant and E.coli F18-sensitive individuals were all significantly different(P<0.05).The expressions of TLR5,IL-1βgenes in duodenum tissues between Meishan E.coli F18-resistant and E.coli F18-sensitive individuals showed extremely significant difference(P<0.01),but FUT2 expression was not significantly different(P>0.05).Combined with previous studies of molecular mechanism of resistance to E.coli F18 in Meishan and foreign pig breeds,this study revealed the differences of hereditary basis in regulating E.coli F18 resistance between Chinese domestic and foreign pig breeds.Toll-like receptor signaling pathway including CD14 and TLR5 probably played immune roles in regulating E.coli F18 resistance in Chinese domestic pig breeds such as Meishan pigs,while the glycosphingolipid biosynthesis-lacto and neolacto series pathway including FUT2 was associated with the formation of E.coli F18 receptor in foreign pigs.
To reveal the regulatory pathways and important candidate genes related to E.coli F18 resistance in Sutai pig(Duroc× Meishan pigs),and further explore the differences of hereditarybasis in regulating E.coli F18 resistance between Chinese domestic and foreign pig breeds,Sutai and Meishan populations previously established with resistance and sensitivity to E.coli F18 were selected as experiment objects.Using transcriptome sequencing,we screened out the regulatory pathways and candidate genes related to E.coli F18 resistance in Sutai pig,then analyzed the expressions of candidate genes(proteins)in different serotype of E.coli F18(F18 ac and F18 ab)-stimulated pig intestinal epithelial cell(IPEC-J2)by qPCR and Western blot.Additionally,the differential expressions of candidate genes in duodenal tissues between E.coli F18-resistant and E.coli F18-sensitive weaned piglets were detected by qPCR.The results showed that:1)there were 238 differentially expressed genes in duodenum between Sutai E.coli F18-resistant and E.coli F18-sensitive weaned piglets,which were mainly involved in toll-like receptor signaling pathway and glycosphingolipid biosynthesis-lacto and neolacto series pathway,including TLR5,IL-1βand FUT2 genes.2)The mRNA expression levels of IL-1β,TLR5,FUT2 were all significantly up-regulated after F18 ac-,F18 ab-stimulated IPEC-J2,respectively(P<0.05),and the corresponding proteins expression were also obviously increased,which indicated that IL-1β,TLR5 and FUT2 might play important regulatory roles in the process of Sutai piglets responsing to E.coli F18 infection.3)Tissue expressions analysis showed that the expression levels of TLR5,IL-1β,FUT2 genes in duodenum tissues between Sutai E.coli F18-resistant and E.coli F18-sensitive individuals were all significantly different(P<0.05).The expressions of TLR5,IL-1βgenes in duodenum tissues between Meishan E.coli F18-resistant and E.coli F18-sensitive individuals showed extremely significant difference(P<0.01),but FUT2 expression was not significantly different(P>0.05).Combined with previous studies of molecular mechanism of resistance to E.coli F18 in Meishan and foreign pig breeds,this study revealed the differences of hereditary basis in regulating E.coli F18 resistance between Chinese domestic and foreign pig breeds.Toll-like receptor signaling pathway including CD14 and TLR5 probably played immune roles in regulating E.coli F18 resistance in Chinese domestic pig breeds such as Meishan pigs,while the glycosphingolipid biosynthesis-lacto and neolacto series pathway including FUT2 was associated with the formation of E.coli F18 receptor in foreign pigs.
引文
[1]王红宁.仔猪腹泻成因及综合防治技术措施[J].中国畜牧杂志,2006,42(6):58-60.WANG H N.The cause and integrated control of piglet diarrhea[J].Chinese Journal of Animal Science,2006,42(6):58-60.(in Chinese)
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[3] VOGELI P,MEIJERINK E,FRIES R,et al.A molecular test for the detection of E.coli F18Receptors:A breakthrough in the struggle against edema disease and post-weaning diarrhea in swine[J].Schweiz Arch Tierheilkd,1997,139(11):479-484.
[4] MEIJERINK E,NEUENSCHWANDER S,FRIES R,et al.A DNA polymorphism influencing alpha(1,2)fucosyltransferase activity of the pig FUT1enzyme determines susceptibility of small intestinal epithelium to Escherichia coli F18adhesion[J].Immunogenetics,2000,52(1-2):129-136.
[5]晏学明,任军,郭源梅,等.猪a1-岩藻糖转移酶基因(FUT1)在26个中外猪种中的遗传变异研究[J].遗传学报,2003,30(9):830-834.YAN X M,REN J,GUO Y M,et al.Research on the genetic variations of a1-fucosytransferase(FUT1)gene in 26pig breeds[J].Acta Genetica Sinica,2003,30(9):830-834.(in Chinese)
[6]施启顺,谢新民,柳小春,等.猪肠毒素大肠杆菌(ETEC)F18受体基因型检测报告[J].遗传,2002,24(6):656-658.SHI Q S,XIE X M,LIU X C,et al.Experimental results on enterotoxigenic E.coli F18 receptor genotypes[J].Hereditas(Beijing),2002,24(6):656-658.(in Chinese)
[7]施启顺,黄生强,柳小春,等.不同猪种E.coli F18受体基因的多态性[J].遗传学报,2003,30(3):221-224.SHI Q S,HUANG S Q,LIU X C,et al.Polymorphism of E.coli F18receptor gene in different pig breeds[J].Acta Genetica Sinica,2003,30(3):221-224.(in Chinese)
[8]刘月环.氟烷基因与FUT1基因对猪生产性能效应的研究[D].杭州:浙江大学,2001.LIU Y H.Study on effects of halothane gene and FUT1gene for the performances of swine[D].Hangzhou:Zhejiang University,2001.(in Chinese)
[9] BAO W B,WU S L,MUSA H H,et al.Genetic variation at the alpha-1-fucosyltransferase(FUT1)gene in Asian wild boar and Chinese and Western commercial pig breeds[J].J Anim Breed Genet,2008,125(6):427-430.
[10]吴正常,董文华,刘颖,等.梅山猪E.coli F18菌株攻毒试验及其表型的鉴定分析[J].畜牧兽医学报,2014,45(10):1608-1615.WU Z C,DONG W H,LIU Y,et al.Attack experiment and phenotype analysis of Meishan piglets by E.coli F18strain[J].Acta Veterinaria et Zootechnica Sinica,2014,45(10):1608-1615.(in Chinese)
[11] WU Z C,LIU Y,DONG W H,et al.CD14in the TLRs signaling pathway is associated with the resistance to E.coli F18in Chinese domestic weaned piglets[J].Sci Rep,2016,6:24611.
[12]吴圣龙,原志伟,鞠慧萍,等.苏太仔猪FUT1基因M307位点多态性与F18大肠杆菌抗病相关性的体外鉴定[J].中国预防兽医学报,2007,29(10):783-787.WU S L,YUAN Z W,JU H P,et al.Polymorphisms of the FUT1gene M307locus in post-weaning Sutai breed piglet and resistance to F18fimbrial Escherichia coli in vitro[J].Chinese Journal of Preventive Veterinary Medicine,2007,29(10):783-787.(in Chinese)
[13] LIU L,WANG J,ZHAO Q H,et al.Genetic variation in exon 10of the BPI gene is associated with Escherichia coli F18 susceptibility in Sutai piglets[J].Gene,2013,523(1):70-75.
[14]钦伟云,甘丽娜,夏日炜,等.LTβR基因在大肠杆菌F18抗性型和敏感型苏太仔猪间的差异表达分析[J].畜牧与兽医,2016,48(7):79-82.QIN W Y,GAN L N,XIA R W,et al.Differential expression analysis of LTβR gene between Sutai E.coli F18-resistant and-sensitive pigs[J].Animal Husbandry&Veterinary Medicine,2016,48(7):79-82.(in Chinese)
[15] DONG W H,DAI C H,SUN L,et al.Expression of key glycosphingolipid biosynthesis-globo series pathway genes in Escherichia coli F18-resistant and Escherichia coli F18-sensitive piglets[J].Anim Genet,2016,47(4):428-435.
[16] WU Z C,XIA R W,YIN X M,et al.Proteomic analysis of duodenal tissue from Escherichia coli F18-resistant and-susceptible weaned piglets[J].PLoS One,2015,10(6):e0127164.
[17]戴超辉,吴嘉韵,孙丽,等.大肠杆菌刺激猪肠上皮细胞后Nramp1基因的表达变化分析[J].中国畜牧杂志,2017,53(6):40-43.DAI C H,WU J Y,SUN L,et al.Analysis of the expression changes of Nramp1gene in pig intestines epithelial cells stimulated by Escherichia coli[J].Chinese Journal of Animal Science,2017,53(6):40-43.(in Chinese)
[18] XIA R W,YIN X M,QIN W Y,et al.Genome-wide analysis of codon usage bias patterns in an enterotoxigenic Escherichia coli F18strain[J].Genes Genom,2017,39(11):1285-1295.
[19] DAI C H,WANG H F,ZHU G Q,et al.Porcine CD14gene silencing partially inhibited the bacterial immune response mediated by TLR4signaling pathway[J].Gene,2017,628:267-274.
[20]孙丽,宗秋芳,吴森,等.猪FUT2基因沉默对其所在通路基因表达及小肠上皮细胞E.coli F18黏附能力的影响[J].畜牧兽医学报,2017,48(11):2034-2045.SUN L,ZONG Q F,WU S,et al.The Effects of FUT2gene silencing on the expression of genes involved in glycosphingolipid biosynthesis-globo series pathway and the adhesion of E.coli F18to small intestinal epithelial cells[J].Acta Veterinaria et Zootechnica Sinica,2017,48(11):2034-2045.(in Chinese)
[21] YOUNG M D,WAKEFIELD M J,SMYTH G K,et al.Gene ontology analysis for RNA-seq:Accounting for selection bias[J].Genome Biol,2010,11(3):R14.
[22] MORTAZAVI A,WILLIAMS B A,MCCUE K,et al.Mapping and quantifying mammalian transcriptomes by RNA-Seq[J].Nat Methods,2008,5(7):621-628.
[23] 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.
[24] CODDENS A,VERDONCK F,TIELS P,et al.The age-dependent expression of the F18+E.coli receptor on porcine gut epithelial cells is positively correlated with the presence of histo-blood group antigens[J].Vet Microbiol,2007,122(3-4):332-341.
[25] DEPREZ P,VAN DEN HENDE C,MUYLLE E,et al.The influence of the administration of sow’s milk on the postweaning excretion of hemolytic E.coli in the pig[J].Vet Res Commun,1986,10(6):469-478.
[26] KAMADA N,N U′EZ G.Regulation of the Immune System by the Resident Intestinal Bacteria[J].Gastroenterology,2014,146(6):1477-1488.
[27] BARTON G M,MEDZHITOV R.Toll-like receptor signaling pathways[J].Science,2003,300(5625):1524-1525.
[28] AKIRA S,UEMATSU S,TAKEUCHI O.Pathogen recognition and innate immunity[J].Cell,2006,124(4):783-801.
[29] CODDENS A,DISWALL M,NGSTR M J,et al.Recognition of blood group ABH type 1determinants by the FedF adhesin of F18-fimbriated Escherichia coli[J].J Biol Chem,2009,284(15):9713-9726.
[30] MOONENS K,BOUCKAERT J,CODDENS A,et al.Structural insight in histo-blood group binding by the F18fimbrial adhesin FedF[J].Mol Microbiol,2012,86(1):82-95.
[31] LONARDI E,MOONENS K,BUTS L,et al.Structural sampling of glycan interaction profiles reveals mucosal receptors for fimbrial adhesins of enterotoxigenic Escherichia coli[J].Biology(Basel),2013,2(3):894-917.
[32] ORIOL R.ABO,Hh,Lewis,and secretion[C]//CARTRON J P,ROUGER P.Molecular bsis of human blood group antigens.Boston,M A:Springer,1995,6:37-73.
[33] WATKINS W M,GREENWELL P,YATES A D,et al.Regulation of expression of carbohydrate blood group antigens[J]. Biochimie,1988,70(11):1597-1611.
[2] BOLDIN B.Persistence and spread of gastro-intestinal infections:The case of Enterotoxigenic escherichia coli in piglets[J].Bull Math Biol,2008,70(7):2077-2101.
[3] VOGELI P,MEIJERINK E,FRIES R,et al.A molecular test for the detection of E.coli F18Receptors:A breakthrough in the struggle against edema disease and post-weaning diarrhea in swine[J].Schweiz Arch Tierheilkd,1997,139(11):479-484.
[4] MEIJERINK E,NEUENSCHWANDER S,FRIES R,et al.A DNA polymorphism influencing alpha(1,2)fucosyltransferase activity of the pig FUT1enzyme determines susceptibility of small intestinal epithelium to Escherichia coli F18adhesion[J].Immunogenetics,2000,52(1-2):129-136.
[5]晏学明,任军,郭源梅,等.猪a1-岩藻糖转移酶基因(FUT1)在26个中外猪种中的遗传变异研究[J].遗传学报,2003,30(9):830-834.YAN X M,REN J,GUO Y M,et al.Research on the genetic variations of a1-fucosytransferase(FUT1)gene in 26pig breeds[J].Acta Genetica Sinica,2003,30(9):830-834.(in Chinese)
[6]施启顺,谢新民,柳小春,等.猪肠毒素大肠杆菌(ETEC)F18受体基因型检测报告[J].遗传,2002,24(6):656-658.SHI Q S,XIE X M,LIU X C,et al.Experimental results on enterotoxigenic E.coli F18 receptor genotypes[J].Hereditas(Beijing),2002,24(6):656-658.(in Chinese)
[7]施启顺,黄生强,柳小春,等.不同猪种E.coli F18受体基因的多态性[J].遗传学报,2003,30(3):221-224.SHI Q S,HUANG S Q,LIU X C,et al.Polymorphism of E.coli F18receptor gene in different pig breeds[J].Acta Genetica Sinica,2003,30(3):221-224.(in Chinese)
[8]刘月环.氟烷基因与FUT1基因对猪生产性能效应的研究[D].杭州:浙江大学,2001.LIU Y H.Study on effects of halothane gene and FUT1gene for the performances of swine[D].Hangzhou:Zhejiang University,2001.(in Chinese)
[9] BAO W B,WU S L,MUSA H H,et al.Genetic variation at the alpha-1-fucosyltransferase(FUT1)gene in Asian wild boar and Chinese and Western commercial pig breeds[J].J Anim Breed Genet,2008,125(6):427-430.
[10]吴正常,董文华,刘颖,等.梅山猪E.coli F18菌株攻毒试验及其表型的鉴定分析[J].畜牧兽医学报,2014,45(10):1608-1615.WU Z C,DONG W H,LIU Y,et al.Attack experiment and phenotype analysis of Meishan piglets by E.coli F18strain[J].Acta Veterinaria et Zootechnica Sinica,2014,45(10):1608-1615.(in Chinese)
[11] WU Z C,LIU Y,DONG W H,et al.CD14in the TLRs signaling pathway is associated with the resistance to E.coli F18in Chinese domestic weaned piglets[J].Sci Rep,2016,6:24611.
[12]吴圣龙,原志伟,鞠慧萍,等.苏太仔猪FUT1基因M307位点多态性与F18大肠杆菌抗病相关性的体外鉴定[J].中国预防兽医学报,2007,29(10):783-787.WU S L,YUAN Z W,JU H P,et al.Polymorphisms of the FUT1gene M307locus in post-weaning Sutai breed piglet and resistance to F18fimbrial Escherichia coli in vitro[J].Chinese Journal of Preventive Veterinary Medicine,2007,29(10):783-787.(in Chinese)
[13] LIU L,WANG J,ZHAO Q H,et al.Genetic variation in exon 10of the BPI gene is associated with Escherichia coli F18 susceptibility in Sutai piglets[J].Gene,2013,523(1):70-75.
[14]钦伟云,甘丽娜,夏日炜,等.LTβR基因在大肠杆菌F18抗性型和敏感型苏太仔猪间的差异表达分析[J].畜牧与兽医,2016,48(7):79-82.QIN W Y,GAN L N,XIA R W,et al.Differential expression analysis of LTβR gene between Sutai E.coli F18-resistant and-sensitive pigs[J].Animal Husbandry&Veterinary Medicine,2016,48(7):79-82.(in Chinese)
[15] DONG W H,DAI C H,SUN L,et al.Expression of key glycosphingolipid biosynthesis-globo series pathway genes in Escherichia coli F18-resistant and Escherichia coli F18-sensitive piglets[J].Anim Genet,2016,47(4):428-435.
[16] WU Z C,XIA R W,YIN X M,et al.Proteomic analysis of duodenal tissue from Escherichia coli F18-resistant and-susceptible weaned piglets[J].PLoS One,2015,10(6):e0127164.
[17]戴超辉,吴嘉韵,孙丽,等.大肠杆菌刺激猪肠上皮细胞后Nramp1基因的表达变化分析[J].中国畜牧杂志,2017,53(6):40-43.DAI C H,WU J Y,SUN L,et al.Analysis of the expression changes of Nramp1gene in pig intestines epithelial cells stimulated by Escherichia coli[J].Chinese Journal of Animal Science,2017,53(6):40-43.(in Chinese)
[18] XIA R W,YIN X M,QIN W Y,et al.Genome-wide analysis of codon usage bias patterns in an enterotoxigenic Escherichia coli F18strain[J].Genes Genom,2017,39(11):1285-1295.
[19] DAI C H,WANG H F,ZHU G Q,et al.Porcine CD14gene silencing partially inhibited the bacterial immune response mediated by TLR4signaling pathway[J].Gene,2017,628:267-274.
[20]孙丽,宗秋芳,吴森,等.猪FUT2基因沉默对其所在通路基因表达及小肠上皮细胞E.coli F18黏附能力的影响[J].畜牧兽医学报,2017,48(11):2034-2045.SUN L,ZONG Q F,WU S,et al.The Effects of FUT2gene silencing on the expression of genes involved in glycosphingolipid biosynthesis-globo series pathway and the adhesion of E.coli F18to small intestinal epithelial cells[J].Acta Veterinaria et Zootechnica Sinica,2017,48(11):2034-2045.(in Chinese)
[21] YOUNG M D,WAKEFIELD M J,SMYTH G K,et al.Gene ontology analysis for RNA-seq:Accounting for selection bias[J].Genome Biol,2010,11(3):R14.
[22] MORTAZAVI A,WILLIAMS B A,MCCUE K,et al.Mapping and quantifying mammalian transcriptomes by RNA-Seq[J].Nat Methods,2008,5(7):621-628.
[23] 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.
[24] CODDENS A,VERDONCK F,TIELS P,et al.The age-dependent expression of the F18+E.coli receptor on porcine gut epithelial cells is positively correlated with the presence of histo-blood group antigens[J].Vet Microbiol,2007,122(3-4):332-341.
[25] DEPREZ P,VAN DEN HENDE C,MUYLLE E,et al.The influence of the administration of sow’s milk on the postweaning excretion of hemolytic E.coli in the pig[J].Vet Res Commun,1986,10(6):469-478.
[26] KAMADA N,N U′EZ G.Regulation of the Immune System by the Resident Intestinal Bacteria[J].Gastroenterology,2014,146(6):1477-1488.
[27] BARTON G M,MEDZHITOV R.Toll-like receptor signaling pathways[J].Science,2003,300(5625):1524-1525.
[28] AKIRA S,UEMATSU S,TAKEUCHI O.Pathogen recognition and innate immunity[J].Cell,2006,124(4):783-801.
[29] CODDENS A,DISWALL M,NGSTR M J,et al.Recognition of blood group ABH type 1determinants by the FedF adhesin of F18-fimbriated Escherichia coli[J].J Biol Chem,2009,284(15):9713-9726.
[30] MOONENS K,BOUCKAERT J,CODDENS A,et al.Structural insight in histo-blood group binding by the F18fimbrial adhesin FedF[J].Mol Microbiol,2012,86(1):82-95.
[31] LONARDI E,MOONENS K,BUTS L,et al.Structural sampling of glycan interaction profiles reveals mucosal receptors for fimbrial adhesins of enterotoxigenic Escherichia coli[J].Biology(Basel),2013,2(3):894-917.
[32] ORIOL R.ABO,Hh,Lewis,and secretion[C]//CARTRON J P,ROUGER P.Molecular bsis of human blood group antigens.Boston,M A:Springer,1995,6:37-73.
[33] WATKINS W M,GREENWELL P,YATES A D,et al.Regulation of expression of carbohydrate blood group antigens[J]. Biochimie,1988,70(11):1597-1611.