【作者】 曹晓华；

【导师】 王金勇；

【作者基本信息】 西南大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 杀菌/通透性增强蛋白(bactericidal/permeability-increasing protein,BPI)是人和哺乳动物的内源性阳离子蛋白质,主要存在于多形核白细胞的嗜苯胺蓝颗粒中。它具有很强的杀菌(主要指革兰氏阴性细菌)、中和内毒素或脂多糖(lipopolysaccharide,LPS)的活性和调理功能,在动物机体天然防御中起到很重要的作用。目前,人、牛、鼠、的BPI基因序列都已清楚。人BPI基因外显子3和4区域存在多态性,猪BPI基因第4和10外显子的RFLP多态性与沙门氏菌的易感性有关,并被确定为抗病育种的候选基因。在我们的前期研究中克隆了荣昌猪BPI基因的全长cDNA序列,并发现荣昌猪BPI基因外显子3和外显子4上存在丰富的多态位点。不同猪种(地方猪种、引进品种和培育品种)之间BPI基因外显子3和外显子4区域的多态性如何,是否存在差异,是本课题需要在前期研究中需要进一步研究的问题。本研究以渝荣Ⅰ号配套猪B系、A系、C系三个专门化品系和荣昌猪为实验对象,应用聚合酶链式反应—单链构象多态性(polymerase chain reaction-single strand conformationpolymorphism,PCR-SSCP)技术,检测四个猪种BPI基因第3和4外显子的单核苷酸多态性(singlenucleotide polymorphism,SNP),并将显示不同带型的PCR扩增产物克隆测序,利用DNAman分子生物学软件对测序结果进行分析比对,找出BPI基因外显子3和外显子4的突变位点,统计不同猪种的基因型频率,计算等位基因频率和各SNP位点的优势碱基。通过试验分析得出外显子3的SNP结果如下:荣昌猪BPI基因外显子3存在两个SNP位点,即第376位的A→C突变和397位的A→G突变变。A376C位突变引起氨基酸由Gln替换为Pro,氨基酸的疏水性由0.28增加到0.60。A397G位引起氨基酸由Gln替换为Arg,且产生带正电荷的Arg。B系猪外显子3存在两个突变,即第371位的T→C突变和445位的A→G,T371C的突变未引起氨基酸的变化。A445 G位引起氨基酸由Lys替换为Arg,疏水性减弱。C系猪产生了一个突变,即435的A→G突变,氨基酸由Ile替换为Val,且产生的氨基酸疏水性降低。A系猪未检测到SNP位点。外显子4上的SNP情况:荣昌猪外显子4区段共检测到6个SNP位点,它们分别是第512位C→T(C512T)、第551位T→G(T551G)、第563位C→T(C563T)、第573位T→C(T573C)、第599位G→A(G599A)和第607位T→C(T607C)突变。其中C512T,T551G,C563T,G599A为无义突变。T551G突变使限制性内切酶MscⅠ或BalⅠ酶切位点(TGG/CCA)消失。第573位的T→C突变,引起了氨基酸由Ser替换为Pro,突变引起氨基酸疏水性降低。第607位T→C的突变引起了氨基酸由Val替换为Ala,使得氨基酸疏水性降低,SunⅠ或SplⅠ酶切位点(C/GTACG)也消失。荣昌猪BPI基因外显子4区段的高度多态性,表明该区段有较大的遗传选择潜力。B系猪存在3个SNP位点,即512位的C→T(C512T)、551位的T→G(T551G)和599位的G→A(G599A)突变。此三个突变未引起氨基酸的变化。T551G突变使限制性内切酶MscⅠ或BalⅠ酶切位点(TGG/CCA)消失。A系C系猪仔外显子4未检测到SNP位点。从四个猪种外显子3和外显子4的SNPs位点的分析可以看出,荣昌猪的多态信息含量丰富,此外B系猪也较丰富的多态性,A系C系多态性不丰富。本试验检测到的外显子3和4区段的某些SNP位点,可能成为猪机体抗某些革兰氏阴性病原菌或其引起的疾病的分子标记。更多还原

【Abstract】 Bactericidal/permeability-increasing protein(BPI)is a highly cationic protein which is located mainly in the primary granules of polymorphonuclear leucocyte(PMN).BPI has a high affinity for LPS of Gram-negative bacteria.Binding of BPI to susceptible bacteria is followed by increasing outer membrane permeability,inhibition of growth and ultimately,irreversible loss of viability.In addition,BPI also possesses an opsonic function.Because of these important functions,BPI plays an important role in host initial defense.At present,the full-length cDNA sequences of BPI gene has been elucidated in several species,including human,bovine,brown rat,mouse,and pig of rongchang. In addition,several polymorphic sites were found in exons 3 and 4 of human BPI gene.It has been shown that the restriction fragment length polymorphism(RFLP)sites in exons 4 and 10 of porcine BPI gene are related to the susceptibility of Salmonella cholerasuis in several pig breeds.The porcine BPI gene was considered as a candidate gene of breeding for disease resistance.we have found several polymorphic sites in exons 3 and 4 of rongchang pig in our investigation we have done.However,Are different of polymorphism of exons 3 and 4 between Rongchang pig and other strain pigs(native,import,crossbreed pigs)BPI gene,and are the differens obvious is unclear.In this study,the pig breeds be investigated are rongchang,A,B and Cstrain pigs which were pure breeds elected by four generations.the SNP sites in exons 3 and 4 of this gene were detected by polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP)analysis.Then. the PCR products showing different patterns were purified and cloned for sequencing.Finally,the frequencies of difference genotypes were accounted and the allelic genes frequencies were calculated according to the former result.Further more,the SNP sites in exons 3 and 4 were found by sequence alignment of different allelic genes.Frequencies of the bases with higher appearance frequency in these SNP sites were calculated according to the distribution of SNP sites in all the allelic genes.In the exon 3 fragment,There was two novel SNPs(A376C,A397G)detected in the exon 3 of Rongchang pig BPI gene by PCR-SSCP.The A376C made Gln94Pro substitution,the hydrophobicity increase from 0.28 to 0.60.the A397G mutation made Arg101 Gin substitution,two novel SNPs(T371C,A445G)were detected in the exon 3 of B strain pigs BPI gene by PCR-SSCP,the T371C mutation was synonymous substitutions.the A445G mutation made Lys 117Arg substitution,and the hydrophobicity drop from -0.89 to -1.05.There was one novel SNP(A435G) detected in the exon 3 of Cstrain pigs BPI gene by PCR-SSCP,it made Lle114Val substitution,the hydrophobicity drop from 0.83 to 0.78.there was no novel SNP be found in A lineage pig.In the exon 4 fragment,six novel(T512C,G551T,C563T,T573C,G599A,T607C)of SNPs were detected,four of them(T512C,G551T,C563T,G599A)were synonymous substitutions.The G551T novel made a restriction endonuclease Msc I orBal I(TGG/CCA)disappear.the novel(T573C) made Ser160Pro substitution,and the hydrophobicity drop from 0.6 to 0.46.the novel(T607C)made Val171Ala substitution,hydrophobicity drop from -1.21 to -1.61.Three SNPs which were same whith three of the rongchang pig’s SNPs were found in B strain of pigs,there were T512C, G551T,G599A.SNPs were not found in A and C strain pigs.The affluent polymorphism of exon 4 indicate its huge potentiality in genetic selection.Some of the SNP sites detected in this study may be important molecular markers of porcine disease resistance,specifically resistant to Gram-negative bacterias and its caused diseseses.更多还原