【作者】 刘洪瑜；

【导师】 昝林森；

【作者基本信息】 西北农林科技大学 ， 动物学， 2009， 博士

【摘要】 本研究以秦川牛、南阳牛、郏县红牛、夏南牛、鲁西牛、安格斯牛、安西牛7个品种的787个个体为材料,采用PCR-SSCP、PCR-RFLP、DNA测序技术及DNA序列分析技术,结合生物信息学方法,研究了影响牛脂肪代谢的leptin、MC4R、ApoA1、ApoA4 4个基因的全序列及MC4R和ApoA4基因的5’非翻译区及3’侧翼区的遗传变异,以探讨试验牛群的遗传结构和遗传多样性。测定了其中101头秦川牛的胴体组成和肉质性状等主要经济指标,对以上基因的遗传突变位点与秦川牛部分经济性状进行关联分析,以期筛选出显著影响秦川牛肉质性状的功能基因或分子标记,并利用生物信息学方法初步分析了部分分子标记的作用机理,为秦川牛胴体、肉质等经济性状选育的分子标记辅助选择和优质高产新品系(种)培育提供科学依据。试验得到以下主要结果:1、采用PCR-SSCP及测序的方法,研究了牛leptin基因第2、3外显子的遗传变异情况。在leptin基因第2外显子发现73 C>T的突变位点,7个试验牛群体在该位点均为中度多态;秦川牛、鲁西牛、南阳牛、郏县红牛四个群体在该位点处于Hardy-Weinberg非平衡状态,夏南牛、安格斯牛和安西牛在该位点处于Hardy-Weinberg平衡状态;该位点多态性与秦川牛宰前活重、背膘厚极显著相关(P<0.01),与眼肌面积显著相关(P<0.05),CC型和CT型的宰前活重、背膘厚极显著高于TT型(P<0.01),CC型的眼肌面积显著高于CT型和TT型(P<0.05);生物信息学研究发现该位点突变导致了编码蛋白的精氨酸突变为半胱氨酸,导致蛋白二级结构24、146处的α螺旋丢失和20、144处的β折叠丢失,改变了三级结构中β转角的空间位置,这些变化可能与该基因功能的改变有关;编码蛋白的氨基酸改变导致了疏水性结构改变,半胱氨酸编码蛋白的疏水性最小值为-1.689,位于第102个氨基酸处,精氨酸编码蛋白的疏水性最小值为-2.222,位于第29个氨基酸处,未引起最大值的改变;突变并未引起该蛋白松散度的改变。leptin基因第3外显子前段扩增片段呈现AA和AB两种带型,AA型在第3外显子95 bp处的C变为T,AB型第3外显子在267 bp处的T变为C,在252 bp和351 bp处的C均变为T;该位点在试验牛群体中均为低度多态、处于Hardy-Weinberg平衡状态,该位点不同基因型秦川牛个体之间与秦川牛肉质性状间无显著差异(P>0.05)。leptin基因第3外显子后段扩增片段没有多态性。2、生物信息学分析表明,不同物种MC4R基因编码蛋白质的氨基酸序列在中间部分的结构较为相似。通过DNA池测序技术发现牛MC4R基因存在5’端-293C>G、-193A>T、-192T>G、-129A>G、-84T>C以及位于外显子1069 C>G的突变位点。通过PCR-RFLP检测发现MC4R基因-293C>G和-129A>G位点为连锁突变,秦川牛、鲁西牛、南阳牛、夏南牛、郏县红牛、安格斯牛在-129A>G位点处于中度多态,安西牛群体处于低度多态;安西牛在该位点处于Hardy-Weinberg非平衡状态,其他牛群均达到平衡状态;-129A>G多态位点与秦川牛宰前活重之间存在显著相关,GG型的个体在宰前活重上显著高于AA型和AG型(P<0.05)。7个试验牛群体在1069 C>G位点处于中度多态,且均处于Hardy-Weinberg平衡状态;1069 C>G多态位点与秦川牛宰前活重、胴体重、背膘厚、大理石花纹等级显著相关。GG型的宰前活重和胴体重显著高于CC型(P<0.05)而CG型与CC、GG型之间差异不显著(P>0.05),GG、CG型的背膘厚显著高于CC型(P<0.05),GG型的大理石花纹等级显著高于CC型(P<0.05),而CG型与CC、GG型之间差异不显著(P>0.05);1069 C>G位点突变导致了编码氨基酸由亮氨酸突变为缬氨酸,缬氨酸编码的蛋白二级结构在280处产生α螺旋丢失,281处β折叠丢失,无规则卷曲没有发生变化,没有改变蛋白的三级结构、疏水性及松散度。3、通过PCR-SSCP及测序分析,发现APOA1基因第411碱基处C>G的突变,该突变位于第二内含子,秦川牛、鲁西牛、夏南牛、郏县红牛在该位点中处于中度多态,南阳牛、安格斯牛、安西牛群体在该位点为低度多态;秦川牛在该位点处于Hardy-Weinberg非平衡状态,其他牛群均达到平衡状态;该位点与肉质和胴体性状无相关性。通过6个随机个体测序及比对发现,在ApoA1基因第1523碱基处T>G的突变,第1572碱基处C>A的突变,均发生在ApoA1基因第3外显子,但未引起氨基酸的变化。通过PCR-RFLP检测发现,ApoA1基因1523 T>G位点在鲁西牛、南阳牛、郏县红牛群体中为中度多态,在秦川牛、夏南牛、安格斯牛、安西牛群体中为低度多态;鲁西牛、南阳牛、郏县红牛在该位点处于Hardy-Weinberg非平衡状态,其他牛群均达到平衡状态;该多态位点与秦川牛背膘厚显著相关,GT型个体显著高于TT型个体(P<0.05)。ApoA1基因1572 C>A位点除安西牛外其他牛种均处于中度多态;秦川牛、夏南牛在该位点处于Hardy-Weinberg非平衡状态,其他牛群均达到平衡状态;该多态位点与秦川牛背膘厚显著相关,CC基因型显著高于AC型和AA型(P<0.05)。4、采用电子克隆(e-PCR)及反转录PCR(RT-PCR),从秦川牛脂肪组织中克隆并鉴定了牛ApoA4基因cDNA的编码区,并用生物信息学方法对ApoA4蛋白进行了预测分析,发现该蛋白没有跨膜结构,在其20～21位氨基酸处很可能存在信号肽区域,并预测了其二级结构与三级结构。通过6个随机个体测序,并未发现该基因存在SNP位点。更多还原

【Abstract】 To identify the genetic characteristics and to explore usable molecular markers with significant effects on economic important traits for efficient selection and improvement of Qinchuan cattle, PCR-SSCP, PCR-RFLP, DNA sequencing and bioinformatics techniques were applied to detect genetic variations of lipid metaboloism related genes among 787 individuals of seven breeds (Qinchuan, Nanyang, Jiaxian Red, Xianan, Luxi, Angus and Anxi). 4 genes (leptin、MC4R、ApoA1、ApoA4) , 5’ untranslation region and 3’ region of MC4R and ApoA4 had been analyzed. 101 indivduals among Qinchuan cattle were slaughted and some economic traits, i.e. meat quality and carcass composition, were determinated. Association analyses were carried out to evaluate the effects of genotypes of candidate genes on economic traits of Qinchuan cattle. And molecular markers function was analysed by bioinformation. The objects of this study were to discover the hereditary characteristics and to explore molecular markers with significant effects on economic important traits for efficient selection and improvement of Qinchuan breed. Finally, possible functions of variations identified in these candidiate genes were predicted by bioinformational methods. Results were shown as follows:1. PCR-SSCP and DNA sequencing method were used to study genetic variations of leptin gene exon 2 and exon 3. A 73C>T mutation was identified in exon 2, this SNP was moderate polymorphic in the seven populations; Qinchuan, Luxi, Nanyang, Jiaxian Red were at Hardy-Weinberg equiliabration and Xianan, Angus, Anxi were at Hardy-Weinberg disequiliabration; this SNP was high significantly associated with live weight and backfat thickness (P<0.01), was significantly associated with loin muscle area (P<0.05); CC type and CT type have high live weight and backfat thickness than TT type (P<0.01), CC type has high live weight and backfat thickness than CT type and TT type (P<0.05). This mutation lead Arg change into Cys, which hadα-helix lost in 24 and 146 position,β-sheet lost in 20 and 144 position in 2D structure and had aβ-turn change in 3D structure. The hydrophobicity structure min value change from -2.222 (29th AA) to -1.689 (102nd AA), and the loose degree did not change. The LP2 fragment in exon3 showed AA and AB type, AA has a mutation of 96 C>T, AB has 252C>T, 267T>C and 351C>T mutations. These SNPs were low polymorphic in the seven populations. The seven populations were at Hardy-Weinberg equiliabration in these SNPs. These SNPs were no significantly associated with economic in Qinchuan cattle (P>0.05). The LP3 fragment in exon3 showed no SNP.2. Bioinformatics analysis showed MC4R protein had highly similary strcture in AA sequence in differenet species. The result of DNA pool sequencing showed that 5’ region (-293C>G, -193A>T, -192T>G, -129A>G, -84T>C) and exon (1069 C>G) SNPs in cattle MC4R gene. The PCR-RFLP result showed that -293C>G and -129A>G are linkage. Qinchuan, Luxi, Nanyang, Xianan, Jiaxian Red, and Angus populations were moderate polymorphic and were at Hardy-Weinberg equiliabration at -129A>G SNP. Anxi was low polymorphic and was at Hardy-Weinberg disequiliabration at -129A>G SNP. The -129A>G SNP was significantly associated with live weight in Qinchuan cattle (P<0.05), GG type had high live weight than AA type and AG type. All the 7 populations were moderate polymorphic and were at Hardy-Weinberg equiliabration at 1069 C>G SNP. The 1069 C>G SNP was significantly associated with live weight, carcass weight, backfat thickness and marbling scorce in Qinchuan cattle (P<0.05). GG type had high live weight, carcass weight than CC type (P<0.05), GG and CG type had high backfat thickness than CC type (P<0.05), GG type had high marbling scorce than CC type (P<0.05). The 1069 C>G mutation lead Leu change into Val, which hadα-helix lost in 280 position andβ-sheet lost in 281 in Val in 2D structure. The 3D structure, hydrophobicity structure and the loose degree did not change.3. PCR-SSCP and DNA sequencing method were used to detect a 411 C>G in APOA1 gene. This SNP located in intron 2 and was moderate polymorphic in Qinchuan, Luxi, Xianan and Jiaxian Red populations, was low polymorphic in Nanyang, Angus and Anxi populations. Qinchuan cattle were at Hardy-Weinberg disequiliabration at this SNP and other 6 populations were at Hardy-Weinberg equiliabration. The SNP was not significantly associated with economic in Qinchuan cattle (P>0.05). Through 6 random samples sequcening and alignment, 1523 T>G and 1572 C>A mutations were detected. These two SNPs were all located in exon 3 and did not lead to AA change. The 1523 T>G SNP was moderate polymorphic in Luxi, Nanyang and Jiaxian Red populations, was low polymorphic in Qinchuan, Xianan, Angus and Anxi populations. Luxi, Nanyang and Jiaxian Red populations were at Hardy-Weinberg disequiliabration at this SNP and other 4 populations were at Hardy-Weinberg equiliabration. The 1523 T>G SNP was significantly associated with backfat thickness in Qinchuan cattle. GT type had high backfat thickness than TT type (P<0.05). The 1572 C>A SNP was moderate polymorphic in all the 6 populations except Anxi population. Qinchuan and Xianan populations were at Hardy-Weinberg disequiliabration at this SNP and other 5 populations were at Hardy-Weinberg equiliabration. The 1572 C>A SNP was significantly associated with backfat thickness in Qinchuan cattle. CC type had high backfat thickness than AC and AA type (P<0.05).4. cDNA sequences of ApoA4 gene, which contained complete CDS, were e-cloned and identified from fat of Qinchuan cattle. ApoA4 protein had no transmembrane, probably has signal peptide in 20th to 21st AA. The 2D and 3D structure has been prediceted by bioinformatics methods. 6 random samples sequcening and alignment method were used to detect SNPs, no SNP was been found in ApoA4 gene.更多还原