【作者】 侯冠彧；

【导师】 许尚忠；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 本研究以西门塔尔牛、安格斯牛、鲁西牛、秦川牛、晋南牛、夏洛莱牛、利木赞牛、海福特牛和西门塔尔牛×蒙古牛9个群体共323个个体为实验材料。利用生物信息学PCR-RFLP、PCR-SSCP、实时荧光定量PCR、DNA测序、DNA序列分析技术,克隆和分析了牛CAV1和CAV3基因,并研究了CAV1、CAV3、CAPN1、CAPN3、CAST、HRSP12和CACNA2D1共7个候选基因的遗传变异,同时探讨了在试验群体中的遗传特征及其与肉质和胴体组成性状(宰前活重、胴体重、屠宰率、净肉率、大理石花纹评分、眼肌面积、肉色、脂肪颜色、背膘厚、嫩度和日增重)的关系,并检测了CAV1、CAV3、CAPN1和CAPN3基因的mRNA表达量,旨在获取相应的分子遗传学信息,找到与牛胴体和肉品质性状相关的DNA标记,为中国地方牛品种选育提高、品种改良及分子标记辅助选择提供科学依据。获得的主要结果如下:1.采用电子克隆(e-PCR)和反转录PCR(RT-PCR)等技术,从西门塔尔牛肌肉组织中克隆分离并得到了牛CAV1基因、CAV3基因cDNA的编码区(CDS)全长和部分非翻译区序列。2.推导CAV1、CAV3蛋白的氨基酸序列,并通过生物信息学方法对其特征和功能进行了预测分析,预测结果表明牛CAV1、CAV3基因分别编码178和151个氨基酸,它们都含有22个氨基酸的跨膜特征功能结构域。结构域序列保守程度极高。3. CAV1、CAV3、CAPN1、CAPN3、CAST、HRSP12和CACNA2D1基因在不同牛品种中,经扩增、克隆、测序和比对发现32个SNPs,其中颠换突变13个,转换突变19个。SNP突变中转换突变占突变的59.4%。4.牛CAV1、CAV3、CAPN1、CAPN3、CAST、HRSP12和CACNA2D1基因遗传变异位点与肉质和胴体组成性状存在效应差异,结果发现:(1)CAPN1-StuⅠ位点在大理石花纹评分和嫩度指标上,AA型显著大于BB型(P<0.05)。CAPN1-AgeⅠ位点在宰前活重指标上,AB、BB型个体显著大于AA型(P<0.05)。CAPN1-MaeⅡ位点在宰前活重指标上,BB型个体显著大于AA型个体(P<0.01);在屠宰率、净肉率、眼肌面积和嫩度指标上,AA型个体极显著大于AB、BB型个体(P<0.01),在日增重指标上,AB、BB型个体显著大于AA型(P<0.05)。SSCP-CAPN1α位点在嫩度上,AA型显著大于AB和BB型(P<0.01);在日增重上,AB和BB型显著大于AA型(P<0.05)。SSCP-CAPN1β位点在大理石花纹评分和嫩度上,AA型个体显著大于AB型(P<0.01);在背膘厚上,AA型个体显著大于BB型(P<0.05)。(2)CAPN3-TaqⅠ位点不同基因型与胴体重之间存在显著相关:AB型个体的胴体重显著高于BB型个体(P<0.05)。(3)CAST基因在大理石花纹评分上,BB型显著高于AA型(P<0.05);在嫩度指标上,BB型显著高于AA型和AB型(P<0.01)。(4)HRSP12-BsrⅠ位点在宰前活重和胴体重指标上,AB型个体显著大于BB型(P<0.01);在大理石花纹评分上,AA型个体显著大于BB和AB型(P<0.05)。(5)CAV1-AvaⅡ位点在宰前活重、胴体重、大理石花纹评分和嫩度指标上,BB型显著高于AA型(P<0.01)。CAV1-HinfⅠ位点在宰前活重和日增重指标上,BB型显著高于AA型(P<0.05)。CAV1-BglⅠ位点在宰前活重指标上,BB型显著高于AA型(P<0.05)。CAV1-MspⅠ位点在宰前活重、胴体重、眼肌面积和嫩度指标上,AA型显著高于AB型和BB型(P<0.05)。(6)CAV3基因在宰前活重指标上,BB型显著高于AB型(P<0.05);在屠宰率指标上,AA型显著高于BB型(P<0.05);在净肉率指标上,BB型显著高于AA型和AB型(P<0.05)。(7)CACNA2D1-HaeIII位点在胴体重和屠宰率指标上,BB型显著高于AB型(P<0.01);在净肉率指标上,AA型显著高于AB型(P<0.01);在背膘厚指标上,BB型显著高于AB型和AA型(P<0.01)。5.采用荧光定量PCR方法分析了CAPN1、CAPN3、CAV1、CAV3 4个基因在胎儿期、12月龄、24月龄和36月龄4个不同时期西门塔尔牛9种组织中的表达情况,结果如下:(1)CAPN1基因在肺脏、肌肉、肾脏、瘤胃、脾脏中胎儿期的mRNA表达量均显著高于其它三个时期(P<0.01)。(2)CAPN3基因在肌肉和心脏组织中胎儿期的表达量显著高于其它三个时期(P<0.01)。(3)CAV1基因在12月龄、24月龄、36月龄的脂肪中mRNA表达量显著高于胎儿期(P<0.01),CAV1基因在24月龄、36月龄的肌肉mRNA表达量显著高于12月龄和胎儿期(P<0.01)。(4)CAV3基因在24月龄、36月龄肌肉中的mRNA表达量显著高于12月龄和胎儿期(P<0.01),CAV3基因在12月龄、24月龄、36月龄小肠中的表达量显著高于胎儿期(P<0.01)。更多还原

【Abstract】 The bioinformatics, PCR-RFLP, PCR-RFLP, RT-PCR, DNA sequencing and DNA sequence analysis techniques were applied to (1) clone bovine CAV1 and CAV3 gene, (2) analyze the genetic variations and characters of CAV1、CAV3、CAPN1、CAPN3、CAST、HRSP12 and CACNA2D1 genes and (3) their association with meat quality traits and carcass composition traits, including Live weight (LW), Carcass weight (CW), Dressing percentage (DP), Meat percent(MP), Marbling score (MS), Loin muscle area (LMA), Meat color (MC), Fat color (FC), Backfat thickness (BF), Meat tenderness(MT) and Average daily gain(ADG) in 323 cattle, which comprised of nine breeds, namely, Simmental (n=110), Angus (n=48), Luxi (n=30), Qinchuan (n=29), Jinnan (n=24), Charolais (n=30), Limousin (n=25), Hereford (n=30) and Simmental×Mongolian, (4) analyze the expression of CAV1、CAV3、CAPN1 and CAPN3 gene. These results will benefit for the application of DNA marker related to the economic traits on marker-assist-selection (MAS), improvement and promotion of local cattle breeds. The main results were shown below:1.Whole coding sequence (CDS) and partial untranslated sequence (UTR) of bovine CAV1 and CAV3 gene were isolated from the muscle of Simmental by e-PCR and RT-PCR techniques.2. The amino acids sequence deduced by ORF of CAV1 and CAV3 cDNA were analyzed while the character and protein domain were predicted by bio-informatics, The results showed that bovine CAV1 and CAV3 protein were consisted of 178 and 151 amino acids respectively. They both contained Transmembrane domains (was consisted of 22 amino acids). The amino acids sequence in the domain showed high conservation in different species.3. 32 SNPs were identified by PCR-RFLP, PCR-SSCP and sequencing in bovine CAV1、CAV3、CAPN1、CAPN3、CAST、HRSP12 and CACNA2D1 gene and 19 of them were transition mutation.4. Genetic variations in bovine CAV1、CAV3、CAPN1、CAPN3、CAST、HRSP12 and CACNA2D1 genes were associated with carcass and meat quality traits. The results showed that:(1) CAPN1-StuⅠsite was significantly associated with MS and MT. Animals with AA genotype were significantly higher than those with BB genotype (P<0.05). CAPN1-AgeⅠsite was significantly associated with LW. Animals with AB and BB genotype were significantly higher than those with AA genotype (P<0.05). CAPN1-MaeⅡsite was significantly associated with LW, DP, MP, LMA, MT and ADG. Animals with BB genotype had significantly higher LW than those with AA (P<0.01). Animals with AA genotype had significantly higher DP, MP, LMA and MT than those with AB and BB (P<0.01). Animals with AA and BB genotype had significantly higher ADG than those with AA genotype (P<0.05). SSCP-CAPN1αsite was significantly associated with MT ADG and FC. Animals with AA genotype had significantly higher MT (P<0.05) and lower ADG and FC (P<0.01) than those with AB and BB genotype. SSCP-CAPN1βsite was significantly associated with BF, MS and MT. Animals with AA genotype had higher MS and MT than those with AB genotype (P<0.01), and had higher BF than those with BB genotype (P<0.01).(2) CAPN3-TaqⅠsite of CAPN3 gene was significantly associated with CW. Animals with AB genotype had larger CW than those with BB genotype (P<0.05).(3) CASTαsite was significantly associated with MS and MT. Animals with BB genotype had larger MS than those with AA genotype (P<0.05). Animals with BB genotype had larger MT than those with AA or AB genotype (P<0.01).(4) HRSP12-BsrⅠsite of HRSP12 gene was significantly associated with LW, CW and MS. Animals with AB genotype had larger LW and CW than those with BB genotype (P<0.01). Animals with AA genotype had larger MS than those with BB and AB genotype (P<0.05).(5) Animals with BB genotype in CAV1-AvaⅡsite had larger LW, CW, MS and MT than those with AA genotype (P<0.01). Animals with BB genotype in CAV1-HinfⅠsite had larger LW and ADG than those with AA genotype (P<0.05). Animals with BB genotype in CAV1-BglⅠsite had larger LW than those with AA genotype (P<0.05). Animals with AA genotype in CAV1-MspⅠsite had larger LW, CW, LMA and MT than those with AB and BB genotype (P<0.05).(6) CAV3 gene was significantly associated with LW, DP and MP. Animals with BB genotype had larger LW than those with AB genotype (P<0.05). Animals with AA genotype had larger DP than those with BB genotype (P<0.05). Animals with BB genotype had larger MP than those with AA and AB genotype (P<0.05).(7) CACNA2D1-HaeIII site was significantly associated with CW, DP, MP and BF. Animals with BB genotype had larger CW and DP than those with AB genotype (P<0.01). Animals with AA genotype had larger MP than those with AB genotype (P<0.01). Animals with BB genotype had larger BF than those with AA and AB genotype (P<0.01) and there was no significant difference between animals with AA and AB genotype. 5. Expression of four candidate genes (CAPN1、CAPN3、CAV1、CAV3) were detected in 9 tissues, which were collected from four different physiological phases: embryonic phase、12 months old、24 months old and 36 months old, by RT-PCR and Real-time Q-PCR, and the results showed that:(1) The CAPN1 mRNA expression in lung, muscle, kidney, rumen and spleen in embryonic phase was significantly higher than the other phases.(2) The CAPN3 mRNA expression in muscle and heart in embryonic phase was higher than the other three phases (P<0.01).(3) The CAV1 mRNA expression in fat tissue in the 12 months old, 24months old and 36months old was significantly higher than the embryonic phase (P<0.01). The CAV1 mRNA expression in muscle in 24months old and 36months old was significantly higher than the 12 months old and embryonic phase (P<0.01).(4) The CAV3 mRNA expression in muscle in the 24months old and 36 months old was higher than the embryonic phase and 12 months old (P<0.01). The CAV3 mRNA expression in small intestine in the 12 months old,24months old and 36months old was significantly higher than the embryonic phase (P<0.01).更多还原