【作者】 邓桂馨；

【导师】 许尚忠；

【作者基本信息】 中国农业科学院 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 本研究以西门塔尔X黄牛、日本和牛X复州牛2个杂交品种共200个个体为试验动物。利用DNA测序、序列分析和PCR-RFLP等技术,结合生物信息学方法,克隆和分析牛Cathepsin家族CTSB、CTSD、CTSF、CTSH、CTSL及其抑制基因CSTB,共6个候选基因的遗传变异,探讨试验群体遗传结构、遗传多样性及其与经济性状的关系,从而获取相应的分子遗传学信息,筛选对牛经济性状有重要影响的分子标记,为牛的分子标记辅助选择研究提供科学依据;同时为牛业生产的优质新品种或杂交新品系培育提供早期检测方法,以节约生产成本提高经济效益。主要研究结果如下:1.采用反转录PCR(RT-PCR)技术,从试验牛肌肉组织中克隆并鉴定了CSTB、CTSL基因cDNA的编码区(CDS)全长。利用生物信息学分析方法,获得CSTB、CTSL蛋白的氨基酸序列,并对其特征和功能进行预测分析,结果表明牛CSTB、CTSL基因分别编码101和334个氨基酸,CSTB蛋白包含1个CY功能结构域,CTSL蛋白包含两个功能结构域,包括:第29到88个氨基酸区域的inhibitor结构域和第114到333个氨基酸区域的(Pept)结构域。2.利用PCR-RFLP方法检测CTSB、CTSF、CTSL基因3个位点的多态性,进行基因型分型,并分析群体遗传结构,计算基因和基因型频率,进行多态信息含量检测和哈代温伯格平衡检测,并建立动物模型与各测定的经济性状进行关联分析。结果发现:(1)CTSB基因与大理石纹评分性状均存在显著相关,与嫩度性状呈极显著相关:其中AA型和AB型个体的大理石纹评分等级明显高于BB型个体(P<0.05);而AB型的剪切力值显著高于AA和BB型,故嫩度明显逊于纯和基因型。(2)CTSF基因不同基因型与实测背膘厚性状存在极显著相关:AA型的背膘厚度明显大于BB型个体和AB型个体(P<0.01)。(3)CTSL不同基因型与净肉率和嫩度性状均存在显著相关:BB型个体的净肉率显著大于AA型和AB型个体(P<0.05);BB型个体的剪切力值显著高于AA型和AB型个体(P<0.05)。3.利用DNA测序的方法检测CTSD基因Exon5、6、8的多态性,检测到的4个SNP,并与各测定经济性状进行关联分析。结果表明:在EXON5上的C581T位点的多态与净肉率之间存在显著相关:CG型个体的净肉率显著大于GG型和CC型个体(P<0.05)。4.利用DNA测序的方法检测到CTSF、CTSH、CTSL基因共计20个SNP。CTSF基因intron12的A22/G;exon14的3个突变,A111G、C152T、A283G;3’下游区的A72G、C92G。CTSH基因intron1的C290G、C467T;intron11的A526G、A559G;exon12的C34T、C292T;5’上游区的598AG插入。CTSL基因intron2的A103G、C134G;exon2的C92T;intron3的C165G、A230G;intron8的C692T、A702G。利用Haploview软件对此20个突变位点进行群体连锁不平衡和单倍型分析,结果如图3-25:20个SNP中共发现7个SNP位点紧密连锁,其他SNP均不连锁,7个位点的决定系数分别为:CTSF的C152T、A283G为0.53,CTSH的C34T和C292T为0.69,CTSL的A103G和C134G为1.0,并均与C165G呈0.3的决定系数。5.对所研究的CTSF、CTSH和CTSL 3个候选基因,在肝脏,心脏,脂肪,小肠,肺,肌肉,卵巢,淋巴8个组织中的表达,进行了荧光定量PCR分析。结果发现CTSF基因在小肠、心脏、肌肉和淋巴组织的表达量显著高于其他组织。CTSH基因除了小肠、肝脏中,其他组织表达量都较高。CTSL基因在肝脏、肌肉和淋巴的表达量均显著地高于其他组织。更多还原

【Abstract】 In this study, a total of 200 cattles , including 50 cattles of Simmental x yellow cattle and 150 cattles of Waygu x Fuzhou cattle, were used to investigate. The RT-PCR、PCR-RFLP、DNA sequencing and bioinformatics methods were put into use of the analysis of bovine cathepsin family genes(CTSB、CTSD、CTSF、CTSH、CTSL) and its inhibitor cystatin B (CSTB)gene. By using general linear model (GLM) we find the genetic variations of these genes and analysis their association with economic trais( comprised of growth、carcass and meat quality traits). We also investigated population genetic structure and distribution of genotype. These will give more choices of DNA marker related to the economic traits of good cross-breed population on marker-assist-selection (MAS) and will improve beef cattle industry economic profit and reduce of the production cost. The main results were shown below:1. By using RT-PCR, DNA sequencing,we cloned complete CDS of CSTB and CTSL gene from skeletal muscle of Simmentalxyellow cattle. By using the Bio-informatics analysis the ORF of CSTB and CTSL cDNA and predict the character and protein domain, the results showed bovine CSTB and CTSL protein were consisted of 101 and 334 Amino acids. The CSTB contained CY domain , CTSL contained an inhibitor domain and a pept domain (which were consisted of 60 and 220 Amino acids).2. Identification for 3 SNPs mutations in the amplified fragments of CTSB、CTSF and CTSL genes by PCR-RFLP. Allele and genotype frequencies of 3 SNPs were analyzed of PIC and HW balance examination in the two cross-breed population. The calculation of least square was used to find association between these SNPs and 8 economic traits. The results showed that: (1) The CTSB SNP was significantly associated with marbling score (MS) and very significantly associated with meat tenderness(MT). Animals with AA and AB genotypes had larger MS than the BB genotype (P<0.05), but AB had a bigger WBS value than AA and BB(P<0.01). (2) The CTSF SNP was very significantly associated with back fat (BF). Animals with the genotype AA had larger BF than the AA and AB genotype (P<0.01). (3) The CTSL SNP was significantly associated with meat percent (MP). Animals with the genotype BB had larger MP than AA and AB genotype (P<0.05).3. 4 SNPs were detected in the exon5、exon6 and exon8 of CTSD gene by DNA sequencing. The calculation of least square was used to employ to association between these SNPs and 8 economic traits. The results showed that: The C581T of exon5 polymorphism was significantly associated with MP. Animals with the genotype CG had larger MP than the CC and GG genotype (P<0.05).4. Parts of fragments of CTSF CTSH CTSL genes were amplified and the 20 SNPs were detected by DNA sequencing: CTSF intron12 A22/G;exon14 A111G、C152T、A283G;3’UTR A72G、C92G;CTSH intron1 C290G、C467T;intron11 A526G、A559G;exon12 C34T、C292T;5’UTR 598AG;CTSL intron2 A103G、C134G;exon2 C92T;intron3 C165G、A230G;intron8的C692T、A702G。The linkage disequilibrium analysis of 20 SNPs were analyzed by using Haploview soft ware. The results showed that: 7 SNPs of them were uncompleted linked and other 13 SNP were not linked. The r~2 of CTSF C152T and A283G is 0.53; The r~2 of CTSH C34T and C292T is 0.69; The r~2 of CTSL A103G and C134 G is 1.0 ,and their r~2 with C165G is 0.3.5. Three candidate genes (CTSF,CTSH and CTSL) were detected by RT-PCR and Real-time Q-PCR, in 8 tissue of Simmental x yellow cattle, which include: liver,heart,fat,small intestines,lung,muscle,ovary and lymph tissue. The results showed that: The expression of CTSF in the small intestines,heart,muscle and lymph tissues are higher than other tissues. The expression of CTSH in the small intestines and liver tissues are lower than other tissues. The expression of CTSL in the liver,muscle and lymph tissues are higher than other tissues.更多还原