【作者】 朱志明；

【导师】 刘榜； 李奎；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2006， 硕士

【摘要】 藏鸡是我国青藏高原在原始饲养状态下保存下来的珍贵家禽品种。藏鸡具有耐寒、抗病、抗缺氧、肉质鲜美、药用价值较高，耐粗放和可观赏性强等优良特性，同时也有生长速度慢、产蛋率低的缺点。如何在保存好这一珍贵遗传资源的同时，一方面在不影响藏鸡优良特性的情况下通过引入杂交改良藏鸡的缺点以培育新藏鸡，另一方面通过经济杂交和产品开发利用好藏鸡就成为当前重要的研究课题。本研究在农业部“948”项目的基础上，针对目前藏鸡研究现状，一方面通过引进国外优良鸡种对藏鸡进行杂交利用，另一方面利用我室所建藏鸡腿肌全长cDNA文库信息及侯选基因策略，采用生物信息学与分子生物学技术相结合的方法，克隆和分离与重要经济性能有关的新基因，为分子标记辅助选择育种提供分子遗传基础。主要取得了如下结果：1．对藏鸡、隐性白羽鸡和藏隐F1代(藏鸡♂×隐性白羽鸡♀)的产蛋性能进行分析，发现藏隐F1代64周龄产蛋数、蛋重及蛋的大小都较藏鸡显著提高(P＜0.05)。同时，对藏鸡、隐性白羽鸡、藏隐F1代及75％藏鸡血液的杂种(藏♂×藏隐F1♀)早期生长发育进行了比较，结果表明：藏隐F1代的各周龄体重、周增重及胫长生长情况都高于75％藏鸡血液的杂种和纯藏鸡，达到显著水平(P＜0.05)。2．运用三种非线性模型Logstic、Gompertz、von Bertalanffy对藏鸡的早期生长发育规律进行了曲线模拟。三种模型都能较好的模拟藏鸡的生长发育规律，但Gompertz更加适宜模拟藏鸡的生长过程。3．依据本实验室所建藏鸡腿肌全长cDNA文库信息及侯选基因策略，分离并鉴定了鸡MyoT基因的全长cDNA序列，并获得了Cacngl和Camk2d基因片段。结合鸡基因组序列信息，进一步对MyoT、Cacng1和Camk2d基因氨基酸序列、蛋白质结构进行了初步分析和预测。4．采用了PCR—RFLP、mp-PCR和M-ASP三种方法检测了MyoT、Cacng1和Camk2d等3个基因7个位点的多态性和Cacng1基因第2内含子长片段的插入／缺失造成的PCR产物长度多态性，分析了部分多态位点在不同鸡种中的基因型频率和基因频率，并分析了各基因型在不同鸡种中的分布差异。5．在我室与西藏大学农牧学院合作所建立的试验群体(藏鸡、隐性白羽鸡、藏隐F1代和75％藏鸡血液的杂种)中采用：疗差分析的最小二乘法分析了上述三个基因的酶切位点多态性与部分经济性状的关联。结果发现：①MyoT基因第10外显子Hin6I多态位点不同基因型与腿肌重、腿肌率、全净膛率和腿肌肉色显著关联(P＜0.05)；3’-UTR区Rsa I多态位点不同基因型与屠体重、皮下脂肪厚显著关联(P＜0.05)；采用mp-PCR方法在3’-UTR引入了—BsuR I多态位点，发现不同基因型与屠体重、肌间脂肪宽、半净膛重、全净膛重间差异显著(P＜0.05)。②Cacngl基因第2内含子的长片段插入／缺失位点LS与SS基因型个体间嫩度差异极显著(P＜0.01)；第4外显子MspⅠ多态位点不同基因型与嫩度和腿肌肉色显著关联(P＜0.05)：Cacngl基因第5外显子M-ASP多态位点不同基因型与肝重、滴水损失显著关联(P＜0.05)，与胸肌肉色极显著相关(P＜0.01)；3’-UTR区MspⅠ多态位点不同基因型与腿肌肉色显著关联(P＜0.05)，并发现该多态位点与滴水损失也存在相关趋势(P=0.072)。③Camk2d基因第15外显子VspⅠ多态位点不同基因型与腹脂重极显著关联(P＜0.01)，与pH值显著关联(P＜0.05)。6．以成年白来航母鸡的心、肝、脾、肺、肾、脑和肌肉七种组织作为研究材料，采用半定量RT-PCR方法研究鸡MyoT和Cacngl基因在各组织的表达谱和表达规律，结果发现MyoT基因在心脏和肌肉中表达；Cacngl基因在心、肝、肾、大脑和肌肉中都表达。同时进一步分析了MyoT和Cacngl基因在不同组织中的表达差异，MyoT，在心脏的表达量高于肌肉的表达量；Cacngl在肌肉的表达量最高，其次是心脏、肾脏、大脑，在肝脏中的表达量最低。更多还原

【Abstract】 Tibetan chicken mainly distributed in Tibetan altiplano, is a rare and excellent chicken breed in China. It has many unique characteristics, such as cold resistance, strong disease resistance, anoxia resistance, delicious meat quality, good officinal value, extensive cultivation and good fancy. At the same time, it has the disadvantage of low growth rate and poor laying performance. How to improve the flaw of Tibetan chicken with un-affecting its virtue for cultivating new Tibetan chicken by introductive crossing, and how to exploit and utilize the Tibetan chicken by economic crossing is an important study task in current.This study is based on the item of National Ministry of Agriculture (948). According to the study actuality of Tibetan chicken, overseas excellent breed was introduced for crossing the Tibetan chicken; In addition., using the information of Full-length cDNA bank and candidate gene strategy, some new genes with important economic performance were cloned and separated by bioinformatics and biotechnology, which could have the basic for searching the molecular assistant marker. The main results are as follows:1. Comparing analysis the laying performance of Tibetan chicken (TC), Recessive White chicken (RW) and F1 (Tibetan chicken♂×Recessive White♀, TR), the egg production、egg weight of the TR with 64 weeks age has significant improvement comparing with TC (P < 0.05). At the same time, the early development of TC, RW, TR and 75%TC (Tibetan chicken♂×♀F1♀) was compared, the result showed TR had good body weight, weekly gain weight and shank length comparing with TC and 75%TC and represent significant level (P < 0.05).2. The growth curve of Tibetan chicken from 0 to 16 weeks old was analyzed and fitted with three kinds of nonlinear models (Gompertz, Logistic, von Bertalanffy). The result showed that the growth curves could be well fitted with three models, but the Gompertz model had the best effect on fitting with the growth curves.3. According to the information of Full-length cDNA library of Tibetan chicken leg muscle and candidate gene strategy, the Full-length cDNA sequence of Myotilin (MyoT) was isolated and identified, and the fragments of Calcium channel voltage-dependent gamma subunit 1 (Cacngl), Calcium/calmodulin-dependent protein kinase II delta 2-subunit (Camk2d) through the PCR amplification. Combining with chicken genome information, the amino acid sequence and protein structure of three genes were analyzed4. Seven polymorphic sites of AfyoT、Cacngl and Camk2d were detected respectively and a long fragment indel polymorphism was detected in the intron 2 of Cacngl by PCR-RFLP, mpPCR and M-ASP. The allele frequencies and genotype frequencies were analyzed in different populations and the distribution difference of genotype was analyzed.5. The associations between genotypes and phenotypes of economic traits of the above polymorphic sites were analyzed in the experimental chicken population constructed under the cooperation of our lab and Tibet Agriculture and Animal Husbandry College. The result showed:①There are significant associations between the genotype of Hin6Ⅰpolymorphie site at exon 10 of MyoT with phenotypes of leg muscle weight, leg muscle weight rate, Eviscerated rate and color of leg muscle (P＜0.05); there are significant associations between the genotype of RsaⅠpolymorphic site at 3’-UTR of MyoT with phenotypes of carcass weight and subcutaneous fat thickness (P＜0.05); there are significant associations between the genotype of BsuRⅠpolymorphic site at 3’-UTR of MyoT with phenotypes of carcass weight, intermuscular fat width, semi-eviscerated weight and eviscerated weight (P＜0.05);②There are significant assoeiatious between the long fragment indel polymorphie site in intron 2 of Cacng1 with phenotype of tenderness (P＜0.01); there are significant associations between the genotype of MspⅠpolymorphic site at exon 4 of Cacng1 with phenotypes of tenderness and color of leg muscle (P＜0.05); there are significant associations between the genotype of M-ASP polymorphic site at exon 5 of Cacng1 with phenotypes of liver weight, drip loss (P＜0.05) and color of breast muscle (P＜0.01); there are significant associations between the genotype of MspⅠpolymorphie site at 3’-UTR of Cacng1 with phenotypes of color of leg muscle (P＜0.05) and there has current correlation between this polymorphie site with phenotypes of drip loss;③There are significant associations between the genotype of VspⅠpolymorphie site at exon 15 of Camk2d with phenotypes of Abdominal fat weight (P＜0.01) and pH value (P＜0.05).6. The semi-quantitative RT-PCR was performed to detect the expression pattern of chicken MyoT and Cacng1 gene in different tissues (heart, liver, spleen, lung, kidney, brain and muscle). The result showed MyoT gene was expressed in heart and muscle and Cacng1 gene was expressed in heart, liver, kidney, brain and muscle. Next, the mRNA expression level was analyzed. The result revealed the expression level of heart was higher than muscle in MyoT, and the expression level of muscle was highest in Cacng1, next was heart, kidney, brain, the lowest expression level was liver.更多还原