【作者】 李娟；

【导师】 朱庆；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 本研究以培育中的地方品种二郎山山地鸡为试验材料,测定了二郎山山地鸡的生长发育性状。克隆了鸡MUSTN1基因和APOBEC2基因的全编码区序列并进行了生物信息学分析。采用荧光定量试验技术分析MUSTN1、APOBEC2基因的组织表达谱；分析骨骼肌组织中MUSTN1、MyoD、MyoG、IGFl、APOBEC2、 FWMvFRM、SM基因在不同品系、性别、日龄、赖氨酸水平的时空表达模式。并以低赖氨酸水平SD02品系公母鸡的胸腿肌为试验材料,采用免疫组化技术和免疫印记实验技术探讨了MUSTN1、APOBEC2两个蛋白的在肌肉组织中的分布和定量表达。通过比较二郎山山地鸡两品系的生长发育的差异,探讨肌纤维生长相关基因之间的关系及可能的分子机制,为地方品种鸡的生长发育和肉品质营养调控以及品种选育提供科学依据。试验结果如下：性能测定发现,不同品系、性别、日龄和赖氨酸水平对二郎山山地鸡部分生长发育性状存在影响。SD02品系母鸡前期均重和平均增重均大于SD03,SD03品系公鸡70日龄的腹脂重均高于SD02品系,SD03品系总蛋白含量高于SD02。结果提示SD02生长速度比SD03快,产肉性能高；SD03品系脂肪沉积高于SD02品系。高赖氨酸水平的母鸡70日龄活重、胸肌肌纤维直径大于低赖氨酸水平,高赖氨酸水平胆固醇含量高于低赖氨酸水平,提示赖氨酸浓度能提高母鸡产肉性能,也有助于胆固醇的浓度的增大。MUSTNl基因和APOBEC2基因在不同组织的表达存在显著差异。MUSTN1基因在心肌和骨骼肌中大量表达,在其他组织中均有分布,但表达量极少。APOBEC2基因大量表达于骨骼肌和心肌中,但在鸡的性腺、肌胃和皮脂组织中也有少量表达,在其他组织不表达。结果推测MUSTN1基因和APOBEC2基因在肌肉发育系统占有重要作用。肌纤维生长相关基因表达量在不同品系、性别、日龄和赖氨酸水平亦存在显著差异。四个时间点,SM基因表达量在1日龄最高；胸肌组织中FWM基因的表达量1日龄最低；随着日龄的增长,SM基因表达量降低,FWM基因表达量升高。结果提示家禽出生后早期是肌纤维类型转变的重要阶段,随着日龄的增长,快白肌含量增加。高浓度赖氨酸水平能上调胸肌组织中MyoD、IGF1基因的表达量,并且也能上调公鸡腿肌组织MUSTN1、MyoD、MyoG、IGF1、APOBEC2、 FWM、FRM基因的表达量。基因表达量的相关性分析表明,胸肌组织中MUSTN1、 MyoD、APOBEC2基因表达量之间均存在着极显著的正相关；腿肌组织中,MUSTN1、MyoD、MyoG、 IGF1、APOBEC2、FWM基因存在极显著的正相关；MUSTN1基因表达量在胸腿肌组织中均和肌纤维直径、密度呈极显著相关。结果提示MUSTN1、APOBEC2和MyoD的表达存在一定的关系,MUSTN1基因和肌纤维的肥大密切相关。通过克隆获得了鸡MUSTN1、APOBEC2基因的全编码区序列,并翻译获得其氨基酸序列(78个和222个)。通过生物信息学分析,确定MUSTN1、APOBEC2蛋白都是亲水的非跨膜蛋白。MUSTN1和APOBEC2氨基酸序列磷酸化位点均为Threonine,分别为1个和12个；MUSTN1蛋白和APOBEC2蛋白糖基化位点分别为7个和2个。免疫组化结果表明MUSTN1蛋白和APOBEC2蛋白均分布在心肌和骨骼肌肌纤维的细胞核。免疫印记结果表明MUSTN1和APOBEC2蛋白分别具有日龄、性别和组织特异性。MUSTN1和APOBEC2表达具有相似性,推测MUSTN1和APOBEC2蛋白可能共同协调肌肉组织发育的过程,但具体机制还不清楚。MUSTN1和APOBEC2转录水平和翻译水平并不完全一致,推测两个水平可能有不同的调节机制,来影响mRNA和蛋白合成的速度。更多还原

【Abstract】 In this study, MUSTNl and APOBEC2gene entire coding sequence were cloned, and the correlations of the bioinformatics in Erlang Mountainous (EM) Chickens were analysed. We measured the growth traits of EM chickens and evaluated the expression tissue-pattern of MUSTN1and APOBEC2by Real-time Quantitative PCR (qPCR) and evaluated the skeletal muscle expression pattern (MUSTN1, MyoD, MyoG, IGF1, APOBEC2, FWM, FRM and SM) in female and male chickens at both two lysine (Lys) levels and two Lines at four growth points. Meanwhile, we evaluated the protein abundance expression of MUSTN1and APOBEC2among muscle tissues at four days of chickens by western blotting and also evaluated protein location of among muscle tissues at70-day-old chickens by immunohistochemistry. The objective of this study was to evaluate the developmental-specific regulation of mRNA and protein abundance expression in chickens. It was good references for study on the local breed chickens growth characteristic, genetic mechanism of muscle regulation, and meat quality regulation of the two lines. The results were as follows:We evaluated the line-, sex-, age-and lysine level-differences in different growth traits in this study. The results indicated that, SD02Line females had greater live weight and weight gain than SD03Line in starter growth period; SD03male chickens had greater abdominal fat weight than SD02at70-day-old, SD03had greater total protein than SD02; It might be greater of muscle growth in Line SD02than SD03, and SD03had greater fat deposited than SD02. Female chickens had greater live weight and breast muscle fiber diameters feeding high lysine level (HL) than low lysine level (LL) at70-day-old. Chickens also had greater cholesterol feeding HL than LL. Thus, it’s better to fed HL to have the superior growth performance and high cholesterol concertration than LL.There were tissue-specific differences in gene expression of different tissues that were examined in this study. MUSTN1mRNA is predominantly expressed in heart and skeletal muscle, with negligible expression in other tissues. APOBEC2mRNA is predominantly expressed in skeletal and cardiac muscle, with negligible expression in tissues such as gonad, gizzard and subcutaneous fat tissues, and not expressed in other tissues. It suggested that MUSTN1and APOBEC2mRNA might be an important role in regulating during chicken post-hatch muscle growth. In addition to tissue-specific differences in gene expression, we also observed line-, gender-, age-and lysine level-specific differences in the two muscle tissues that were examined in this study. The expression of SM mRNA was highest at day1; the expression of FWM mRNA was lowest at day1. It implies the possibility that muscle fiber switched in early post-growth. High lysine concentration could upregulation the expression of MyoD and IGF1mRNA in pectoralis major and upregulation the expression of MUSTN1, MyoD, MyoG, IGF1, APOBEC2, FWM and FRM mRNA in male thigh muscle.There was a positive correlation among MUSTN1, MyoD and APOBEC2mRNA expression in pectoralis major. There was also a positive correlation among MUSTN1, FWM, APOBEC2, MyoD, MyoG and IGF1mRNA expression in thigh muscle. MUSTN1and APOBEC2mRNA might be related with the expression of MyoD expression during chicken post-hatch muscle growth. There was a correlation among expression MUSTN1and muscle fiber traits in both pectoralis major and thigh muscle tissue, suggesting a role in muscle development.We successfully obtained the coding sequence of MUSTN1and APOBEC2gene. Two amino acid sequences were obtained from these two CDS sequences, their lengths were78aa and222aa. The result indicated that MUSTN1and APOBEC2maybe a hydrophilic but Non-transmembrane protein. MUSTN1and APOBEC2have one and twelve threonine of phosphorylation site, respectively. MUSTN1and APOBEC2have seven and two glycosylation sites, respectively.Immunohistochemical analysis revealed that MUSTN1and APOBEC2localizes to nuclei in skeletal and cardiac muscle myofibers. There was tissue-, gender-and age-specific differences in gene expression. MUSTN1and APOBEC2protein might be synergy regulated during chicken post-hatch muscle growth during muscle development. Changes in mRNA abundance were reflected by similar changes in abundance of MUSTN1and APOBEC2protein in Line SD02which consumed the LL, there were also differences between mRNA abundance and protein abundance. Maybe different regulation mechanisms have taking part in the processing, affect the amount of the two molecules.更多还原