【作者】 王淑辉；

【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， 2008， 博士

【摘要】 本试验首先以同龄、同场的60头国内、外肉牛品种为研究对象,利用超声波活体检测技术采用最小二乘法建立了肉牛宰后肉质性状的预测模型,显示超声波技术不仅可以准确、快速、低成本的检测肉牛背膘厚、眼肌面积和大理石花纹情况,还提供了通过检测活体性状预测宰后性状的可能。为研究肉牛生长发育和脂肪沉积的分子机制,本试验以西门塔尔牛公牛和阉牛各3头作为试验材料,因为阉割是肉牛生产中常用的技术手段,阉割后肉牛在习性、外观和生长发育过程都发生了很大的改变,这些发展的速度很慢,暗示这些发展是通过基因表达差异的改变进行的,因此采用Affymetix公司的基因芯片技术,研究了二者之间肝脏和脂肪组织的基因表达谱,经过数据分析找到了因阉割引起的生长发育减缓,脂肪沉积速度加快的有关的差异表达基因。并通过荧光定量PCR技术对芯片结果进行验证。结果如下:(1)本研究在国内首次建立了超声波技术活体检测技术预测牛肉产量的数学模型:屠宰EMA=30.6032+1.0383×8月龄EMA ,屠宰EMA=8.3520+0.8873×20月龄EMA ;屠宰RIB12=0.3777+0.3470×8月龄RIB12,屠宰RIB12=0.2856+0.4203×20月龄RIB12;高档肉质量=0.3616×8月龄EMA +40.4824×8月龄RIB12+0.065×8月龄体重,高档肉质量=0.6765×8月龄EMA +39.0974×8月龄RIB12,高档肉质量=0.4451×20月龄EMA +1.3847×20月龄RIB12+0.0177×20月龄体重,高档肉质量=0.5621×20月龄EMA +2.7208×20月龄RIB12;后部肉质量=1.1316×8月龄EMA +24.4678×8月龄RIB12+0.0985×8月龄体重,后部肉质量=1.6034×8月龄EMA +22.3924×8月龄RIB12,后部肉质量=0.9869×20月龄EMA -9.5036×20月龄RIB12+0.0148×20月龄体重,后部肉质量=1.0849×20月龄EMA -8.3843×20月龄RIB12;优质肉质量=1.4932×8月龄EMA +64.9502×8月龄RIB12+0.1642×8月龄体重,优质肉质量=2.8384×8月龄EMA +61.4898×8月龄RIB12,优质肉质量=1.4320×20月龄EMA -8.1190×20月龄RIB12+0.0325×20月龄体重,优质肉质量=1.6470×20月龄EMA -5.6636×20月龄RIB12;胴体质量=8.3397×8月龄EMA -204.1618×8月龄RIB12+0.3270×8月龄体重,胴体质量=9.8856×8月龄EMA -198.8285×8月龄RIB12,胴体质量=0.9547×20月龄EMA -93.4699×20月龄RIB12+0.5753×20月龄体重,胴体质量=4.4994×20月龄EMA +7.6870×20月龄RIB12。模型都达到显著水平( p<0.05),可以在实际生产中灵活运用。通过我们实践中测得的牛数据对模型的验证,表明所得模型计算方便,精确度高,具有较强的实用性,提供了从幼龄牛或成年牛预测宰后性状的可能。(2)对西门塔尔牛公牛和阉牛各5头的生长性状和肉质性状进行分析,发现育肥初公牛和阉牛的生长发育性状(体重、体高、体斜长、胸围和管围)无差异(p>0.05),肥育14个月后公牛体斜长、出栏重、屠宰重、胴体深显著高于阉牛(p<0.05);肉质性状中公牛胴体重、蹄重、前腿肉、脂色、眼肌面积显著高于阉牛,但系膜网膜油、大理石花纹、背膘厚、水分和含钙量显著低于阉牛(p<0.05),且公牛脖肉、里脊和上脑重极显著高于阉牛(p<0.01),结果可以看出,与生长相关的性状阉牛发育较慢,但脂肪沉积速度较公牛快,阉牛肉质性状较公牛好。(3)随机选取健康无病公牛和阉牛各3头,利用Affymetrix基因芯片对其肝脏组织进行差异表达分析发现,公牛与阉牛相比代表334个基因的362个探针发生转录变化(大于等于2倍或小于等于0.5倍),其有251个已知基因发生转录改变,上调195条、下调56条,它们主要20个参与免疫系统过程、26个参与刺激应答、22个参与发育过程、19个参与多细胞有机过程、9个参与生物粘连生物学过程,多位于胞外区(13个),具有结合功能(77个)。没有分类信息的基因132个。结合关键字方法共获得直接影响生长发育和脂质代谢的基因173个在肝脏中发生转录变化。涉及酶调控功能、结合功能、催化功能、抗氧化剂功能、分子转运功能、结构分子功能、转录因子功能和运输功能。通路分析结果显示阉割后引起生长发育迟缓的机理可能是阉割后雄激素分泌的减少会引起肝脏IGF1上调和IGFBP5转录下调,从而引起胰岛素通路上FBP2和PPARGC1a、肌动蛋白调控通路上ACTG2、BAIAP2、MGC127421、ITGB2和SCIN基因发生转录变化,从而导致肌肉合成速度减慢。并且阉割后引起大量免疫相关基因BOLA-DQA1、BOLA-DQA2、BOLA-DRB13、SELL、JSP.1、EVA1、LRRC25、CD53、LOC783725、FCGR3A发生转录变化,揭示阉割后可能免疫基因的转录变化也影响了生长发育和脂质代谢。(4)对阉牛和公牛脂肪基因芯片筛选发现,有1925个已知基因发生转录改变,上调103条、下调1822条,它们主要参与代谢过程(406个)、生物调节(176个)、定位(138个)、巨大细胞过程(95个)、刺激应答(91个)、发育过程(89个)、生长(16个)、免疫过程(41个)和生物粘连(38个),多位于细胞器官(301个)、胞外区(64个)和外膜(36个)上,多数为涉及结合功能(524个)、催化功能(326个)和酶调控功能(57个)的基因。经分析推测由于阉割,引起雄性激素和雌性激素代谢途径中的HAD11B1、UGT1A1和HSD基因差异表达,引起激素水平中的LHCGR、PRL、PRLR和GHR转录下调,从而调节ApoE,影响脂类代谢。阉割可能引起脂肪细胞自身分泌LEP基因减少,并且乳糖通路所有基因在转录水平上均发生变化。引起免疫通路中IL2/3和EPO发生变化,从而引起一系列反应,影响蛋白质水解过程。从而影响脂肪沉积过程。(5)肝脏和脂肪差异表达基因结果各选择了10个代表性基因进行FQ-PCR验证,结果显示除脂肪LEPR基因外所有的基因表达变化趋势与芯片杂交反映的结果一致,基因的表达变化情况符合生理学特征,也从另外一个角度证明本研究筛选的生长发育和脂肪沉积相关基因准确可靠、具有潜在的应用价值。LEPR基因检测结果不符合也证明了选择(0.5,2)作为筛选标准具有实践性,采用倍数法不应任意缩小差异标准。总之,本研究获得的差异表达基因为今后进一步深入研究牛乃至整个哺乳动物生长发育和脂肪沉积提供了一个有价值而重要的候选基因库。更多还原

【Abstract】 60 cattle at same age and same factory were used to establish prediction modes of pos slaughter traits by ultrasound measurement in live cattle. The results showed that ultrasound technology can not only accurate, rapid, low-cost testing beef cattle surface fat between the 12th and 13th ribs over the longissimus muscle(RIB12),ribeeye muscle area(EMA) and marbling but also provided the chance to forecast pos slaughter traits by living cattle.The liver and lipid samples from 3 bull and 3 steer-Simmental cattle were collected to study the growth and fat deposition of molecular mechanisms in Beef cattle by genechip. Castration is a commonly technology used in beef cattle production. the habits, appearance and growth of cattle have taken great changes after emasculation. Such development was very slow suggesting that these developments was through gene expression changes differently. So the genechip technology was used to research the difference gene expression profiling of the liver and adipose tissue to find the growth rate decrease and fat deposition rate increase mechanism in our experiment. The results were verified by fluorescence quantitative PCR.The results were as follows:(1) The prediction models of pos slaughter traits by ultrasound measurement in live cattle were established first in China:EMA after slaughter =30.6032+1.0383×8Month EMA, EMA after slaughter =8.3520+0.8873×20Month EMA, RIB12 after slaughter =0.3777+0.3470×8Month RIB12, RIB12 after slaughter =0.2856+0.4203×20 MonthRIB12, high-grade meat quality =0.3616×8 Month EMA +40.4824×8 Month RIB12+0.065×8 Months Weight, high-grade meat quality =0.6765×8 Month EMA +39.0974×8 MonthRIB12, high-grade meat quality =0.4451×20Month EMA+1.3847×20Month RIB12+0.0177×20 Month Weight, high-grade meat quality=0.5621×20Month EMA +2.7208×20 Month RIB12, rear meat quality=1.1316×8 Month EMA +24.4678×8 Month RIB12+0.0985×8 Month Weight, rear meat quality =1.6034×8 Month EMA +22.3924×8 Month RIB12, rear meat quality =0.9869×20 Month EMA -9.5036×20 Month RIB12+0.0148×20 Month Weight, rear meat quality =1.0849×20 Month EMA -8.3843×20 MonthRIB12, fine quality meat quality=1.4932×8 Month EMA +64.9502×8 Month RIB12+0.1642×8 Month Weight, fine quality meat quality =2.8384×8 Month EMA +61.4898×8 Month RIB12, fine quality meat quality =1.4320×20 Month EMA -8.1190×20 Month RIB12+0.0325×20 Month Weight, fine quality meat quality =1.6470×20 Month EMA -5.6636×20 Month RIB12, carcass quality=8.3397×8 Month EMA -204.1618×8 Month RIB12+0.3270×8 Month Weight, carcass quality =9.8856×8 Month EMA -198.8285×8 Month RIB12, carcass quality =0.9547×20 Month EMA -93.4699×20 Month RIB12+0.5753×20 Month Weight, carcass quality =4.4994×20 Month EMA +7.6870×20 Month RIB12.It is significant in all models(p<0.05).After demonstration all models could be used flexible in actual production.So the models were convenient, high-precision, greater practicality and could provid the possible to predict the pos slaughter traits from young cattle or adult cattle.(2) The growth traits and meat traits by 5 bull and 5 steer were analysed the different between them. The results showed that the growth traits(body weight,withers high, diagonal length,heart girth,pastern girth) were no different at 8 month(p>0.05) but the body weight and diagonal length of bull after 14 month breeding were higher than steer(p<0.05). After slaughter, the slaughter weight and naked body depth of bull were higher than steer too(p<0.05). The naked body weight,feet weight,shank weight,fat colour, EMA, and Ca content of bull were higher than steer ,but the retina lipid, marbling, RIB12, water content and Ca content of steer were higher than bull (p<0.05).The chudr weight, tenderloin weight and highrid weight of bull were significant higher than steer(p<0.01).So It can be concluded that the growth rate was decreased but fat depositon rate was increased afer emasculation.(3) 3 healthy disease-free bull and steer were selected randomly to analysis the liver differentially expressed genes by Affymetrix genechip.The results showed that 362 probe , represent 334 gene were changes. There were 251 known gene changed, 195 up, 56 down. They participate in immune system process(20), response to stimulus(26), development process(22), multi-cell organic process(19), biological adhesion(9), more in the extracellular region(13), binding(77)., 132 unclassified. With the keyword method 139 genes were different transcript in liver of growth and lipid metabolism. They were involved in Enzyme regulator activity, binding, catalytic activity, antioxidant activity, molecular transducer activity, structural molecule activity, transcription factor activity and transporter activity. Pathway analysis showed that the mechanism of the emasculation in growth rate decreasing may be related with male hormone secretion reduced after castrated caused liver IGF1 and IGFBP5 transcription down, thus leading to the insulin pathway FBP2 and PPARGC1a, actin regulatory pathway on ACTG2, BAIAP2, MGC127421, ITGB2 and SCIN changes in gene transcription, leading to slow down the speed of muscle synthesis. After castration caused massive immune-related genes BOLA-DQA1, BOLA-DQA2, BOLA-DRB13, SELL, JSP.1, EVA1, LRRC25, CD53, LOC783725, FCGR3A changes in transcription. Revealed the immune gene transcription changes caused by emasculation have also affected the growth and lipid metabolism.(4) there were 1,925 known gene transcription changes( up 103, down 1,822) using genechip to analysis the different transcription of lipid organ between bull and steer.They mainly involved in metabolic process(406), biological regulation(176),localization(138), multicellular organismal process (95), response to stimulus(91), developmental process (89), growth (16), immune system process (41) and biological adhesion (38). Most of them located in organelle (301), extracellular region(64) and envelope(36), most molecular function were related to binding(524), catalytic activity(326) and the enzyme regulator activity(57). Then it was supposed that the reason due to lipid deposition change after castrate might be the HAD11B1, UGT1A1 and HSD gene expression different in androgen and estrogen metabolism pathway. That caused LHCGR, PRL, PRLR and GHR transcription downturn at hormone levels to regulate ApoE, impact on lipid metabolism. Castration likely to cause fat cells to reduce its own secrete of LEP. All gene of leptin access roads transcription in the level of change. And IL2 / 3 and EPO gene expression were changend in immune pathway, thus leading to a series of reactions, affect protein hydrolysis process. Thus affect fat deposition process.(5) 10 representative genes in liver and fat were selected to verified the result of genechip by FQ-PCR. The results showed that all genes changed consistent with the result of genechip except LEPR gene.Gene expression changes consistent with the physiological characteristics, From another point of view, this study proved that the growth of screening and fat deposition related genes are accurate and reliable, have potential value. LEPR genes do not meet the test results also proved that the choice (0.5, 2) as a screening standards are practical, using multiples of law should not arbitrarily narrow the differences in standards.Different expressed genes in the study provided a valuable and important candidate gene pool for the future further in-depth study cattle and even the mammals growth and fat deposition.更多还原