【作者】 冯琳；

【导师】 周小秋；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2010， 博士

【摘要】 本研究通过生长试验与原代细胞培养模型相结合,研究了18.0-74.7g幼建鲤不同体重阶段体成分以及组织器官中蛋白质合成调控信号分子TOR基因表达量变化规律,并在此基础上通过生长试验研究了不同水平Thr对幼建鲤生长性能、体成分沉积、血清、肝胰脏和肌肉中GOT.GPT酶活力、消化器官生长发育、消化酶和肠刷状缘酶活力及肌肉和前中后肠中TOR基因表达量的影响；通过生长试验和攻毒试验探索了不同水平的Thr对幼建鲤非特异性和特异性免疫功能的影响；通过细胞培养模型研究了不同水平的Thr对幼建鲤肠道上皮细胞增殖分化的影响；通过细胞培养模型结合同位素示踪技术研究了Thr对肠道蛋白质合成能力的影响及对蛋白质调控信号分子TOR基因表达量的影响。主要的研究内容及结果如下：1不同体重幼建鲤体成分以及组织器官中蛋白质合成调控信号分子TOR基因表达量变化规律研究选择体重相近(18.0±0.2g)的健康建鲤750尾进行生长试验。整个试验分为五个阶段,饲喂相同的日粮,每14天采样一次,用于测定幼建鲤体蛋白、体脂肪、体灰分及体水分的含量,考察血清、肌肉和肝胰脏中GOT和GPT活力以及血氨含量的变化情况,并测定肌肉、肝胰脏、头肾、后肾、脾脏、心脏和鳃中TOR相对表达量。研究发现：18.0—74.7g的幼建鲤随体重增加,水分含量显著下降(P<0.01),脂肪含量显著增加(P<0.01)；幼建鲤随体重增加体蛋白含量先显著增加(P<0.01),体重达31.4g以后达到稳定值；体灰分含量随体重增加显著下降(P<0.05),体重达40.1g后达到稳定值(P>0.05)。血氨浓度和肝胰脏GPT和GOT活力随体重的增加极显著升高(P<0.01),且与体重呈极显著的线性关系(Y1=1083.781+16.457X,R2=0.964；Y2=1249.597+16.190X,R2=0.947;Y3=2198.725+29.567X,R2=0.957,P<0.01,其中X为体重g),肌肉中的GPT和GOT活力也随体重增加极显著升高(P<0.01)；而血清中GOT活力随体重增加先极显著增加(P<0.01),体重达40.1g后极显著下降(P<0.01),GPT活力随体重增加先保持稳定值,体重达74.7g后极显著升高；18.0—74.7g的幼建鲤头肾、后肾、脾脏和鳃TOR的表达量随体重的增加呈线性增加(Y1=0.112+0.03X,R2=0.776,P<0.05;Y2=0.036+0.06X,R2=0.862, P＜0.05；Y3=0.255+0.002X,R2=0.770,P:0.05;Y4=0.132+0.002X,R2=0.774,P<0.05,其中X为体重g)；肌肉中TOR基因表达随体重增加而增加,并分别与体蛋白含量和肌肉蛋白含量呈显著的正相关(r1=+0.890；r2=+0.848,P<0.05)；而肝胰脏中TOR的表达量随体重增加呈显著的线性下降(Y=0.486-0.02X,R2=0.854,P<0.05)；心脏中TOR基因表达在体重达40.1g后显著(P<0.05)下降后达稳定值。研究结果说明20-80g幼建鲤随体重增加体蛋白质和脂肪含量增加,而体水分和灰分含量下降；随体重增加幼建鲤肝胰脏和肌肉中的氨基酸代谢增强；幼建鲤肌肉、脾脏、头肾、后肾、鳃、心脏和肝胰脏中均有蛋白质合成调控信号分子TOR的表达,肌肉、脾脏、头肾、后肾和鳃中TOR的表达量随体重增加而增加,而肝胰脏和心脏中TOR的表达量随体重增加而减少。2 Thr对幼建鲤生长性能、体成分沉积、血清、肝胰脏和肌肉中GOT、GPT酶活力及肌肉中TOR基因表达的影响研究选择体重相近(13.60±0.03g)的健康建鲤1050尾,平均分成7个处理,每处理150尾,每处理设3个重复,每个重复50尾,组间初始体重差异不显著(P>0.05)。7个处理组分别饲喂Thr水平为0.74、0.91、1.22、1.57、1.86、2.23和2.52%的饲料,考察不同水平Thr对幼建鲤生长性能、体成分沉积、血清、肝胰脏和肌肉中GOT、GPT活力及肌肉中TOR基因表达量的影响。结果发现：Thr能极显著提高幼建鲤特异性增长率和饲料效率(P<0.01),且建鲤特异性增长率和饲料效率分别与Thr含量呈极显著(P<0.01)和显著(P<0.05)的二次回归关系(Y1=-0.3627x2+1.1765x+1.3705, R2=0.927；Y2=-5.8180x2+19.7028x+51.4993,R2=0.790,X代表Thr含量%),当Thr含量分别为1.62%和1.69%时,特异性增长率和饲料效率分别达到最佳值；Thr能极显著提高幼建鲤蛋白质沉积率和脂肪沉积率(P<0.01),但是对灰分的沉积率没有显著影响(P>0.05),蛋白质沉积率与Thr水平呈显著的二次回归关系(Y=-4.8344x2+15.2266x+21.3823,R2=0.820,P<0.05,X代表Thr含量%),当Thr含量为1.57%时,蛋白质沉积率达到最佳值；Thr能显著提高幼建鲤肝胰脏和肌肉中的GOT和GPT活力(P<0.05),且肝胰脏中的GOT和GPT活力分别与Thr含量呈显著或极显著的二次回归关系(Y1=-1632.7x2+5584.86x-578.64,R2=0.787,P <0.05:Y2=-586.98x2+1575.15x+2101.41,R2=0.961,P<0.01；Y3=-126.06x2+ 421.100x+703.604,R2=0.948,p<0.01:Y4=-174.89x2+564.995x+590.875,R2=0.813,P<0.05,X代表Thr含量%),当Thr含量分别为1.71、1.34、1.67和1.62%时,幼建鲤肝胰脏和肌肉中的GOT和GPT活力达到最大值；Thr对幼建鲤血氨浓度有显著的影响(P<0.01),且血氨浓度与日粮Thr含量呈显著的二次回归关系(Y=1349.88x2-4183.5x+4392.23,R2=0.908,P<0.01)；Thr对幼建鲤肌肉中的TOR基因表达量有显著的影响(P<0.05),当Thr含量为0.91%时,与缺乏组相比,肌肉中TOR基因表达量显著降低(P<0.05),饲料中Thr含量达到1.57%时肌肉中TOR基因表达显著增加,随Thr水平的增加肌肉TOR的表达量呈增加趋势,但是差异不显著(P<0.05)。对结果进一步分析发现：幼建鲤特异性增长率与摄食量、饲料效率、蛋白质沉积率和脂肪沉积率呈极显著(P<0.01)的正相关(r1=+0.976；r2=+0.883；r3=+0.923；r4=+0.932)。研究结果说明：Thr促进了幼建鲤的生长,生长的促进与摄食量、饲料效率、蛋白质沉积率和脂肪沉积率的提高有关；Thr能提高幼建鲤肌肉和肝胰脏中的氨基酸代谢,并显著影响肌肉中TOR的表达量；以特异性增长率为指标确定的幼建鲤(20-60g)Thr需要量为1.62%或5.07g/100gCP。3 Thr对幼建鲤消化器官生长发育、消化吸收能力及肠道中蛋白质合成调控信号分子TOR基因表达量的影响选择体重相近(13.60±0.03g)的健康建鲤1050尾,平均分成7个处理,每处理150尾,每处理设3个重复,每个重复50尾,组间初始体重差异不显著(P>0.05)。7个处理组分别饲喂Thr水平为0.74、0.91、1.22、1.57、1.86、2.23和2.52%的饲料,考察Thr对幼建鲤肝胰脏和肠道的生长发育、消化酶活力极肠道刷状缘酶活力、肠道微生物及肠道中蛋白质合成调控信号分子TOR基因表达的影响。研究发现：Thr能显著提高幼建鲤肝体指数和肝胰脏蛋白含量(P<0.01),显著提高幼建鲤肝胰脏中胰蛋白酶、糜蛋白酶、脂肪酶的活力(P<0.01),肝胰脏糜蛋白酶和脂肪酶活力分别与Thr呈显著(P<0.05)或极显著(P<0.01)的二次相关(Y1=-4.0220x2+13.8680x-5.3260,R2=0.896,Y2=-189.38x2+624.789x+158.988,R2=0.915,X代表Thr含量%),当Thr含量分别为1.72和1.65%时,肝胰脏糜蛋白酶和脂肪酶的活力达到最高值；Thr能显著提高幼建鲤肠体指数、肠长指数、肠蛋白含量和前中后肠皱襞高度(P<0.01),显著提高肠道胰蛋白酶、糜蛋白酶、脂肪酶和淀粉酶的活力(P<0.01,),肠道胰蛋白酶和脂肪酶活力与Thr含量呈显著(P<0.05)的二次相关(Y1=-0.7157x2+2.3674x+1.0750,R2=0.895,Y2=-140.57x2+454.669x+16.3032,R2=0.879,X代表Thr含量%),当Thr含量分别达1.65和1.62%时肠道胰蛋白酶和脂肪酶活力最高；Thr显著提高了肠道肌酸激酶和前中后肠Na+-K+ATP酶、碱性磷酸酶、γ-谷氨酰转肽酶的活力(P<0.01),且肠道肌酸激酶和前中后肠Na+-K+ATP酶、碱性磷酸酶、γ-谷氨酰转肽酶的活力与Thr含量呈显著或极显著的二次相关(Y1=-25.088x2+81.4732x-20.459,R2=0.894,P<0.05;Y2=-106.30x2+363.988x-68.602,R2= 0.945,P＜0.01;Y3=-127.01x2+430.508x-92.817,R2=0.943,P<0.01；Y4=-70.337x2+226.023x-31.678,R2=0.938,P＜0.01；Y5=-11.769x2+37.5870x-6.4615, R2=0.868,P＜0.05；Y6=-5.2309x2+16.5667x+1.5345,R2=0.924,P<0.01；Y7=-1.0622x2+3.5065x+0.9637,R2=0.883,P＜0.05；Y8=-22.440x2+71.1129x-9.7408,R2=0.9 71,P<0.01;Y9=-22.757x2+74.3064x+8.8138,R2=0.917,P<0.01; Y10=-25.413x2+81.2129x+15.3137,R2=0.910,P<0.01,X代表Thr含量%),当Thr含量分别为1.62、1.71、1.69、1.61、1.60、1.58、1.65、1.58、1.63、1.60%时,肠道肌酸激酶、前中后肠Na+-K+ATP酶、碱性磷酸酶和γ-谷氨酰转肽酶的活力达到最大；Thr显著影响了肠道乳酸杆菌、大肠杆菌和嗜水气单孢菌的数量(P<0.01),当Thr含量为1.57%时,肠道中乳酸杆菌数量达最大值,继续添加Thr乳酸杆菌数量呈下降趋势,而大肠杆菌和嗜水气单孢菌的数量变化与乳酸杆菌恰好相反；Thr显著影响了幼建鲤前中后肠的TOR基因表达量,肠蛋白含量与中肠TOR基因表达量呈极显著负相关(r=—0.917,P<0.01)。试验结果说明：Thr促进了幼建鲤肝胰脏的生长发育,促进了肝胰脏分泌消化酶的能力；Thr促进了肠道的生长发育,提高肠道消化蛋白质、脂肪和淀粉的能力,增加了幼建鲤对营养物质的吸收能力,同时优化了幼建鲤肠道微生物区系,增加了有益菌的数量,减少了有害菌的数量；Thr能显著影响幼建鲤前中后肠TOR的表达,Thr缺乏或过量TOR的基因表达均上升,在接近需要量时下降；根据消化酶活力和肠道刷状缘酶活力确定的幼建鲤(20-60g)Thr需要量为1.58-1.65%,与根据生长速度确定的需要量1.62%相近。4 Thr对鲤鱼肠上皮细胞增殖分化的影响采用单因子实验设计,以建鲤原代贴壁肠道长皮细胞体外培养模型考察不同Thr水平对鲤鱼IEC增殖和分化的影响。试验设计为6个处理,每个处理设4个重复。每个处理分别添加Thr浓度为：0、135、170、205、240和275mg／L。结果发现：Thr显著影响鲤鱼IEC的MTT OD值(P<0.05),随Thr浓度的增加,鲤鱼IEC的MTT OD显著增加(P<0.05),当Thr浓度为205mg／L时,MTT OD值最大,继续增加Thr浓度,MTT OD值显著下降(P<0.05)；Thr显著影响了鲤鱼IEC碱性磷酸酶、Na+-K+ATP酶活力及细胞蛋白含量(P<0.05),当Thr浓度为205mg/L时,鲤鱼IEC碱性磷酸酶、Na+-K+ATP酶活力分别达到最大值,继续增加Thr浓度,两种酶活力显著下降(P<0.05),细胞蛋白含量则随Thr浓度的增加而增加；Thr显著降低了培养液中蛋白质的消化量(P<0.05),Thr浓度为170mg/L时,蛋白质消耗量最低；Thr显著降低了培养液中乳酸脱氢酶活力(P<0.05),当Thr浓度为205 mg/L时,培养液中乳酸脱氢酶活力最低；Thr显著影响细胞中GOT和GPT活力,显著影响培养液中氨浓度(P<0.05),当Thr浓度为205 mg/L时,细胞中GOT活力显著高于其余各处理,而GPT活力则先显著增加(P<0.05),Thr浓度为170mg/L后又显著下降(P<0.05),Thr浓度为135mg/L时,培养液中的氨浓度显著下降(P<0.05),当Thr浓度为205mg/L时,氨浓度最低。研究结果说明：Thr促进了鲤鱼IEC的增殖和分化,促进了鲤鱼IEC的氨基酸代谢,保证了IEC结构和功能的正常。5 Thr对鲤鱼肠细胞蛋白合成率和TOR基因表达的影响本研究进行了3小试验。第一个小试验设2个处理组,研究Thr对肠细胞蛋白质合成的影响,每个处理组设10个重复,每个重复1孔,培养时间为240min。2个处理组分别含Thr 0mg／L和205mg/L。第二个小试验研究了205mg/L Thr对肠细胞TOR基因表达的影响,设2个处理组,分别含Thr Omg/L和205mg/L,每个处理设6个重复,培养时间30min。第三个小试验研究了205mg/L Thr对肠细胞TOR基因表达影响的时间效应,设5个处理组,设5个时间点,分别为15min,30min, 100min,170min和240min,每个时间点设6个重复。结果发现：Thr显著提高了鲤鱼肠道上皮细胞的蛋白质合成率(P<0.05),显著提高了幼建鲤IEC中TOR基因表达量(P<0.05),提高幅度达到55%；随着试验时间的延长,Thr显著提高了幼建鲤IEC中TOR的表达量(P<0.05),Thr作用30min后IEC中TOR的表达量显著增加(P<0.05),30—100min之间TOR的表达量保持稳定,当作用时间达到170分钟后IEC中TOR的表达量显著增加(P<0.05),之后保持稳定。结果说明Thr提高了幼建鲤IEC的蛋白质合成能力,蛋白质合成能力的增加与Thr提高IEC中蛋白质合成调控信号分子TOR的表达量有关；Thr对IEC中TOR的表达量随时间增加,表达量逐步增加,到170分钟达到平台值。6 Thr对幼建鲤非特异性和特异性免疫指标的影响选择体重相近(13.60±0.03g)的健康建鲤1050尾,平均分成7个处理,每处理150尾,每处理设3个重复,每个重复50尾,组间初始体重差异不显著(P>0.05)。7个处理组分别饲喂Thr水平为0.74、0.91、1.22、1.57、1.86、2.23和2.52%的饲料,生长试验60d结束以后用嗜水气单孢杆菌攻毒17d,考察Thr对幼建鲤免疫器官生长发育、疾病抵抗能力、非特异性免疫和特异性免疫功能的影响。结果发现：Thr显著提高了幼建鲤攻毒后成活率(P<0.01),当Thr含量为1.57%时幼建鲤攻读后成活率为100%；Thr显著提高了幼建生长试验期鲤头肾指数和脾体指数(P<0.01),显著提高其红细胞和白细胞数量(P<0.01)；Thr显著提高了幼建鲤白细胞吞噬率(P<0.01),且白细胞吞噬率与Thr含量呈极显著(P<0.01)的二次相关(Y=-26.408x2+88.0857x-21.261,R2=0.969),当Thr水平达1.67%时,白细胞吞噬率达最高；Thr极显著提高了幼建鲤IgM含量和抗嗜水气单孢菌抗体效价(P<0.01)当Thr含量为1.57%时,IgM含量最高,且抗嗜水气单孢菌抗体效价与Thr含量呈显著(P<0.05)的二次相关(Y=-54.651x2+179.867x-99.372,R2=0.969),当Thr含量为1.65%时嗜水气单孢菌抗体效价达到极值；Thr显著影响了幼建鲤血清补体C3和C4含量、溶菌酶活力、酸性磷酸酶活力、总铁结合力和凝集素效价(P<0.01),且血清补体C3和C4含量、酸性磷酸酶活力、总铁结合力呈显著(P<0.05)的二次相关(Y1=-105.55x2+352.445x-127.43,R2=0.800；Y2=-14.066x2+46.1787x-12.768,R2=0.805；Y3=-186.01x2+594.124x-168.16,R2=0.883；Y4=-31.992x2+99.9297x+22.3843,R2=0.793,),当Thr水平分别达1.67、1.64、1.60、1.56%时血清补体C3和C4含量、酸性磷酸酶活力、总铁结合力达到极值。对结果相关分析表明：幼建鲤攻毒后成活率与头肾重、脾脏重、白细胞吞噬率、血清补体C3含量、血清IgM水平呈极显著(P<0.01)的正相关(r1=+0.936,r2=+0.981,r3=+0.9841,r4=+0.932,r5=+0.934)。研究结果说明Thr提高了幼建鲤的疾病抵抗能力,疾病抵抗能力的提高与Thr提高幼建鲤免疫器官的生长发育、提高特异性和非特异性免疫能力有关。以免疫功能指标确定的Thr需要量为1.56-1.69%,与生长性能确定的需要量1.62%和以消化吸收功能指标确定的1.58-1.65%接近。综上所述：20-80g的幼建鲤幼建鲤肌肉、脾脏、头肾、后肾和鳃中TOR的表达量随体重增加而增加,而肝胰脏和心脏中TOR的表达量随体重增加而下降；Thr能够促进幼建鲤生长,提高体蛋白和体脂肪含量,体蛋白含量与肌肉中TOR基因表达有关；Thr了促进幼建鲤消化器官的生长发育,提高了消化酶的分泌能力和肠道吸收能力,肠蛋白含量与前中后肠的TOR基因表达量有关；Thr能够促进幼建鲤肠细胞的增殖和分化,提高蛋白合成力和TOR基因表达；Thr提高了幼建鲤的特异性和非特异性免疫力,非特异性免疫能力提高与其提高了对病原菌的识别、粘附调理、抑菌和溶菌能力有关。20-60g幼建鲤Thr的需要量为1.62%或5.07g/100gCP。更多还原

【Abstract】 Six experiments were carried out in this paper. Firstly, we studied the regularity of juvenile Jian carp body composition and the TOR gene expression in different organ and tissue at different weight stage. Based on the results of first study,we studied the effect of dietary threonine levels on growth performance, deposit ration of nutrients,activities of GOT and GPT in serum, hepatopancreas and muscle,development of digestive organ,activities of digestive enzyme, intestinal brush border enzyme activities, TOR gene expression in muscle and intestine, immue fuction of juvenile Jian carp;then we studied the effect of Thr concentration on cell proliferation and differentiation of Jian carp’s IECs with cell culture research model;studied the effect of Thr concentration on protein synthesis ability of Jian carp’s IEC,and the effect on TORgene expression of Jian carp’s IEC.The content and results are as follows:1 Study on body composition and TOR gene expression of variable weight juvenile Jian carp which weight between 18.0-74.7gA total of 750 Jian carp (initial mean weight,18.0±0.2g) were randomly distributed into five groups of each three replicates, feeding the same diet, for the 0,14th,28th,42nd and 56th day sampling, to explore the effect of various weights on body composition of protein,lipid,ash and water, activities of GOT and GPT in serum,muscle and hepatopancreas, TOR gene expression in muscle, hepatopancreas,head kidney,hind kidney,spleen,heart and gill. The result shows:moisture content significantly decreased (P＜0.01) while fat content significantly increased(P<0.01)with the increasing of weight of juvenile Jian carp which weight between 18.0-74.7g; and the protein content increased firstly, after weight was 31.4g the protein content keep a stable level, while ash content decreased significantly (P<0.05), when the weight was 40.1g, the ash content keep a stable level (P>0.05)。The weight of juvenile Jian carp shows highly significant effect (P<0.01) on blood ammonia content, activities of hepatopancreas GPT and GOT. Blood ammonia content, activities of hepatopancreas GPT and GOT showed a highly line regression with weight respectively (Y1= 1083.781+16.457X,R2=0.964; Y2=1249.597 +16.190X, R2=0.947; Y3=2198.725+29.567X, R2=0.957,P<0.01).Activities of GPT and GOT in muscle significantily advanced with the increasing of weight of Jian carp (P<0.01); but activity of serum GOT significantily increased first after the weight reached 40.1 g significantly decreased(P<0.01), activity of serum GPT keep a stable value until weight reached 74.7g activity of GPT significantly increased(P<0.01); TOR gene expression of Jian carp’s weight between 18.0 to 74.7g in head kidney, hind kidney, spleen and gill significantly increased with the increasing of weight (Y1=0.112+0.03X,R2= 0.776, P<0.05; Y2=0.036+0.06X, R2=0.862, P<0.05; Y3=0.255+0.002X, R2=0.770, P=0.05; Y4= 0.132+0.002X, R2=0.774, P<0.05);TOR gene expression in muscle increased with the increasing of weight and showed significantily direct correlation with body protein content and muscle protein content (r1=+0.890; r2=+0.848, P<0.05);while TOR gene expression of hepatopancreas significantily decreased with increasing of Jian carp’s weight (Y=0.486-0.02X, R2=0.854,P<0.05), TOR gene expression of heart keep a stsble value until the weight reached 40.1g significantly decreased (P<0.05)。The result of study shows:juvenile Jian carp which weight between 18.0-74.7g, body protein content and fat content increased with the increasing of weight while moisture content and ash content decreased; the amino acid metabolism of hepatopancreas and muscle enhanced with increasing of weight; muscle,spleen, hepatopancreas, head kidney, hind kidney, gill and heart of juvenile Jian carp showed TOR gene expression, TOR gene expression of muscle,spleen, gill, head kidney, hind kidney increased with the weight increasing while hepatopancreas’s and heart’s deceased.2 Effects of dietary threonine on growth performance, body component, activities of GOT and GPT of serum, hepatopancreas and muscleA total of 1050 Jian carp (13.60±0.03g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Thr (0.70,0.95, 1.25,1.55,1.85,2.15,2.45%,) for 60 days to investigate the effects of Thr on growth performance, body deposition and the GPT and GOT activities in serum, hepatopancreas and muscle. Results showed:Threonine improved highly siginificantly juvenile Jian carp’s specific growth rate and feed efficiency (P<0.01) and specific growth rate, feed efficiency and content of threonine, respectively,showed highly significantly (P<0.01) or significantly(P<0.05)quadratic regression(Y1=-0.3627x2+1.1765x+1.3705, R2=0.927; Y2=-5.8180x2+19.7028x+51.4993, R2=0.790), when threonine content was 1.62%and 1.69% respectively,specific growth rate and feed efficiency reached optimum value; Thr can significantly improve the deposition of protein and fat deposition rate of juvenile Jian carp (P<0.01), but the ash deposition rate of had no significant effect (P> 0.05), protein deposition rate and Thr content showed significant quadratic regression relationship (Y=-4.8344x2+15.2266x+21.3823, R2=0.820, P<0.05, X:Thr%), when the Thr content was 1.57%, the protein deposition rate achieved the best value; Thr can significantly increase activities of GOT and GPT in juvenile Jian carp’s hepatopancreas and muscle (P<0.05), activity of GOT and GPT in hepatopancreas between Thr content showed significant or highly significant quadratic regression relationship (Yi=-1632.7x2+5584.86x-578.64, R2=0.787, P<0.05; Y2=Y=-586.98x2+1575.15x+2101.41, R2=0.961,P<0.01;Y3=-126.06x2+421.100x+703.604,R2=0.948,P<0.01;Y4=-174.89x2+56 4.995x+590.875,R2=0.813,P<0.05,X:Thr%), When Thr contents were 1.71,1.34,1.67 and 1.62%, GOT and GPT activity in hepatopancreas and muscle of juvenile Jian carp reached its maximum; Thr content showed significant effect to blood ammonia concentration (P<0.01), and the ammonia concentration and dietary Thr content showed a significant quadratic regression relationship(Y=1349.88x2-4183.5x+4392.23, R2=0.908, P＜0.01); dietary Thr content significantly effected TOR gene expression of juvenile Jian carp in muscle (P＜0.05), when dietary Thr reached 0.91%, TOR gene expression in muscle significantly decreased(P<0.05)and TOR gene expression significantly increased when Thr content reached 1.57%, but it had no significant difference with 0.70% group, with increasing of Thr content,TOR gene expression in muscle showed a increasing tendency, but had no significant difference (P<0.05)。Correlation analysis showed highly direct correlation between specific growth rate and feed intake, feed efficiency, protein deposition rate, Fatty deposition rate(r1=+0.976; r2=+0.883; r3=+0.923; r4=+0.932, P<0.01)。Presentation of results:Thr improved juvenile Jian carp’s growth, and improment of growth had a direct correlation of increasing of feed intake, feed efficiency, protein and fatty deposition rate; Thr evalulated amino acid metabolism of juvenile Jian carp’s muscle and hepatopancreas, and it significantly effected TOR gene expression in muscle; the optimum dietary Thr requirement for optimal specific growth rate of juvenile Jian carp(13.6-55.6g) was determined to be 1.62%or 5.07g/100gCP。3 Effect of dietary threonine on growth and development of digestive organ, function of digest and absorb ability, TOR gene expression of intestineA total of 1050 Jian carp (13.60±0.03g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Thr (0.70,0.95, 1.25,1.55,1.85,2.15,2.45%,) for 60 days to investigate the effects of Thr on growth and development of digestive organ, function of digest and absorb ability, TOR gene expression of intestine. Results showed:Thr can significantly increased juvenile Jian carp’s hepatopancreas weight and protein content (P<0.01), significantly increased activities of hepatopancreas trypsin, chymotrypsin, lipase (P<0.01), hepatopancreas chymotrypsin activity, lipase activity and Thr content respectively showed significantly (P<0.05) or highly significantly (P<0.01) quadratic regression relationship (Yi=-4.0220x2+13.8680x-5.3260, R2=0.896, Y2=-189.38x2+624.789x+158.988, R2=0.915, X:Thr%), When Thr content reached 1.72 and 1.65%, chymotrypsin and lipase activity of hepotopancreas reached the highest value; Thr can significantly increase juvenile Jian carp’s intestine index, intestinal length index, intestinal protein content and folds height in proximal,middle and distal intestine (P<0.01), significantly increased intestinal trypsin, chymotrypsin, lipase and amylase activity (P<0.01), intestinal pancreatic protease,lipase activity and Thr content showed a significantly (P<0.05) quadratic regression relationship(Y1=-0.7157x2+2.3674x+1.0750, R2=0.895, Y2=-140.57x2+454.669x+ 16.3032, R2=0.879, X:Thr%), When Thr content reached 1.65 and 1.62% respectively, intestinal trypsin and lipase activity reached the highest value; Thr significantly increased intestinal creatine kinase activity and Na+-K+ATP enzyme, alkaline phosphatase, y-glutamyltranspeptidase of proximal,middle and distal intestine (P<0.01), and between activities of these enzyme and content of Thr showed significant or highly significant quadratic regression relationship (Y1=-25.088x2+81.4732x-20.459, R2=0.894, P＜0.05; Y2=-106.30x2+363.988x-68.602; R2=0.945, P＜0.01; Y3=-127.01x2+430.508x-92.817; R2=0.943, P＜0.01; Y4=-70.337x2+226.023x-31.678, R2=0.938, P<0.01; Y5=-11.769x2+37.5870x-6.4615, R2=0.868,P＜0.05; Y6=-5.2309x2+16.5667x+1.5345;R2 =0.924,P＜0.01;Y7=-1.0622x2+3.5065x+0.9637,R2=0.883,P＜0.05;Y8=-22.440x2+71.1129x-9.7408,R2=0.971,P<0.01;Y9=-22.757x2+74.3064x+8.8138,R2=0.917,P<0.01;Y10=-25.413 x2+81.2129x+15.3137,R2=0.910,P<0.01,X:Thr%), when content of Thr reached 1.62. 1.71.1.69.1.61.1.60.1.58.1.65.1.58.1.63.1.60% respectively, Intestinal creatine kinase, proximal,middle and distal intestinal Na+-K+ATPase, alkaline phosphatase and y-glutamyl transferase enzyme activity reached the maximum value. Thr content significantly affect the number of intestinal Bacterium acidi lactici, E. coli and Aeromonas of juvenile Jian carp (P＜0.01),when Thr content reached 1.57%, number of intestinal Bacterium acidi lactici reached a peak, continue to add Thr, number of intestinal Bacterium acidi lactici showed a descend tendency, while the number chang of E. coli and Aeromonas showed a opposite tendency; Thr significantly affected intestinal TOR gene expression of juvenile Jian carp, intestinal protein content and middle intestinal TOR gene expression showed significant negative correlation (r=-0.917, P<0.01)。The results demonstrated that Thr improved Jian carp hepatopancreas growth and development, increased hepatopancreas’s ability of secreting digestive enzyme; Thr improved Jian carp intestinal growth and development, increased intestinal ability of digesting protein.fatty and starch; improved Jian carp’s absorb ability, at the same time Thr optimized intestinal microflora of juvenile Jian carp, increased the number of beneficia bacterium, decreased number of harmful bacterium; Thr content significantly affected intestinal TOR gene expression of Jian carp; the optimum dietary Thr requirement for optimal activities of digestive enzyme and intestinal brush border enzyme of juvenile Jian carp(13.6-55.6g) was determined to be 1.58～1.65%, which close to 1.62% determined by specific growth rate.4 Effects of threonine on intestinal enterocyte cell multiplication and differentiation of Jian carpUsing single-factor experiment design in order to sdudy the effects of threonine on intestinal enterocyte cell multiplication and differentiation of Jian carp by in vitro cell culture model.Experimental Design 6 treatment, with 4 replicates for each treatment. Threonine were added for each treatment concentration:0,135,170,205,240, and 275mg/ L. The results showed that:threonine levels significantly affected the MTT OD values of IEC of carp (P<0.05), with Thr concentration increasing, MTT OD of carp IEC increased significantly (P<0.05), when the Thr concentration reached 205mg/L, MTT OD value reached the max, when increased threonine concentration continually, MTT OD values decreased significantly (P<0.05); Thr significantly effected on alkaline phosphatase, Na+-K+ATP enzyme activity and cell protein content of carp IEC (P<0.05), when the Thr concentration reached 205mg/L, alkaline phosphatase, Na+-K+ATP enzyme activity reaches its maximum, continued to increase threonine concentration, both of these two enzyme activity decreased significantly (P<0.05),while the protein content increased with the increasing of threonine concentration;Thr significantly reduced the consumption of protein in culture medium (P<0.05), when Thr concentration reached 170mg/L, the protein consumption was the lowest; Thr significantly reduced the activity of lactate dehydrogenase in culture medium (P<0.05), when Thr concentration reached 205 mg/L, the lactate dehydrogenase activity in culture medium reached minimum; Thr significant effected GOT and GPT activity of carp IEC, significantly affected the ammonia concentration in the culture medium (P<0.05), when the Thr concentration reached 205 mg/L, GOT activity of carp’s IEC was significantly higher than the rest of the treatments, while the GPT activity was first increased significantly (P<0.05), when Thr concentration reached 170mg/L then decreased significantly (P<0.05), when Thr concentration reached 135mg/L, ammonia concentration in the culture medium decreased significantly (P＜0.05), when the Thr concentration reached 205mg/L, the ammonia concentration reachede the lowest value.Research results indicate that:Thr promoted the proliferation and differentiation of carp IEC, promoted amino acid metabolism, thus ensuring the normal structure and function of IEC.5 Effect of threonine on protein synthesis and TOR gene expression of carp intestinal epithelial cells3 small pilot study was carried out in this study. the First one seted two treatment to study the effects of threonine on protein synthesis of carp IEC, with 10 replicates for each treatment, incubation time was 240min. threonine concentration of two treatment were: Omg/L and 205mg/L Thr; the second small study investigated the effects of TOR gene expression of carp IECs, seted two treatment respectively, Omg/L and 205mg/L Thr,6 replicates for each treatment, incubation time was 30min. The third study examined the time effect of TOR gene expression of carp IECs by threonine with 205mg/L, seted up five treatment 15min,30min, 100min,170min and 240min), at each time point seted 6 replicaation. The results showed that:Thr significantly increased protein synthesis rate of carp intestinal epithelial cells (P<0.05), significantly increased TOR gene espression of Jian Carp’s IEC (P<0.05), increasment rate reached 55%; with the time extension, Thr significantly increased TOR gene expression of Jian carp IECs (P<0.05), when the culture time reached 30 min, TOR gene expression of Jian carp IECs significantly increased (P<0.05),when the culture time between 30min to 100min,TOR gene expression remained a stable level, when the cultue time reached 170 min, TOR gene expression increased significantly (P<0.05), then keep a stable level again. The results suggested that threonine increased the protein synthesis of Jian carp’s IECs, the improvement of protein synthesis concerned with the increase of TOR gene expression by threonine,the effects of threonine on TOR gene expression of carp’s IECs were significantly affected by culture time,when the culture time reached 170min, TOR gene expression of carp’s IECs reached a stable value.In summary:TOR gene expression in muscle, spleen, head kidney, kidney and gill of juvenile jian carp weighted 20 to 80g increased with weight gain, while the hepatopancreas and heart decreased; Thr could promote growth performence, enhance body protein and body fat content, protein content concerned with TOR gene expression in muscle; Thr could promote the growth and development of juvenile Jian carp’s digestive organs, improve its capacity of the secretion of digestive enzymes and improve intestinal absorptive capacity, intestinal protein content concerned with the TOR gene expression of intestine.Threonine could promote proliferation and differentiation of juvenile Jian carp’s IECs, elevate the ability of protein synthesis and TOR gene expression of Jian carp’s IECs; Threonine could improve nonspecific immunity and specific immunity ability, and the improvement of nonspecific immunity ability was responsed to its improvement of identify, sticking, bacteriostasis and bacteriolysis to pathogenic bacterium. According our study recult, the requirement of juvenile Jian carp(20-60g) was determined to be 1.62% or 5.07g/100gCP。6 Effect of Thr on immune function of juvenlile Jian carpThis study was carried out with juvenile Jian carp to investigate the effect of dietary threonine on the disease resistance and immune response. purified diets with seven levels (threonine content:0.70,0.95,1.25,1.55,1.85,2.15,2.45%) were fed to Jian carp (initial weight13.60±0.03g g, mean±SD). After 60 days feeding trial, the challenge experiment with Aeromonas hydrophila (AH) was conducted to determine the impact of the graded threonine levels on the disease resistance of Jian carp. Recults showes:Survival after vaccination was direct correlation with the increasing threonine levels(P＜0.01). when content reached 1.57% Survival after vaccination was 100%; threonine had a positive effect on the weight of head kidney and spleen (P＜0.05), significantly increased Blood counts of red cell and white cell(P<0.01); threonine also increased the Phagocytic activity of leukocyte significantly (P＜0.01), Phagocytic activity of leukocyte and threonine content showed a significantly quadratic regression relationship (Y=-26.408x2+88.0857x-21.261, R2=0.969, P＜0.01),when content of threonine reached 1.57%, Phagocytic activity of leukocyte reached the max; Serum immunoglobulin M (IgM) level and agglutination antibody titer to A. hydrophila were significantly increased (P＜0.05) in juvenile Jian carp fed the diets with the increasing of dietary threonine levels,when dietary content reached 1.57%, IgM level reached max, agglutination antibody titer to A. hydrophila and content of threonine showed a significantly quadratic regression relationship (Y=-54.651x2+179.867x-99.372, R2=0.969,P<0.05), when content of threonine reached 1.65%, agglutination antibody titer to A. hydrophila reached the max; thronine also significantly affected content of serum complement 3 and complement 4, lectin potency, activity of lysozyme (LA) and acid phosphatase (ACP) and total iron-binding capacity (TIBC) (P＜0.01), serum complement 3 and complement 4, acid phosphatase, total iron-binding capacity and content of thronine showed significantly quadratic regression relationship (Y1=-105.55x2+352.445x-127.43, R2=0.800; Y2=-14.066x2+46.1787x-12.768,R2=0.805;Y3=-186.01x2+594.124x-168.16,R2=0.883; Y4=-31.992x2+99.9297x+22.3843, R2=0.793, P<0.05, X:Thr%). Correlation analysis showed that there are highly significantly direct correlation between Survival after vaccination and weight of head kidney.spleen, Phagocytic activity of leukocyte, erum complement 3, IgM level (r1=+0.936, r2=+0.981, r3=+0.9841,r4=+0.932, r5=+0.934, P<0.01). These results suggested that threonine could enhance disease resistance of juvenile Jian carp,which response to the improvement of immune organ growth and development, nonspecific immunity and specific immunity ability, the optimum dietary Thr requirement for optimal immune function of juvenile Jian carp(20-60g) was determined to be 1.56-1.69%。更多还原