小肽对肉鸡MyoD基因及肌形成的营养作用及调控机理研究

Application of nutritional regulation is considered as one of the simplest and most economical methods to solve the problems related to feed shortage, food safety, low carbon and environmental protection in animal production. Small peptides being bioactive molecules as well as nutrients perform both nutritional function and physiological regulation. This study using the methods of cell culture, TaqMan real time RT-PCR, western blot, flow cytometry, in ovo feeding and animal feeding trial is to investigate ① the effect of amino acid and dipeptide on the morphology and MyoD expression of broiler myoblast; ② the effect of different peptide concents on the proliferation and differentiation of myoblast; ③ the effect of different small peptide on the cell cycle, expression of myosin heavy chains (MHC) and myoblast determination (MyoD) in myoblast for exploring their nutritional difference; ④ the effect of amino acid, dipeptide and their mixture by yolk sac injection during embryogenesis period on the development of skeletal muscle,the expression of MHC, MyoD, myogenin;and ⑤ the effect of the diet containing dipeptide or tripeptide on the growth and myofiber type of skeletal muscle in the breast and gastrocnemius of broilers, and the profile of MHC, MyoD and myogenin in slow-twitch, red fast-twitch and white fast-twitch. These results will be very helpful for the establishment of the regulation mechanism of small peptide-MyoD- myogenesis-protein accretion and the application of small peptide nutritional regulation in animal production.

动物营养调控技术是解决畜牧业饲料紧缺、食品安全、低碳环保等问题最简单、最经济的办法。小肽是营养物质，更是生物活性分子，具有营养和生理调节双重功能。本项目拟采用细胞培养、TagMan荧光定量RT-PCR、Western blot、流式细胞、鸡胚给养以及饲养试验等技术手段，研究：①氨基酸和二肽对鸡成肌细胞形态及MyoD表达的影响；②不同浓度二肽对鸡成肌细胞增殖与分化的影响；③不同种类二肽对鸡胚成肌细胞周期、MHC和MyoD表达的影响；④鸡胚卵黄囊注射氨基酸、二肽、二者混合物，比较对胚胎期肌肉发育及MHC、MyoD、生肌素表达的影响；⑤二肽和三肽日粮饲喂肉鸡，对胸肌、腿肌生长及慢肌、快红肌、快白肌中MHC、MyoD、生肌素表达的影响。拟初步构建小肽营养-MyoD-肌形成-体蛋白沉积的调控机理，为建立小肽营养调控技术提供理论基础。

小肽是营养物质，更是生物活性分子，具有营养和生理调节双重功能。生肌因子调控着机体肌肉的发育过程，同时其调节作用又受到营养等外部因素的影响。DNA甲基化是基因型与表型联系的纽带，能调控特定基因的表达，进而影响动物的生产性能。研究结果：1）亮氨酸及其二肽对鸡成肌细胞生长的比较研究，发现二肽处理组成肌细胞的生长曲线增长点均早于氨基酸处理组对应时间的增长点，表明二肽对细胞生长提供的营养优于氨基酸营养。氨基酸及二肽混合物组细胞的生长优于纯氨基酸或二肽组。2）亮氨酸及其二肽对鸡成肌细胞MyoD的比较研究，发现亮氨酸、二肽和二者混合物对鸡胚成肌细胞MyoD转录和翻译水平的表达有显著影响，以二肽组、混合组、氨基酸组依次降低。3）不同种类二肽对鸡成肌细胞周期分布规律的影响，发现三种二肽都能促使G0期和G1期细胞跨越限制点，开始分裂增殖。4）不同种类二肽对肉鸡成肌细胞MHC表达的影响，Di-Leu能够促进肌形成，处理时间为32.20min、处理浓度为24.87μM时的影响最为显著。5）小肽对成肌细胞MyoD表达的影响，亮氨酸、二肽和三肽对细胞迁移呈依次显著递增作用，Di-Leu 组MyoD表达量显著高于Leu-Gly和Gly-Gly组。6）胚胎给养二肽显著提高鸡胚孵化效果，但是对肉仔鸡生产性能和养分消化率没有影响；对雏鸡早期肠道形态学有显著影响，但是氨基酸和二肽之间差异不显著；出壳后12h，处理对腿肌重、MHC、MyoD以及生肌素的转录表达有显著影响。7）胚蛋给养对雏鸡RPS6KB1启动子甲基化的影响，胸肌和腿肌之间甲基化水平相似，处理组甲基化程度低于同时期对照组甲基化程度。结论：与氨基酸相比，小肽在成肌细胞生长、生肌因子活性、胚胎发育、肌肉组织发育等方面都呈现优势，但是不同氨基酸残基组成的小肽之间存在差异，小肽与氨基酸混合氮源营养效果最好，本研究结果为营养调控体蛋白合成的机制以及精准营养供给技术的建立提供了宝贵的理论基础和开创性思路。