牦牛小肽转运载体（PepT1）克隆表达及其转运调控研究

Yak, a dominated livestock species in the Qinghai-Tibet plateau, mainly utilized dietary amino acids in the form of small peptide from mesenteric system, and had more efficient in retention of nitrogen than cattle, which presented the specific mechanism of non-protein utilization, and this mechanism also comprises a peptide absorption mechanism, but the research is still blank. Furthermore,small peptide nutrition is paid more and more attention in the ruminant protein nutrition, and characterization of expression and regulation of gastrointestinal peptide transporter (PepT1) gene can reflect micro-mechanism on absorption and transportation of small peptide. Therefore, a proposal was disigned to study the mechanism of regional expression of yak; clone,express and purify yak PepT1; construct cell model of PepT1 in CHO cell and study the effect of regulative factors on cell model transportation. ExperienceⅠwill be establish the RT-PCR methods to quantitative investigate peptide transporter PepT1 mRNA expression rate in yak small Intestine; And based on established Real-Time PCR method, evaluate expression of yak PepT1 in the different gastrointestinal tisse (Epithelium of rumen, reticulum, omasum and abomasum; Mucous membranes of duodemum, jejunum, ileum and colon).ExperienceⅡ is to construct the vector of yak intestinal peptide transporter PepT1, and analyze gene sequence. Experiment Ⅲ will include three trials. Construction of the expression vector of yak intestinal peptide transporter PepT1 and its transfecting condition optimization; Cloning and constructing recombinant yak PepT1 in CHO cell; Construct yak PepT1 gene in eukaryotic expression vector and express it in CHO cells, detecting transport activity of recombinant yak PepT1 protein in vitro. So, if above molecular biology techniques will be developed successfully, it helps to clarify nutrition physiological adaptive mechanism on alpine environments for yak and tibetan sheep. Therefore, it also will provide theoretical basis and technical support for sustainable development of grassland stockbreeding in the Qinghai-Tibetan plateau.

牦牛在青藏高原生态系统具有举足轻重的作用，其氮代谢高寒适应性机制是学术界关注的重点问题。早期研究证明牦牛主要经非肠系膜系统以小肽的形式吸收氨基酸，我们前期研究亦发现牦牛比黄牛更能高效利用氮，这种差异性与肽的转运机制密不可分，但目前对其小肽转运的分子基础尚不清楚。本项目拟建立牦牛消化道小肽转运载体PepT1基因表达RT-PCR检测方法，并依此技术评定牦牛消化道各部位PepT1基因表达水平；对牦牛消化道PepT1基因进行克隆测序，分析其功能；建立细胞外源表达的方法，将牦牛PepT1在仓鼠卵母细胞中表达，进一步研究不同培养体系下该模型中PepT1对同位素标记小肽转运量的影响，探索牦牛小肽转运调控机理。预期研究成果可以揭示牦牛小肽吸收转运的分子基础，同时分子生物学技术在该畜种的研发与利用，利于从微观层次明晰高原反刍家畜营养适应性机制，为藏区草地畜牧业可持续发展提供理论依据与技术支持。

牦牛是青藏高原生态系统的特有畜种，其体貌特征、生理特点、牧食习性均有别于其他同类家畜。近年来研究发现牦牛具有适应高寒营养胁迫的独特氮代谢机制，而小肽作为蛋白质的降解产物和参与合成蛋白的营养物质，是否具有特殊转运吸收的营养学特征以适应高寒营养胁迫的环境，目前尚不清楚。鉴于此，本项目主要研究内容为：1）牦牛小肽转运载体PepT1的克隆及生物学分析；2）放牧牦牛PepT1表达分布特性及牦牛与本地黄牛PepT1mRNA表达对日粮氮水平的响应研究；3）牦牛与本地黄牛PepT1转运调控差异研究。结果表明：1）牦牛小肽转运载体yPepT1全长为2805bp，编码707个氨基酸，包含5个N-glycosylation 位点、2个PKA位点、3个PKC位点，存在12个跨膜区，yPepT1与黄牛bPepT1在第258、515、536、544氨基酸存在差异。yPepT1氨基酸与其他物种同源性分别为：黄牛99%，绵羊95%，猪86%，人类83%，小鼠81% 和家兔79%；2）Real-time PCR检测结果显示放牧牦牛PepT1mRNA在各组织中均有表达。其中，空肠表达量最高，显著高于其他组织(P < 0.001)，回肠和十二指肠次之，其他组织表达量由高到低依次为网胃、乳腺、瘤胃、瓣胃、结肠、皱胃、盲肠、肝脏及肾脏。舍饲试验（于青藏高原乌鞘岭牧区选取牦牛和本地黄牛各12头，两个基因型动物分为4组，分别饲喂低、中低、中高和高氮全价饲粮）结果显示牦牛与本地黄牛瘤胃、瓣胃、十二指肠、空肠、回肠和肝脏组织PepT1 mRNA存在基因型之间的差异(P < 0.05)。同时，随着氮水平的增加，牦牛与本地黄牛瘤胃、网胃、瓣胃、十二指肠、回肠和肝脏组织PepT1 mRNA存在氮水平之间的差异(P < 0.05)。此外，牦牛与本地黄牛在瘤胃、网胃、瓣胃、十二指肠、空肠、回肠和肝脏组织PepT1 mRNA均在基因型和氮水平交互之间的差异(P < 0.05)；3）通过不同底物小肽刺激含有pcDNA3.1-yPepT1和pcDNA3.1-bPepT1转染的CHO细胞，发现PepT1mRNA表达量对两基因型牛不同底物小肽的响应存在差异。综上，通过研究牦牛小肽转运的分子基础以及活性调控机理，完善了高原反刍动物营养调控理论，为后期研究牦牛适应高寒营养胁迫肽吸收机制奠定基础。