动物营养学报    2021, Vol. 33 Issue (7): 4030-4037    PDF    
引用本文
周苗苗, 刘桂芹, 刘文强, 朱明霞, 王长法. 驴小肽转运载体1基因的克隆、序列分析及组织表达研究[J]. 动物营养学报, 2021, 33(7): 4030-4037.  
ZHOU Miaomiao, LIU Guiqin, LIU Wenqiang, ZHU Mingxia, WANG Changfa. Cloning, Sequence Analysis and Tissue Expression of Donkey (Equus asinus) Oligopeptide Transporter 1 Gene[J]. Chinese Journal of Animal Nutrition, 2021, 33(7): 4030-4037.  
驴小肽转运载体1基因的克隆、序列分析及组织表达研究
周苗苗 , 刘桂芹 , 刘文强 , 朱明霞 , 王长法     
聊城大学农学院毛驴高效繁育与生态饲养研究院, 聊城 252000
收稿日期: 2020-12-30
基金项目: 国家自然科学基金项目（31902177）；山东省"泰山产业领军人才"项目（LJNY201713）；聊城大学博士科研启动基金（318051816）
作者简介: 周苗苗(1983-), 女, 山东东阿人, 副教授, 博士, 从事草食动物营养研究。E-mail: zhoumm0329@163.com.
摘要: 小肽转运载体1（PepT1）在动物肠道小肽的摄取过程中发挥重要作用。本试验旨在研究驴（Equus asinus）PepT1（ePepT1）基因的分子克隆、序列分析和组织表达。提取驴肠道组织总RNA，PCR克隆ePepT1基因片段，测序验证并进行序列分析；采用实时荧光定量PCR（real-time qPCR）方法分别测定驴肾脏、脾脏、心脏、肝脏、肺脏、胃、十二指肠、空肠和回肠组织中ePepT1基因的相对表达量。结果表明：ePepT1开放阅读框为2 124 bp，共编码707个氨基酸残基。蛋白质理化性质预测显示，ePepT1蛋白分子质量为78.6 ku，等电点为7.52，具有11个跨膜区（TMD），且TMD 9和TMD 10之间有1个大的细胞外环；ePepT1蛋白有5个胞外N-糖基化位点，5个胞外蛋白激酶C（PKC）作用位点和3个蛋白激酶A（PKA）作用位点。组织分布研究发现，ePepT1基因在各组织均有表达，但在肠道（十二指肠、空肠和回肠）中相对表达量最高。综上所述，本研究首次克隆了ePepT1基因，研究了其组织表达规律，为进一步研究驴肠道小肽吸收提供了基础。
关键词: 驴    小肽转运载体1    克隆    序列分析    组织表达    
Cloning, Sequence Analysis and Tissue Expression of Donkey (Equus asinus) Oligopeptide Transporter 1 Gene
ZHOU Miaomiao , LIU Guiqin , LIU Wenqiang , ZHU Mingxia , WANG Changfa     
Liaocheng Research Institute of Donkey High-Efficiency Breeding and Ecological Feeding, College of Agriculture, Liaocheng University, Liaocheng 252000, China
Abstract: Oligopeptide transporter 1(PepT1) plays an important role in the uptake of small peptides from animal intestine. The aim of this study was to study the molecular cloning, sequence analysis and tissue expression of PepT1 gene in donkey (Equus asinus) (ePepT1). Firstly, total RNA was extracted from donkey intestines, and ePepT1 gene fragment was cloned by PCR, sequenced and sequence analyzed. Then, total RNA was extracted from kidney, spleen, heart, liver, lung, stomach, duodenum, jejunum and ileum of donkey, and the relative expression level of ePepT1 gene was determined by real-time quantitative PCR. The results showed that the cloned ePepT1 cDNA sequence was 2 124 bp at full length encoding a 707 amino acids peptide with molecular of 78.6 ku and theoretical soelectric point (pI) of 7.52. Bioinformatics analysis showed that the deduced peptide sequence possessed all the characteristic features of ePepT1 identified. The ePepT1 had 11 potential transmembrane domains (TMD), with a large extracellular loop between TMD 9 and TMD 10. Five putative extracellular N-glycosylation sites, five putative extracellular protein kinase C (PKC) sites, and three protein kinase A (PKA) sites were identified in the ePepT1 sequence. The study of tissue distribution showed that ePepT1 gene was expressed in all tissues, but the relative expression level was the highest in intestinal tract (duodenum, jejunum and ileum). In conclusion, ePepT1 gene is cloned for the first time in this study, and its tissue expression is studied, which provides a basis for further study on the absorption of small peptide in donkey intestine.
Key words: donkey    oligopeptide transporter 1    cloning    sequence analysis    tissue expression    

蛋白质是动物必不可少的营养元素，饲粮蛋白质在动物体内水解为氨基酸(amino acids, AA)或小肽后被肠道吸收。其中，小肽(二肽和三肽)是动物摄取蛋白质的主要形式[1]。小肽的吸收利用在动物营养物质代谢和动物健康方面有重要意义。肠道小肽的吸收主要依赖小肽转运载体1(oligopeptide transporter 1, PepT1)。PepT1是一种低亲和力、高容量的膜转运蛋白，属于质子偶联寡肽转运体(proton-coupled oligopeptide transporter, POT)家族，在质子电化学梯度的驱动下，能跨膜转运寡肽和肽类药物[2-4]。PepT1主要在小肠中表达，此外，在大肠[5-6]、瓣胃和瘤胃[7]、肾脏和肝脏[5, 8]、胰腺[9]、性腺[5, 10]中亦有表达。PepT1可以转运数千种寡肽和多种肽结构类似药物，如氨基头孢菌素、血管紧张素转化酶抑制剂以及抗病毒和抗肿瘤药物[11-12]。PepT1的表达和功能已在人类[13-14]、兔[8, 15]、大鼠[16]、小鼠[17]、猪[18]、鸡[19]、牛[20-21]、绵羊[22]、牦牛[23]、斑马鱼[10]、大西洋鳕鱼[24]、大西洋鲑鱼[25]和双髻鲨[9]等多种动物中进行了研究。众多研究表明，PepT1在物种间高度保守，PepT1蛋白由大约708个AA残基组成，有12个跨膜区(transmembrane domains, TMD)，且在TMD 9和TMD 10之间有1个大的细胞外环[13, 16-18, 21-22]。目前关于PepT1功能的研究主要集中在拟肽类药物转运以及胃肠道生理病理学等方面[2, 11-12, 26]。此外，PepT1的生物活性受底物、激素、年龄和生理条件等多种因素的调节[6, 27-28]。PepT1在动物的生长和代谢中起着重要的生理作用。然而，PepT1在驴中的表达和功能研究还未见报道。因此，本试验拟研究驴(Equus asinus)PepT1(ePepT1)基因的克隆、序列分析及组织表达，以期为进一步研究驴的营养物质转运提供理论依据。

1 材料与方法 1.1 总RNA的提取和全长cDNA克隆

采集健康驴新鲜十二指肠组织，Trizol法提取总RNA，分光光度计测OD260 nm/OD280 nm值，琼脂糖凝胶电泳检测其完整性。根据PrimeScript® RT反转录试剂盒(TaKaRa)提供的方法进行反转录，Pfu高保真聚合酶(transgen biotech)特异扩增ePepT1 cDNA。PCR引物见表 1，PCR反应体系见表 2。PCR反应条件为：94 ℃ 3 min，98 ℃ 10 s，53 ℃ 15 s，72 ℃ 2.5 min (扩增35个循环)，72 ℃ 5 min。PCR产物进行电泳检测，然后使用美基胶回收试剂盒(Magen)回收目的产物并纯化。胶回收产物连接克隆载体pTOPO-Blunt vector (Azbiochem)，连接产物转化DH5α感受态细胞，涂布LB (Luria-Bertani)平板(氨苄，100 μg/mL)，并于37 ℃培养过夜。挑取6个阳性菌落，进行PCR鉴定，并进行测序[生工生物工程(上海)股份有限公司]。

表 1 试验中所用引物 Table 1 Primers used in the experiment
表 2 PCR反应体系 Table 2 PCR reaction system 
1.2 ePepT1的AA序列和系统发育分析

用ORF Finder推导AA序列(http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi)；利用计算机pI/Mw工具(ExPASy Proteomics Server; https://web.expasy.org/compute_pi/)预测ePepT1蛋白的分子质量和理论等电点(theoretical isoelectric point, pI)；利用在线软件(http://scratch.proteomics.ics.uci.edu/)预测ePepT1蛋白质的二级结构；利用生物序列分析(Lyngby TMHMM Server; http://www.cbs.dtu.dk/services/TMHMM-2.0/)预测ePepT1的跨膜区；利用NetNglyc和NetPhos服务器(http://www.cbs.dtu.dk/services/NetNGlyc/; http://www.cbs.dtu.dk/services/NetPhos/)预测潜在的N糖基化、蛋白激酶A(protein kinase A, PKA)和蛋白激酶C(protein kinase C, PKC)位点的AA序列；利用MEGA7构建系统进化树[29]。

1.3 实时荧光定量PCR

试验选用2岁左右的健康德州驴公驴5头，屠宰后取肾脏、脾脏、心脏、肝脏、肺脏、胃、十二指肠、空肠和回肠组织，Trizol法分别提取各组织的总RNA。取1 mg RNA，用PrimeScript® RT试剂盒(TaKaRa)合成cDNA。然后用SYBR® PrimeScriptTM试剂盒(TaKaKa)在ABI 7500 (Applied Biosystems, Singapore)上测定ePepT1基因相对表达量。实时荧光定量PCR反应体系为20 μL。PCR引物序列见表 1。以甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)为内参，采用2-△△Ct的方法[30]计算ePepT1基因相对表达量。每个样品重复3次，超纯水替代样品做阴性对照。

1.4 统计分析

本研究所有数据用SAS 9.2进行分析，多组数据间采用单因素方差分析，数据用柱状图表示，误差线为标准差。当P < 0.05时认为差异显著。

2 结果与分析 2.1 ePepT1的AA序列和系统发育分析

PCR特异性扩增ePepT1基因，然后将PCR扩增产物进行电泳检测并切胶回收。目的基因的PCR产物进行琼脂糖凝胶电泳后可见一条约2 000 bp大小的条带(图 1)。经测序验证，成功扩增ePepT1基因。序列分析表明，ePepT1的开放阅读框为2 124 bp，可编码707个AA残基组成的多肽链，该蛋白分子质量为78.6 ku，等电点为7.52。ePepT1的AA序列与马(99.15%)、羊驼(86.58%)、山羊(86.28%)、牛(86.14%)、绵羊(85.43%)、猪(84.60%)、人类(83.47%)、狗(83.36%)、小鼠(83.36%)和大鼠(82.82%)的PepT1的AA序列高度保守。对ePepT1蛋白进行预测得出，二级结构中α-螺旋(Hh)占32.39%，β-折叠(Ee)占27.58%，β-转角(Tt)占9.76%，无规则卷曲(Cc)占30.27%。亲水性和疏水性分析预测ePepT1有11个潜在的TMD，且在TMD 9和TMD 10之间有1个大的细胞外环(图 2)。如图 3所示，ePepT1的AA序列中有5个胞外N-糖基化位点(Asn50、Asn438、Asn508、Asn530、Asn541)，5个胞外PKC作用位点(Ser8、Ser249、Ser252、Thr356、Ser357)和3个PKA作用位点(Ser8、Ser29、Ser275)(图 3)。根据不同动物PepT1的AA序列构建的系统进化树如图 4所示。

1：DL15 000标记物; 2和3：ePepT1扩增产物。 1: DL15 000 marker; 2 and 3: ePepT1 amplified products. 图 1 PCR扩增产物电泳结果 Fig. 1 Electrophoresis results of PCR products
横轴：氨基酸序数；纵轴：疏水系数。 Horizontal axis: amino acid number; longitudinal axis: hydrophobicity coefficient. 图 2 ePepT1蛋白的跨膜区分析 Fig. 2 Analysis of transmembrane domains of ePepT1 protein
N-glyc: N糖基化位点glycosylation site；PKA: 蛋白激酶A protein kinase A; PKC：蛋白激酶C protein kinase C。 图 3 ePepT1蛋白的跨膜区模型 Fig. 3 Transmembrane domain model of ePepT1 protein
图 4 基于不同动物PepT1的氨基酸序列的系统进化树 Fig. 4 Phylogenic tree constructed based on amino acid sequences of PepT1 from different animals
2.2 ePepT1基因的组织表达

以GAPDH做内参基因，分别检测了ePepT1基因在驴肾脏、脾脏、心脏、肝脏、肺脏、胃、十二指肠、空肠、回肠组织中的表达。结果显示，ePepT1在组织中广泛表达，其在肠道(十二指肠、空肠和回肠)和肺脏中的相对表达量较高，显著高于肾脏、脾脏、胃、心脏、肝脏(P < 0.05，图 5)。

数据柱标注不同字母表示差异显著(P＜0.05)。 Data bars with different letters mean significant difference (P < 0.05). 图 5 ePepT1基因在各组织中的相对表达量 Fig. 5 Relative expression level of ePepT1 gene in different tissues
3 讨论

PepT1在从细菌到人类的不同物种中高度保守[31]。本研究发现，ePepT1 cDNA开放阅读框为2 124 bp，可以编码由707个AA残基组成、分子质量为78.6 ku的多肽链。ePepT1蛋白大小与马、绵羊和牛(707个AA残基)[21-22]相同，但比人类和猪(708个AA残基)[13, 18]、小鼠(709个AA残基)[17]以及大鼠(710个AA残基)[16]小。疏水性分析预测ePepT1有11个潜在的TMD，这与奥奈达希瓦氏菌(Shewanella oneidensis)(TMD 14)[32]和其他哺乳动物如人和牛(TMD 12)不同[13, 21]。同其他物种一样，ePepT1蛋白在TMD 9和TMD 10之间有1个大的细胞外环。此外，ePepT1蛋白TMD 9和TMD 10之间的胞外环上有5个推测的N-糖基化位点(Asn50、Asn438、Asn508、Asn530、Asn541)。N-糖基化对寡肽转运蛋白的表达、稳定性和底物转运至关重要[33-35]。Chan等[34]研究结果显示，人PepT1的TMD 9和TMD 10之间的胞外环中所有的天冬酰胺残基(asparagine, Asn)都可以被N-糖基化修饰，且N-糖基化位点的突变可以降低PepT1对甘氨酰肌氨酸(Gly-Sar)转运的Vmax值。Wang等[35]研究亦发现牛PepT2的转运功能受N-糖基化的影响。此外，ePepT1蛋白还具有5个胞外PKC作用位点(Ser8、Ser249、Ser252、Thr356、Ser357)和3个PKA作用位点(Ser8、Ser29、Ser275)。据报道，PKA和PKC可以通过调节葡萄糖转运蛋白2(GLUT2)和小肽转运蛋白PepT1来改变营养素的摄取[36]。其他研究亦证明小肽转运蛋白的转运功能受PKC磷酸化的调控[22, 37-38]。这些磷酸化位点可能在PKC和PKA调节ePepT1的转运功能中发挥重要作用。

研究发现PepT1主要在肠道中表达，其组织分布规律不同于PepT2(主要在肾脏中表达)[39]。众多研究表明，PepT1在动物消化道中广泛表达，且在十二指肠、空肠和回肠中表达量最高[5-6, 9, 24]。此外，PepT1的表达受生理状态，如发育时期、营养状况和疾病状态等多种因素的调控[6, 28, 40-41]。很多研究发现，5/6肾脏切除、小肽、甲状腺激素等均可增加PepT1的基因丰度[5, 27]。本试验中，ePepT1基因在驴肾脏、脾脏、心脏、肝脏、肺脏、胃、十二指肠、空肠、回肠组织中均有表达，且在肠道(十二指肠、空肠和回肠)中的相对表达量显著高于其他组织，其组织表达规律与以往研究结果一致。PepT1在动物体内的组织分布与其生理功能相适应，PepT1主要在消化道小肽结合氨基酸的摄取中发挥重要作用[7, 18, 21]。畜禽可通过调节PepT1的表达来改变机体对小肽的摄取，以满足不同生长阶段对营养物质的需求。本研究结果提示，ePepT1在驴肠道小肽类营养物质的吸收中发挥重要生理作用。ePepT1的表达调控及其在驴营养物质代谢中的具体作用仍需进一步研究。

4 结论

① 本试验成功扩增出ePepT1基因，序列分析表明ePepT1的开放阅读框为2 124 bp，可编码707个AA残基组成的多肽链。

② 研究证实ePepT1基因在驴各组织中普遍表达，且在十二指肠、空肠和回肠中高表达。

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