CAJ | 학술논문

采用同源克隆和 RACE 技术克隆草鱼(Ctenopharyngodon idellus)PepT1基因的全长 cDNA 序列.该 cDNA全长为2762 bp,包含141 bp 的5′UTR 序列,479 bp 的3′UTR 序列,2142 bp 开放阅读框,编码713个氨基酸;草鱼与鲫(Carassius auratus)、斑马鱼(Danio rerio)的核苷酸同源性分别为77.6%和74.0%,氨基酸同源性分别为78.0%和76.7%;与其他物种的核苷酸同源性为53.9%~59.1%,而氨基酸的同源性为57.2%~61.8%.经预测,其编码蛋白的分子量为79.29 kD,等电点为5.87,该蛋白具有与哺乳动物十分相似的11个螺旋跨膜结构,跨膜区氨基酸高度保守;系统进化分析表明,草鱼 PepT1基因与鲫鱼和斑马鱼的亲缘关系最近;利用 Real-time PCR 技术检测了该基因的时空表达,结果显示, PepT1在草鱼前肠组织表达量最高,其次是肌肉组织;草鱼出膜7 d 后 PepT1 mRNA表达量相对稳定;昼夜节律研究发现,肠道 PepT1基因夜间的表达量较白天高.本研究旨在为小肽转运载体 PepT1介导肠道转运小肽调控草鱼对饲料蛋白消化吸收的分子机理提供理论基础.
채용동원극륭화 RACE 기술극륭초어(Ctenopharyngodon idellus)PepT1기인적전장 cDNA 서렬.해 cDNA전장위2762 bp,포함141 bp 적5′UTR 서렬,479 bp 적3′UTR 서렬,2142 bp 개방열독광,편마713개안기산;초어여즉(Carassius auratus)、반마어(Danio rerio)적핵감산동원성분별위77.6%화74.0%,안기산동원성분별위78.0%화76.7%;여기타물충적핵감산동원성위53.9%~59.1%,이안기산적동원성위57.2%~61.8%.경예측,기편마단백적분자량위79.29 kD,등전점위5.87,해단백구유여포유동물십분상사적11개라선과막결구,과막구안기산고도보수;계통진화분석표명,초어 PepT1기인여즉어화반마어적친연관계최근;이용 Real-time PCR 기술검측료해기인적시공표체,결과현시, PepT1재초어전장조직표체량최고,기차시기육조직;초어출막7 d 후 PepT1 mRNA표체량상대은정;주야절률연구발현,장도 PepT1기인야간적표체량교백천고.본연구지재위소태전운재체 PepT1개도장도전운소태조공초어대사료단백소화흡수적분자궤리제공이론기출.
@@@@To study the molecular mechanism of the small peptide transporter PepT1-mediated protein digestion and absorption, a full-length cDNA sequence of the PepT1 gene was cloned from Ctenopharyngodon idellus using RT-PCR and RACE techniques. The full-length cDNA sequence of PepT1 had 2762 nucleotides, including 141 nucleotides at 5′UTR and 479 nucleotides at 3′UTR. Its open reading frame had 2142 nucleotides encoding a 713-amino-acid peptide. The PepT1 gene sequences from C. idellus were most similar to those of Carassius au-ratus and Danio rerio at 77.6% and 74.0%, respectively; the deduced amino acid similarities were 78.0% and 76.7%, respectively; but the PepT1 gene sequence varied in similarity to other animals from 53.9% to 59.1%, and the deduced amino acid similarities were 60.5%—61.6%. The encoded protein molecular weight was predicted at 79.29 kD with pI at 5.87. The PepT1 protein had 11 helix trans-membrane regions; its amino acid sequence was highly homologous to those of other vertebrates. Phylogenetic analysis showed that the PepT1 gene sequence clustered with C. auratus and D. rerio as its closest neighbor. The abundances of PepT1 mRNA assayed by real-time PCR were differentially expressed by tissue type; the highest expression was in the foregut tissue and the second was in the muscle. However, PepT1 mRNA expression was relatively stable after incubation for 7 days. The PepT1 gene expressed rhythmically in the small intestine of C. idellus. Its expression was higher during the night and lower during the day. This work provides a theoretical basis for the molecular mechanism of protein digestion and absorption by the small peptide transporter PepT1, which mediates intestinal transport of small pep-tides in vivo.