树鼩中APP基因特征描述及可变剪接体的分型鉴定

Characterization of tree shrew APP gene and identification of its alternatively spliced variants

目的区分并鉴定树鼩中APP基因mRNA的多种可变剪接体,对APP基因特征进行描述,并确定其在各组织中的表达分布。方法参考已知人的和树鼩基因组预测的APP基因序列,设计树鼩APP基因mRNA剪切体外显子的共同特异性引物。分别从树鼩不同组织中提取总RNA,反转录为c DNA,利用高保真酶扩增目的剪切体DNA。根据PCR扩增产物电泳条带的有无和大小初步判断剪接体的型别,最后将PCR产物胶回收进行测序鉴定,对获得的基因进行特征描述,并结合定量PCR的结果确定了各剪接体在不同组织中分布情况。结果结果表明树鼩APP剪接体的全长为3514 bp,有一个109 bp的5’-UTR,1092bp的3’-UTP。APP基因在调查的9个物种中高度同源保守,显示树鼩与灵长类存在一个较近的亲缘关系。通过三维建模获得了树鼩和人的APP基因共同拥有的4个结构域。同时确认这4种通过外显子跳跃产生的APP可变剪接体在不同组织中的分布和表达。4种检测到的剪接体APP770,APP695,APP751,APP677均表达于肺、肾和肠,表达量最高的是肺、肌肉和睾丸。结论对树鼩APP基因可变剪接体的表达研究,有助于推动树鼩作为阿尔茨海默病模型深入研究疾病的发生机制和药物研发。

Objective The aim of this study was to differentiate and identify all the variants of APP mRNA in tree shrew,describe the characteristics of APP genes,and determine its distribution in different tissues. Method Based on the known human APP sequence and predicted tree shrew APP gene,we designed a pair of common specific primer. We extracted total RNA from different tissues respectively,using RT-PCR to get the targeted c DNA. We differentiated the variants by electrophoresis. Finally we recollected the RT-PCR products for sequencing. Combined with the q PCR results,we confirmed the quantitative distribution of the variants in different tissues. Results Our sequencing results showed that the length of the tree shrew APP spliceosome was 3514 bp with a 109 bp 5＇-UTR and a 1092 bp3＇-UTP. APP genes in the investigated 9 species were highly homologous and conservative,tree shrews and primates show a very close genetic relationship. The tree shrew APP genes shared four domains by 3D modeling. We also confirmed the distribution pattern of 4 spliceosomes derived from exon jumping. All existed APP variants,including APP770,APP695,APP751,and APP677,were all expressed in the lung,kidney and intestine.The highest expression levels were in the lung,muscle,and testicles.Conclusions This study helps to promote the studies of tree shrews as an Alzheimer’s disease model and the mechanism of its pathogenesis and drug development.