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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: To distinguish and identify a variety of APP gene mRNA variants in tree shrews, to describe the characteristics of APP gene and to determine its distribution in different tissues. Methods Aiming at the APP gene sequence predicted from human and tree shrew genomes, common primers specific for APP gene mRNA exosomal cuttings were designed. Total RNA was extracted from different tissues of tree shrew, reverse transcribed into c DNA, and amplified by high fidelity enzyme. According to the presence or absence of the electrophoresis band of the PCR amplification product, the type of the spliceosome was initially judged. Finally, the PCR product gel was recovered and sequenced for identification. The obtained gene was characterized and combined with the results of the quantitative PCR to confirm that each splice body Distribution in different organizations. The results showed that the full-length of the spider APP strain was 3514 bp with a 109 bp 5’-UTR and a 1092 bp 3’-UTP. The APP gene is highly homologous conservative among the nine species investigated, indicating a close genetic relationship between tree shrew and primates. Through the three-dimensional modeling, four domains co-owned by tree shrew and human APP gene were obtained. At the same time, we confirmed the distribution and expression of these four kinds of APP splicing in different tissues by exon skipping. The four detected spliceosomes, APP770, APP695, APP751 and APP677, were expressed in the lung, kidney and intestine, respectively. The highest expression levels were lung, muscle and testis. Conclusion The study on the alternative splicing of APP gene in tree shrews will help to promote the tree shrew as a model of Alzheimer disease to study the pathogenesis and drug development of APP.