动物造模 z-bio 2021-11-30 372

　　Purpose: To distinguish and identify multiple alternative spliceosomes of APP gene mRNA in tree shrews, describe the characteristics of APP gene, and determine its expression distribution in various tissues.

　　Method: Refer to the APP gene sequence predicted by the known human and tree shrew genome, and design the common specific primers for the tree shrew APP gene mRNA to cut the exons. Total RNA was extracted from different tissues of tree shrews, reverse transcribed into cDNA, and the target spliced DNA was amplified by high-fidelity enzymes. Preliminarily judge the type of spliceosome according to the presence and size of the electrophoretic bands of the PCR amplification product. Finally, the PCR product gel is recovered for sequencing and identification, the gene obtained is characterized, and the results of quantitative PCR are used to determine each spliceosome Distribution in different organizations.

　　Results: The results showed that the total length of the tree shrew APP spliceosome is 3514 bp, with a 5'-UTR of 109 bp and a 3'-UTP of 1092 bp. The APP gene is highly homologous and conserved among the nine species investigated, indicating that tree shrews and primates are closely related. Through three-dimensional modeling, four structural domains shared by the tree shrew and the human APP gene were obtained. At the same time, confirm the distribution and expression of these four APP alternative spliceosomes produced by exon skipping in different tissues. The four detected spliceosomes APP770, APP695, APP751, and APP677 were all expressed in the lung, kidney and intestine, and the highest expression was in the lung, muscle and testis.

　　Conclusion: The study on the expression of the alternative spliceosome of the tree shrew APP gene will help to promote the tree shrew as a model of Alzheimer's disease in-depth study of the mechanism of disease and drug development.