The 5′-UTR intron of the midgut-specific BmAPN4 gene affects the level and location of expression in transgenic silkworms

Abstract Introns are important for regulating gene expression. BmAPN4 , which has a 5′-UTR upstream intron (5UI), is specifically expressed in the entire silkworm midgut. In our previous study, the promoter region upstream of the 5UI of BmAPN4 was cloned and identified as the P3 promoter (P3P) with activity only in the anterior midgut. In this study, the sequence consisting of the P3P and the 5UI was cloned and named as P3P+5UI. A transgenic vector was constructed in which EGFP was controlled by P3P+5UI. Transgenic P3+5UI silkworms were generated by embryo microinjection. RT-PCR showed P3P+5UI activity throughout the larval stage. Intense green fluorescence was seen only in the entire midgut of P3+5UI silkworms and expression was confirmed by RT-PCR. qPCR revealed that expression of EGFP in the anterior midgut of P3+5UI silkworms was 64% higher than in P3 silkworms, indicating the 5UI sustained intron-mediated enhancement of gene expression. These results suggested that the BmAPN4 5UI affected the level and site of expression. The 5UI was cloned and added behind P2P, another specific promoter with activity only in the anterior midgut of silkworm, to construct the P2P+5UI and transgenic P2+5UI silkworms. Expression patterns were the same for P2P+5UI and P2P, suggesting that the 5UI of BmAPN4 did not affect P2P. This study found that the BmAPN4 5UI affected the amount and location of gene expression. Its influence appeared to be dependent on a specific promoter.