title: 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 (5 UI), is specifically expressed in the entire silkworm midgut. In our previous study, the promoter region upstream of the 5 UI 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 5 UI was cloned and named as P3P+5 UI. A transgenic vector was constructed in which EGFP was controlled by P3P+5 UI. Transgenic P3+5 UI silkworms were generated by embryo microinjection. RT-PCR showed P3P+5 UI activity throughout the larval stage. Intense green fluorescence was seen only in the entire midgut of P3+5 UI silkworms and expression was confirmed by RT-PCR. qPCR revealed that expression of EGFP in the anterior midgut of P3+5 UI silkworms was 64% higher than in P3 silkworms, indicating the 5 UI sustained intron-mediated enhancement of gene expression. These results suggested that the BmAPN4 5 UI affected the level and site of expression. The 5 UI was cloned and added behind P2P, another specific promoter with activity only in the anterior midgut of silkworm, to construct the P2P+5 UI and transgenic P2+5 UI silkworms. Expression patterns were the same for P2P+5 UI and P2P, suggesting that the 5UI of BmAPN4 did not affect P2P. This study found that the BmAPN4 5 UI affected the amount and location of gene expression. Its influence appeared to be dependent on a specific promoter.
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, PR China.
authors.forename: Liang
authors.initials: L
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, PR China.
authors.forename: Chunlin
authors.initials: C
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, PR China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, PR China.
authors.forename: Huizhen
authors.initials: H
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, PR China.
authors.forename: Tingcai
authors.initials: T
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, PR China.
authors.forename: Zhengwen
authors.initials: Z
authors.lastname: Peng
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, PR China.
authors.forename: Yinghui
authors.initials: Y
authors.lastname: Dang
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, PR China.
authors.forename: Weiqiang
authors.initials: W
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, PR China.
authors.forename: Guowen
authors.initials: G
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, PR China. Electronic address: xiaqy@swu.edu.cn.
authors.forename: Qingyou
authors.initials: Q
authors.lastname: Xia
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D030801:Animals, Genetically Modified; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism; D018826:CD13 Antigens / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D017344:Genes, Insect; D005822:Genetic Vectors; D007438:Introns; D007814:Larva / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic
chemical_list: D020121:5' Untranslated Regions; D018826:CD13 Antigens
keywords: 5′-UTR intron; IME; Midgut; Promoter; Spatial; Transgenic silkworm