Gene Silencing of Mink (Neovison vison) Agouti Induced by 601 bp SINE Insertion Mutation
LI Lan-Hui1,3*, DU Xiao-Long1*, WANG Qi1, ZHANG Le-Chao1, GE Lin-Han1, ZHOU Rong-Yan1, LI Xue-Mei1, LI Xiang-Long2**
1 College of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, China; 2 Hebei Normal University of Science & Technology, Qinhuangdao 066600, China; 3 Research Center of Cattle and Sheep Embryo Engineering Technique of Hebei, Baoding 071001, China
Abstract：Agouti-signaling protein (ASIP), encoded by Agouti and acted as an antagonist of Melanocortin 1-receptor (MC1R), plays a key role in inhibiting deposition of eumelanin and forming the coat color of tan striped character. Mink (Neovison vison) has a colorful coat and is an important economic animal for fur. However, the role of Agouti in mink coat color is unclear. To get the Agouti gene sequence of mink and its structure character, the distribution of 601 bp insertion mutation and the relationship between transcription of fragment with insertion and Agouti gene was explored. The genome of black mink, white mink and coffee mink and the RNA from skin of black mink and coffee mink were used to amplify Agouti gene sequence and transcriptome sequencing, respectively. PCR cloning and sequencing, and bioinformatics analysis were used to get Agouti gene sequence and mutations, respectively. And the spliceosome types were predicted. Population analysis was done for the 601 bp insertion in the three-color-type mink, and the luciferase promoter activity was detected for fragments in both directions with and without the 601 bp insertion. The transcripts of Agouti gene were analyzed based on the transcriptome of mink skin. The mink Agouti gene sequence for 25 478 bp was submitted to NCBI (GenBank No. KP981640.1) and 7 spliceosomes were predicted. 60 SNPs and Short interspersed nuclear element (SINE), a 601 bp insertion mutation were found in Agouti gene. The wild genotype frequency in black mink was significantly higher than white mink and coffee mink (P<0.05). The promoter activity of the 1 605 bp fragment in forward direction, which included the SINE and the up and down stream sequence of the insertion locus, was significantly higher than that of the fragments in inverse direction and the deletion fragments in both directions (P<0.01). A 973 bp transcript contained the 601 bp SINE was 100% covered and similar with the intron 3 sequence of Agouti gene. However, none of the Agouti transcripts was detected. It was concluded that the Agouti transcription was silenced by the 601 bp SINE insertion in the intron 3 of Agouti gene, and the insertion fragments had promoter activity and the transcript. The research discovered that Agouti gene might not take part in the coat color formation of mink, which provides some data for further studying relationship between expression regulation of transposable elements and gene function.
李兰会, 杜小龙, 王麒, 张乐超, 葛琳涵, 周荣艳, 李雪梅, 李祥龙. 601 bp SINE插入突变导致水貂Agouti基因沉默[J]. 农业生物技术学报, 2019, 27(2): 297-306.
LI Lan-Hui, DU Xiao-Long, WANG Qi, ZHANG Le-Chao, GE Lin-Han, ZHOU Rong-Yan, LI Xue-Mei, LI Xiang-Long. Gene Silencing of Mink (Neovison vison) Agouti Induced by 601 bp SINE Insertion Mutation. 农业生物技术学报, 2019, 27(2): 297-306.
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