The Creation Of Zebrafish Indel Mutants And The Expression Differences Of Igfbp-1a/Igfbp-1b Genes Responding To Stress

IGFBP-1, the first identified member of the IGFBP family is highly induced under a variety of catabolic conditions, such as food deprivation, malnutrition, stress and injury. Lacking a single amino acid, such as arginine, cysteine and leucine is sufficient to induce IGFBP-1 expression in vitro. Other catabolic conditions, including endoplasmic reticulum (ER) stress and hypoxia, could also regulate IGFBP-1 expression. Two igfbp-1s of zebrafish(Danio rerio) named igfbp-1a and igfbp-1b are co-orthologs of the human IGFBP-1 gene. Phylogenetic analysis and colinearity data show that teleost fish has undergone genome-wide gene duplications (2R and 3R) during evolution. The differences in function of the duplicated genes need to be studied deeply. It has been found that nutritional stress and hypoxia could highly induce zebrafish igfbp-1a and igfbp-1b genes expression. Some results about the relationship among the growth, the inhibition of IGF-1 activity and the response to environment under stress condition have been reported. The covered regulatory mechanisms for igfbp-1a 、igfbp-1b under starvation in vivo need to be investigated.Phylogenetic analysis reveals that there are two conservative AARE elements in igfbp-1a intron and the covering promoter region. One of the AARE is TGATGCAAC located at-1454/-1446, and the other is ATTTCATCA located at +483/+491. No conservative AARE sequence is found in the igfbp-1b. The pGL3-basic, p1128Luc and p2025Luc were constructed for testing the AARE activity responding to leucine deficiency in HepG2 cells. The results show that the AARE in the promoter region of igfbp-1a caused a modest but statistically significant increase under the leucine deficiency condition. The igfbp-1a mRNA expression did not increase under leucine deficiency condition and no expression of igfbp-1b was detected in ZF4 cell. Igfbp-la and Igfbp-1b protein expression did not increase under leucine deficiency condition in ZF4 cell.In this study, the homozygous mutants of igfbp-1a、igfbp-1b were constructed by TALENs and CRISPR/Cas9 techniques. The mutants could be helpful models for further study of igfbp-1a、igfbp-1b gene function during the whole life cycle of zebrafish. It also provides a platform for further mechanism investigation of igfbp-1a、 igfbp-1b under stress conditions and the functional differences.