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| Construction of IGF1 Gene Overexpression Vector in Goats (Capra hircus) and Its Effect on Uterine Epithelial Cells |
| TANG Wen, CHEN Xiang*, HONG Lei, ZHOU Zhi-Nan, YANG Pei-Fang |
| College of Animal Science, Guizhou University/Key Laboratory of Breeding and Breeding of Plateau Mountain Animals, Ministry of Education/Guizhou Key Laboratory of Animal Genetics and Breeding, Guiyang 550025, China |
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Abstract Insulin like growth factor 1 (IGF1) is a type of single-chain polypeptide hormone, which can promote embryo development and cell proliferation and differentiation in the animal body and plays an important role in improving animal reproduction performance. This study was based on the previous research of the prior research group found that the birth rate of Nubian goats (Capra hircus) can be improved by feeding 0.07% N-acetylcysteine (NAC), which also has the best impacts. Meanwhile, filtering out IGF1 which is a significantly different gene based on the transcriptome sequencing of goat uterine horn. This experimental studied and collected the uterus, fallopian tube, pituitary, hypothalamus, ovary tissues of a Nubian goat and extracted total RNA from each tissue, reverse transcribed into cDNA and used qRT-PCR to verify the result of transcriptome sequencing. At the same time, bioinformatics analysis was performed on the CDS region of the cloned Nubian goat IGF1 gene; the eukaryotic expression vector of IGF1 gene was constructed and then transfected into uterine epithelial cells and detected the expression level of the homeobox genes related to reproductive traits of Nubian goats by the qRT-PCR test, which were homeobox A10 (HOXA10), leukemia inhibitory factor interleukin 6 family cytokine (LIF), C-X-C motif chemokine ligand 14 (CXCL14). The results showed that IGF1 gene was expressed in all 5 tested tissues. In contrast, the IGF1 gene in the uterus, fallopian tubes, hypothalamus and ovary of the experimental group was significantly higher than that of the blank group (P<0.01). The IGF1 gene CDS region of the cloned Nubian goat was 471 bp and encoding 153 amino acids. The protein prediction results showed that the molecular formula of Nubian goat IGF1 protein was C739H1178N212O215S15, the relative molecular mass was 16.953 67 kD and the isoelectric point was 9.36. The phylogenetic tree analysis showed that the Nubian goat IGF1 gene and sheep (Ovis aries) had the closest genetic distance. The result of qRT-PCR showed that the expression level of IGF1 gene mRNA in the cells of the successfully transfected recombinant plasmid group was significantly higher than that of the blank control group. The mRNA expression levels of HOXA10, LIF, and CXCL14 genes of the successfully transfected recombinant plasmid group (P<0.01) were significantly higher than those in the blank control group (P<0.05). The result showed that overexpression of the IGF1 gene would promote the expression of lambing-related genes which were HOXA10, LIF, and CXCL14. This study successfully constructed the eukaryotic expression vector of pEGFP-N3-IGF1 and detected the effect of the IGF1 gene on the expression of Nubian goat reproduction-related genes. It provides more information for further research on the effect of the IGF1 gene on Nubian goat reproduction traits.
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Received: 10 September 2020
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Corresponding Authors:
* xchen2@gzu.edu.cn
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