Construction of Eukaryotic Expression Vector of HSD3B1 Gene and Its Effect on Ovarian Granulosa Cells of Qianbei Ma Goat (Capra hircus)
HONG Lei1, 2, CHEN Xiang1, 2, *, AO Ye1, 2, ZHOU Zhi-Nan1, 2, TANG Wen1, 2, WEI Shi-Nan1, 2
1 Key Laboratory of Breeding and Breeding of Plateau Mountain Animals, Ministry of Education, Guizhou University/Guizhou Key Laboratory of Animal Genetics and Breeding, Guiyang 550025, China; 2 College of Animal Science, Guizhou University, Guiyang 550025, China
Abstract 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) is a key member of the steroid hormone family and is closely related to animal reproductive traits. In order to clone the HSD3B1 gene of the Qianbei Ma goat (Capra hircus), construct a eukaryotic expression vector, and explore its effect on the expression of genes, in this study, Qianbei Ma goats ewes were selected to collect ovarian tissue for ovarian granulosa cell culture and extract the total RNA in the tissue for reverse transcription into cDNA. The full-length sequence of HSD3B1 (GenBank No. NM_001135932.1) gene CDS region of Qianbei Ma Goats was cloned and analyzed by bioinformatics. Meanwhile, eukaryotic expression vector of HSD3B1 gene was constructed and transfected into ovarian granulosa cells. mRNA expression levels of HSD3B1, bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9), and bone morphogenetic protein receptor type 1B (BMPR-1B) genes were detected by qRT-PCR. The results of protein prediction analysis showed that the molecular formula of HSD3B1 was C1940H3002N506O543S15, and the relative molecular weight was 42.583 14 kD, isoelectric point 7.96; The genetic tree analysis showed that the genetic distance between the HSD3B1 gene and the cattle (Bos taurus) was the closest. The results of qRT-PCR showed that the expression of HSD3B1 and GDF9 gene in the transfection experimental group were significantly higher than those in the blank group (P<0.01), and the mRNA expression level of BMP15 gene was significantly higher than that of the control group (P<0.05). The results showed that the overexpression of HSD3B1 gene could promote the expression of GDF9 and BMP15. In this study, the pEGFP-N3-HSD3B1 eukaryotic expression vector was successfully constructed to detect the effect of HSD3B1 gene on the expression of genes related to lambing traits of Qianbei Ma goats, and provide basic data for further research on the effect of HSD3B1 gene on lambing traits of Qianbei Ma Goats..
HONG Lei,CHEN Xiang,AO Ye, et al. Construction of Eukaryotic Expression Vector of HSD3B1 Gene and Its Effect on Ovarian Granulosa Cells of Qianbei Ma Goat (Capra hircus)[J]. 农业生物技术学报, 2020, 28(9): 1604-1613.
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