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
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.
[1] 曹海洋. 2013. 小尾寒羊ADPN和IGF1基因多态性及其遗传效应分析[D].硕士学位论文, 山东农业大学, 导师: 王建民、王桂芝, pp.10-35. (Cao H Y.2013. Polymorphisms and genetic effects of ADPN and IGF1 genes in small Tailed Han Sheep[D]. Thesis for M.S., Shandong Agricultural University, superviser: Wang J M, Wang G Z, pp. 10-35.) [2] 丁宁, 刘思嘉, 田菲, 等. 2020. 本土绵羊品种GH与IGF-1基因多态性研究[J/OL].基因组学与应用生物学(https://kns.cnki.net/kcms/detail/45.1369.Q.20200610.1412.002.html. (Ding N, Liu S J, Tian F, et al.2020. Study on GH and IGF-1 gene polymorphism in native sheep varieties[J/OL]. Genomics and Applied Biology.) [3] 郭敏银. 2018. 孕期补充N-乙酰半胱氨酸对镉诱导的胎盘内质网应激和胎鼠生长发育迟缓的保护效应[D].硕士学位论文, 安徽医科大学, 导师: 徐德祥, pp.3-26. (Guo M Y.2018. The protective effect of N-acetylcysteine supplementation during pregnancy on cadmium-induced stress of placental endoplasmic reticulum and growth retardation of fetal rats[D]. Thesis for M.S., Anhui Medical University, Superviser: Xu D X, pp. 3-26.) [4] 樊庆泊, 边旭明, 郎景和. 1999. 胰岛素样生长因子与胎儿发育[J]. 中华妇产科杂志, 034(004): 251. (Fan Q B, Bian X M, Lang J H.1999. Insulin-like growth factor and fetal development[J]. Chinese Journal of Obstetrics and Gynecology, 034(004): 251.) [5] 胡倡华, 许梓荣. 2000. 胰岛素样生长因子结合蛋白的研究进展[J]. 吉林畜牧兽医, 39(5): 38-41. (Hu C H, Xu Z R.2000. Research progress of insulin-like growth factor binding protein[J]. Jilin Animal Husbandry and Veterinary Medicine, 39(5): 38-41.) [6] 侯永清, 王蕾, 易丹. 2014. N-乙酰半胱氨酸对猪肠道功能的保护作用[J]. 动物营养学报, 26(10): 3064-3070. (Xu Y Q, Wang L, Yi D.2014. The protective effect of N-acetylcysteine on pig intestinal function[J]. Journal of Animal Nutrition, 26(10): 3064-3070.) [7] 刘军泽. 2017. miR-182对奶山羊子宫内膜上皮细胞中HOXA10基因的靶向调控作用研究[D]. 硕士学位论文, 西北农林科技大学, 导师: 宋宇轩, pp. 20-42. (Liu J Z.2017. Study on the targeted regulation of Mir-182 on HOXA10 gene in dairy goat endometrial epithelial cells[D]. Thesis for M.S., Northwest A&F University, Superviser: Song Y X, pp. 20-42.) [8] 宋晓然, 詹凡玢, 王卫民. 2019. 泥鳅二倍体与四倍体trim63a基因的克隆及表达分析[J]. 华中农业大学学报, 38(005): 17-25. (Song X R, Zhan F F, Wang W M.2019. Cloning and expression analysis of diploid and tetraploid TRIM63A gene of loach[J]. Journal of Huazhong Agricultural University, 38(005): 17-25.) [9] 韦炳耐, 俸祥仁, 何立贵, 等. 2017. 努比亚山羊人工授精繁殖效果试验[J]. 贵州畜牧兽医, 41(6): 4-6. (Wei B N, Feng X R, He L G, et al.2017. Artificial insemination and reproduction effect of Nubian goats[J]. Guizhou Animal Husbandry and Veterinary, 41(6): 4-6.) [10] 王华平, 张科, 李发玉, 等. 2018. 重庆市黑山羊杂交组合试验初报[J]. 中国畜牧业, (2): 58-59. (Wang H P, Zhang K, Li F Y, et al. 2018. A preliminary report on the hybrid combination test of black goat in Chongqing[J]. China Animal Husbandry, (2): 58-59.) [11] 张磊. 2017. 奶山羊子宫内膜容受期差异表达miRNA和lncRNA调控作用的研究[D]. 硕士学位论文, 西北农林科技大学, 导师: 曹斌云, pp. 40-120. (Zhang L.2017. Study on the regulatory effects of differentially expressed miRNA and lncRNA in endometrial receptance of dairy goats[D].Thesis for M.S., Northwest A&F University, Superviser: CAO B Y, pp. 40-120.) [12] 周佳勃, 张宇霆, 岳巍, 等. 2017. N-乙酰半胱氨酸对玻璃化冻存小鼠GV期卵母细胞成熟和后续胚胎发育能力影响[J]. 东北农业大学学报, 48(2): 76-82. (Zhou J B, Zhang Y T, Yue W, et al.2017. Effect of N-acetylcysteine on the maturation and subsequent embryonic development of mouse GV stage oocytes in vitrification[J]. Journal of Northeast Agricultural university, 48(2): 76-82.) [13] 周佳, 李鹏, 程强, 等. 2018. N-乙酰半胱氨酸灌胃对超早期断奶仔猪血浆中游离氨基酸及小肠形态结构与功能的影响[J].中国畜牧杂志, 54(04): 50-54. (Zhou J, Li P, Cheng Q, et al.2018. Effects of N-acetylcysteine gavage on free amino acids in plasma and small intestine morphology and function of ultra-early weaned piglets[J]. Chinese Journal of Animal Husbandry, 54(04): 50-54.) [14] Bagot C N, Troy P J, Taylor H S.2000. Alteration of maternal Hoxa10 expression by in vivo gene transfection affects implantation[J]. Gene Therapy, 7(16): 1378. [15] Caufriez A, Frankenne F, Hennen G, et al.1993. Regulation of maternal IGF-I by placental GH in normal and abnormal human pregnancies[J]. The American Journal of Physiology, 265: 572-577. [16] Chernicky C L, Redline R W, Tan H Q, et al.1994. Expression of insulin-like growth factor Ⅰ and Ⅱ in conceptuses from normal and diabeticmice[J]. Molecular Reproduction and Development, 37: 382-390. [17] Docchio M J, Baruselli S, Campanile G.2019. Influence of nutrition, body condition, and metabolic status on reproduction in female beef cattle: A review[J].Theriogenology, 125: 277-284. [18] Gendron R L, Paradis H, Hsieh-Li H M, et al.1997. Abnormal uterine stromal and glandular function associated with maternal reproductive defects in Hoxa-11 null mice[J]. Biology of Reproduction, 56(5): 1097-1105. [19] Guevara A, Jaime.1996. Insulin-like growth factor I-An important intrauterine growth factor[J]. New England Journal of Medicine, 335(18): 1389-1391. [20] Haengseok S, Hyunjung L, Das S K, et al.2000. Dysregulation of EGF family of growth factors and COX-2 in the uterus during the preattachment and attachment reactions of the blastocyst with the luminal epithelium correlates with implantation failure in LIF-deficient mice[J]. Molecular Endocrinology, 14(8): 1147-1161. [21] Horikoshi Y, Takeo T, Nakagata N.2016. N-acetyl-cysteine prolonged the developmental ability of mouse two-cell embryos against oxidative stress at refrigerated temperatures[J]. Cryobiology, 72(3): 198-204. [22] Kimber S J.2005. Leukaemia inhibitory factor in implantation and uterine biology[J]. Reproduction, 130(2): 131-145. [23] Kuang H, Chen Q, Fan X, et al.2010. CXCL14 inhibits trophoblast outgrowth via a paracrine/autocrine manner during early pregnancy in mice[J]. Journal of Cellular Physiology, 221(2): 448-457. [24] Lei Z, Liu X R, Liu J Z, et al.2017. The developmental transcriptome landscape of receptive endometrium during embryo implantation in dairy goats[J]. Gene, 633: 82-95. [25] Lin R, Zhao C, Jing L, et al.2015. Identification of LncRNAs/mRNAs related to endometrium function regulated by homeobox A10 in Ishikawa cells[J]. Cell Biology International, 39(7): 842-851. [26] Liu J L, Grinberg A, Westphal H, et al.1998. Insulin-like growth factor-i affects perinatal lethality and postnatal development in a gene dosage-dependent manner: Manipulation using the Cre/loxP system in transgenic mice[J]. Molecular Endocrinology, 12(9): 1452. [27] Maura A, Marco L, Nicoletta B, et al.1990. Effects of propranolol on GH responsiveness to repeated GH-releasing hormone stimulations in normal subjects[J]. Acta Endocrinologica-Bucharest, 122(6): 735. [28] Nipkow M, Wirthgen E, Luft P, et al.2018. Characterization of igf1 and igf2 genes during maraena white fish (Coregonusmaraena) ontogeny and the effect of temperature on embryogenesis and igf expression[J]. Growth Hormone & IGF Research, 40: 32-43. [29] Simone M, Patrick S, Regula S R, et al.2007. Murine CXCL14 is dispensable for dendritic cell function and localization within peripheral tissues[J]. Journal of Molecular Cell Biology, 27(3): 983-992. [30] Sroga J M,Fei G, Xinghong M, et al.2012. Overexpression of cyclin D3 improves decidualization defects in Hoxa-10 mice[J]. Endocrinology, 153(11): 5575-5586. [31] Trbulo P, Jumatayeva G, Lehloenya K, et al.2018. Effects of sex on response of the bovine preimplantation embryo to insulin-like growth factor 1, activin A, and WNT7A[J]. BMC Developmental Biology, 18(1): 16. [32] Verschure P J, Van C J, Van J, et al.1996. Articular cartilage destruction in experimental inflammatory arthritis: Insulin-like growth factor-1 regulation of proteoglycan metabolism in chondrocytes[J]. The Histochemical Journal, 28(12): 835-857. [33] Wang H S, Chard T.1992. The role of insulin-like growth factor-1 and insulin-like growth factor-binding protein-1 in the control of human fetal growth[J]. Journal of Endocrinology, 132(1): 11-19. [34] Ware C B, Horowitz M C, Renshaw B R, et al.1995. Targeted disruption of the low-affinity leukemia inhibitory factor receptor gene causes placental, skeletal, neural and metabolic defects and results in perinatal death[J]. Development. 121(5): 1283-1299. [35] Won C, Jeong Y M, Kang S, et al.2015. Hair-growth-promoting effect of conditioned medium of high integrin α6 and low CD 71 (α6bri/CD71dim) positive keratinocyte cells[J]. International Journal of Molecular Sciences, 16(3): 4379-4391.
