Expression of COL1A1 Gene in the Gonad Axis of Guizhou Black Goat (Capra hircus) and Its Effect on Lambing Related Genes
ZHOU Ming-Shuai, WEN Xiao-Yan, ZHANG Yan, LI Yong, XU Chang, YANG Xiu-Yuan, ZHAO Jia-Fu*
Key Laboratory of Plateau Mountain Animal Genetics, Breeding and Reproduction, Ministry of Education/College of Animal Science, Guizhou University, Guiyang 550025, China
Abstract：Collagen typeⅠα1 chain (COL1A1) gene as an important component of typeⅠcollagen, plays a role in regulating follicular development and ovarian cell proliferation, and is closely related to animal reproductive traits. In order to explore the relationship between COL1A1 gene and fertility of Guizhou black goats (Capra hircus) and the effects of different expression levels on related genes in lambing, in this study, gonad axis tissues (uterus, ovary, pituitary, hypothalamus, fallopian tube) of monotocous and polytocous Guizhou black goats were collected. The expression of COL1A1 gene and its protein in the gonadal axis of monotocous and polytocous Guizhou black goats were detected by qPCR, and then ovarian granulosa cells were isolated and cultured. The interference vector of the target gene was constructed by RNA interference technology and transfected into ovarian granulosa cells. The expression level of COL1A1 gene and its protein was detected by qPCR and Western blot to screen the best interference group. qPCR was used to detect the effects of COL1A1 gene interference on the expression of lambing related genes bone morphogenetic protein 15 (BMP15), bone morphogenetic protein 1B (BMPR-1B), growth differentiation factor 9 (GDF9) and follicle stimulating hormone β-subunit (FSHβ). The results showed that COL1A1 gene was expressed in various tissues of Guizhou black goats. The expression level in ovary and hypothalamus of polytocous group was the highest, and was extremely significantly higher than that of monotocous Guizhou black goats group (P<0.01), significantly higher in pituitary gland and oviduct than in monotocous group (P<0.05), there was no significant difference in utero; Immunofluorescence results showed that the cells isolated and cultured in the experiment were ovarian granulosa cells. qPCR and Western blot results showed that shRNA-COL1A1-2 was the best interference group. After inhibiting the expression of COL1A1 gene, qPCR results showed that the expressions of lambing related genes BMP15, BMPR-1B, GDF9 and FSHβ were significantly lower than those of shRNA-NC group (P<0.01). This study revealed the difference of COL1A1 gene expression in gonad axis of monotocous and polytocous Guizhou black goat. The high expression of COL1A1 gene in the ovarian tissue of polytocous lambs group and the suppression of lambing related genes by silencing COL1A1 gene indicated that COL1A1 gene was closely related to ovarian development and follicle growth. It can be used as a candidate gene for the breeding of Guizhou black goat.
周明帅, 温晓艳, 张艳, 李永, 徐畅, 杨秀远, 赵佳福. COL1A1基因在贵州黑山羊性腺轴中的表达及其对产羔相关基因的影响[J]. 农业生物技术学报, 2022, 30(11): 2152-2162.
ZHOU Ming-Shuai, WEN Xiao-Yan, ZHANG Yan, LI Yong, XU Chang, YANG Xiu-Yuan, ZHAO Jia-Fu. Expression of COL1A1 Gene in the Gonad Axis of Guizhou Black Goat (Capra hircus) and Its Effect on Lambing Related Genes. 农业生物技术学报, 2022, 30(11): 2152-2162.
[1] 陈克飞, 黄路生, 李宁, 等. 2000. 猪FSHβ及ESR合并基因型对产仔数性状的影响[J]. 科学通报, 45(18): 1963-1966. (Chen K F, Huang L S, Li N, et al.2000. Effect of pig FSHβ and ESR combined genotype on litter number traits[J]. Science Bulletin, 45(18): 1963-1966.) [2] 寸静宇, 刘秋月, 王翔宇, 等. 2019. 小尾寒羊FSHβ和LHβ基因在生殖轴的表达研究[J]. 中国畜牧杂志, 55(01): 37-41. (Chun J Y, Liu Q Y, Wang X Y, et al.2019. Expression of FSHβ and LHβ genes in the reproductive axis in small tail Han sheep[J]. China Journal of Animal Husbandry, 55(01): 37-41.) [3] 付旭煌. 2020. 维生素C通过COL1A1调控牛卵丘细胞增殖及凋亡的研究[D]. 硕士学位论文, 吉林大学, 导师: 陈承祯, pp. 43-45. (Fu X H.2020. Vitamin C regulation of bovine ovulus cell proliferation and apoptosis through COL1A1[D]. Thesis for M.S., Jilin University, Supervisor: Chen C Z, pp. 43-45.) [4] 高可心. 2018. RUNX1和RUNX2在奶山羊卵巢颗粒细胞中的表达调控机理及功能研究[D]. 博士学位论文, 西北农林科技大学, 导师: 曹斌云, pp. 22-23. (Gao K X.2018. Study on the mechanism and function of runx1 RUNX1 and RUNX2 expression regulation in ovarian granule cells of dairy goats[D]. Thesis for Ph.D., Northwest A&F University, Supervisor: Cao B Y, pp. 22-23.) [5] 郭慧慧, 李俊, 罗惠娣, 等. 2018. 杜寒绵羊多胎性能候选基因BMPR-1B和BMP15的研究[J].中国畜牧兽医, 45(05): 1312-1318. (Guo H H, Li J, Luo H D, et al.2018. Study of BPRR-1B and BMP15 as candidate genes for prolificacy Du han Sheep[J]. China Animal Husbandry and Veterinary Medicine, 45(05): 1312-1318.) [6] 洪磊, 陈祥, 唐文, 等, 2020. 黔北麻羊SRD5A2基因干扰载体构建及其对产羔相关基因表达的影响[J].畜牧兽医学报, 51(12): 2980-2990. (Hong L, Chen X, Tang W, et al.2020. Construction of SRD5A2 gene interference vector and its effect on the expression of lambing related genes in Qianbei Ma goats[J]. Journal of Animal Husbandry and Veterinary Medicine, 51(12): 2980-2990.) [7] 李佳蓉, 贾超, 陈洋, 等. 2020. 绵羊生殖器官GDF9基因的表达研究[J]. 中国兽医科学, 50(08): 1050-1057. (Li J R, Jia C, Chen Y, et al.2020. Expression of GDF9 gene in sheep reproductive organs[J]. Chinese Veterinary Science, 50(08): 1050-1057. ) [8] 刘霜, 郑杰, 卢建远, 等. 2016. 山羊卵巢中BMP15、 GDF9和BMPR-1B基因的表达量研究[J]. 畜牧与兽医, 48(5): 66-70. (Liu S, Zheng J, Lu J Y, et al.2016. Expression of BMP15, GDF9 and BMPR-1B genes in goat ovary[J]. Animal Husbandry and Veterinary Medicine, 48(5): 66-70.) [9] 刘建斌, 郎侠, 杨博辉, 等. 2010. BMPR-1B基因和FSHβ基因多态性及其与小尾寒羊产羔数关联性分析[J]. 中国畜牧杂志, 46(19): 1-6. (Liu J B, Lang X, Yang B H, et al.2010. Polymorphisms of BMPR-1B gene and FSHβ gene and their correlation with lambing number in small-tailed Hhan sheep[J]. Chinese Journal of Animal Husbandry, 46(19): 1-6) [10] 钱若文轩, 杨柳青, 石子晴, 等. 2020. COL1A1敲除抑制卵巢癌细胞ES-2增殖[J]. 中国病理生理杂志, 36(01): 05 (Qian R W X, Yang L Q, Shi Z Q, et al.2020. COL1A1 knockout inhibits ES-2 proliferation of ovarian cancer cells[J]. Chinese Journal of Pathophysiology, 36(01): 05). [11] 覃玉凤, 李维, 陈瑶生, 等. 2017. BMP15基因研究进展[J]. 农业生物技术学报, 25(2): 324-334. (Qin Y F, Li W, Cheng Y S, et al.2017. Research progress on BMP15 gene[J]. Journal of Agricultural Biotechnology, 25(2): 324-334.) [12] 王继卿, 胡江, 罗玉柱 .2010. FSHβ基因CDS区遗传特征及其与小尾寒羊产羔数关系的研究[J].中国畜牧杂志, 46(11): 5-9. (Wang J Q, Hu J, Luo Y Z.2010. Relationship between genetic characteristics of FSHβ gene at CDS regions and litter size of small-tail Han sheep[J]. Chinese Journal of Animal Husbandry, 46(11): 5-9.) [13] 王翔宇, 狄冉, 刘秋月, 等. 2020. 小鼠和绵羊BMP15和GDF9突变在卵泡发育中作用及其对排卵数的影响[J]. 农业生物技术学报, 28(10): 1870-1880. (Wang X Y, Di R, Liu Q Y, et al.2020. The effect of BMP15 and GDF9 gene mutations on follicular development and ovulation in mouse (Mus musculus) and sheep (Ovis aries)[J]. Journal of Agricultural Biotechnology, 28(10): 1870-1880.) [14] 王子玉. 2019. 体况影响山羊卵泡发育的机制及哺乳期母仔一体化技术研究[D]. 博士学位论文, 南京农业大学, 导师: 王锋, pp. 27-29. (Wang Z Y.2019. Mechanism of physical condition affecting the development of goat follicles and the integration technology of mother and child during lactation[D]. Thesis for Ph.D, Nanjing Agricultural University, Supervisor: Wang F, pp. 27-29.) [15] 杨新月, 周多恩, 李斌, 等. 2018. 湖羊和川中黑山羊GDF9、BMPR-1B、GnRHR基因多态性及其与产羔数的关联分析[J]. 黑龙江畜牧兽医, (15): 106-110. (Yang X Y, Zhou D E, Li B, et al. 2018. Gene polymorphisms of GDF9, BMPR-1B and GnRHR gene in lake sheep and Sichuan black sheep and their association with lambing number[J]. Heilongjiang Animal Husbandry and Veterinary Medicine, (15): 106-110.) [16] 赵要风, 李宁, 陈永福, 等. 1996. 猪FSHβ亚基基因5′端调控区的PCR克隆测序及变异分析[J]. 自然科学进展, (03): 97-102. (Zhao Y F, LI N, Chen Y F, et al. 1996. Cloning sequencing and variation analysis of PCR clones in the regulated zone of porcine FSHβ subunit gene 5' terminal[J]. Progress in Natural Science, (03): 97-102.) [17] Bodin L, Di Pasquale E, Fabre S, et al.2007. A novel mutation in the bone morphogenetic protein 15 gene causing defective protein secretion is associated with both increased ovulation rate and sterility in Lacaune sheep[J]. Endocrinology, 148(1): 393-400. [18] Chronowska E.2014. High-throughput analysis of ovarian granulosa cell transcriptome[J]. BioMed Research International, 2014: 213570. [19] Cruz M, Requena A, Agudo D, et al.2017. Type of gonadotropin used during controlled ovarian stimulation induces differential gene expression in human cumulus cells: A randomized study[J]. European Journal of Obstetrics & Gynecology and Reproductive Biology, 215: 124-133. [20] Franchi F F, Hernandes M P, Coalho Ferreira A L, et al.2020. Fractal analysis and histomolecular phenotyping provides insights into extracellular matrix remodeling in the developing bovine fetal ovary[J]. Biochemical and Biophysical Research Communications, 523: 823-828. [21] Galloway S M, Gregan S M, Wilson T, et al.2002. BMP15 mutations and ovarian function[J]. Molecular and Cellular Endocrinology, 191(1): 15-18. [22] Galloway S M, McNatty K P, Cambridge L M, et al.2000. Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner[J]. Nature Genetics, 25(3): 279-283. [23] Hatzirodos N, Hummitzsch K, Irving-Rodgers H F, et al.2014. Transcriptome profiling of granulosa cells from bovine ovarian follicles during atresia[J]. BMC genomics, 15(1): 40. [24] Ilhan-Mutlu A, Siehs C, Berghoff AS, et al.2016. Expression profiling of angiogenesis-related genes in brain metastases of lung cancer and melanoma[J]. Tumor Biology, 37(1): 1173-1182. [25] Iva A, Giovanni L, Francesca M, et al.2017. An in vitro prototype of a porcine biomimetic testis-like cell culture system: A novel tool for the study of reassembled Sertoli and Leydig cells[J]. Asian Journal of Andrology. 20(2): 160-165. [26] Li H, Chang H M, Shi Z, et al.2020. The p38 signaling pathway mediates the TGF-β1-induced increase in typeⅠ collagen deposition in human granulosa cells[J]. FASEB Journal, 34(11): 15591-15604. [27] Li H, Xu H, Akhatayeva Z, et al.2021. Novel indel variations of the goat FecB gene and their effects on litter size[J]. Gene, 767(30): 145176. [28] Mulsant P, Lecerf F, Fabre S, et al.2001. Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes[J]. Proceedings of the National Academy of Sciences of the USA, 98(9): 5104-5109. [29] Prockop D J.1990. Mutations that alter the primary structure of typeⅠcollagen. The perils of a system for generating large structures by the principle of nucleated growth[J]. Journal of Biological Chemistry, 265(26): 15349-15352. [30] Shen P L, Xu J, Wang P, et al.2020. A new three-dimensional glass scaffold increases the in vitro maturation efficiency of buffalo (Bubalus bubalis) oocyte via remodelling the extracellular matrix and cell connection of cumulus cells[J]. Reproduction in Domestic Animals, 55(2): 170-180. [31] Tian F, Wang J, Zhang Z, et al.2019. miR-107 modulates chondrocyte proliferation, apoptosis, and extracellular matrix synthesis by targeting PTEN[J]. International Journal of Clinical and Experimental Pathology, 12(2): 488-497. [32] Tu J, Cheung A H, Chan C L, et al.2019. The role of microRNAs in ovarian granulosa cells in health and disease[J]. Frontiers in Endocrinology, 10: 174. [33] Wang Q, Yu J.2018. MiR-129-5p suppresses gastric cancer cell invasion and proliferation by inhibiting COL1A1[J]. Biochemistry and Cell Biology, 96(1): 19-25. [34] Yao Y L, Reheman A, Xu Y F, et al.2018. miR-125b contributes to follicular granulosa cells apoptosis through targeting BMPR-1B, a major gene for goat prolificacy[J]. Reproductive Sciences, 26(2): 295-305. [35] Zhang Z, Wang Y, Zhang J, et al.2018. COL1A1 promotes metastasis in colorectal cancer by regulating the WNT/PCP pathway[J]. Molecular Medicine Reports, 17(4): 5037-5042.
