Col1a1 Regulates The Biological Function Of Ovarian Granulosa Cells And Affects The Kidding Traits Of Guizhou Black Goats

Guizhou black goat(Capra hircus)is a famous local goat breed in Guizhou Province.It has the characteristics of good adaptability,strong disease resistance,resistance to roughage and excellent meat quality.It is the product of artificial breeding and natural selection,and has high research value.However,the low litter size and high breeding cost of Guizhou black goat seriously restrict the development of mutton sheep industry.It has been found that COL1A1(Collagen type I alpha 1 chain)gene,as an important component of extracellular matrix type I collagen,plays a role in maintaining the normal function of human and animal ovaries and is closely related to animal reproductive traits.However,the role of COL1A1 gene in affecting follicular development and ovarian cell proliferation and apoptosis in Guizhou black goats is not clear.Therefore,in order to study the relationship between COL1A1 gene and the fertility of Guizhou black goat,and to analyze its effect on the proliferation and apoptosis of ovarian granulosa cells,Guizhou black goat was used as the research object in this thesis.The m RNA level of COL1A1 gene in the gonadal axis of Guizhou black goat was detected by RT-q PCR.Immunohistochemistry and Western blot were used to detect the expression and localization of COL1A1 protein in ovarian tissues of monotocous and polytocous Guizhou black goats.Subsequently,ovarian granulosa cells were isolated and cultured,and COL1A1 gene interference and overexpression vectors were constructed and transfected into ovarian granulosa cells.The effects of different expression levels of COL1A1 gene on lambing-related genes,granulosa cell proliferation,migration,cycle,ROS level,apoptosis index protein and PI3 K / AKT / m TOR pathway protein expression were detected by RT-q PCR,Western blot,CCK-8,cell scratch and flow cytometry.The main results are as follows:1.The expression level of COL1A1 gene in the gonadal axis of Guizhou black goat with polytocous kids was significantly higher than that of monotocous kid group.Under the premise of simultaneous estrus treatment,from the morphological point of view,the ovary tissue volume of Guizhou black goat in the polytocous kid group was significantly larger than that in the monotocous kid group,and the follicular development was more mature.The results of RT-q PCR showed that the expression of COL1A1 gene in ovary,hypothalamus,pituitary and oviduct tissues of polytocous group was significantly higher than that of monotocous group(P<0.01),and the expression level in ovary of polytocous group was the highest.The results of immunohistochemistry and Western blot showed that COL1A1 protein was expressed in follicles,ovarian cells and connective tissues at all levels of ovarian tissues in monotocous and polytocous kids,and was highly expressed in ovaries of polytocous kids(P<0.01).The above results suggest that the expression of COL1A1 may be positively correlated with goat reproduction rate and play an active role in maintaining ovarian tissue structure and follicular development.2.The overexpression vector and interference vector of COL1A1 gene were successfully constructed.The overexpression vector of COL1A1 gene was constructed by overlap extension PCRenzyme digestion and ligation.The results of double enzyme digestion,sequencing,RT-q PCR and Western blot showed that pc DNA3.1(+)-COL1A1 eukaryotic expression vector was successfully constructed.According to the principle of sh RNA design,four interference vectors were constructed to interfere with the expression of COL1A1 gene,and the inhibition efficiency of different interference vectors on COL1A1 gene and protein was detected by green fluorescent protein luminescence intensity,RT-q PCR and Western blot.The results showed that sh RNACOL1A1-2 was the best interference vector to inhibit the expression of COL1A1 gene.The above results lay a foundation for the subsequent verification of the biological function of granulosa cells affected by different expression levels of COL1A1 gene.3.Overexpression and interference of COL1A1 gene seriously affected the phenotype of granulosa cells and the expression of kidding-related genes.The results of CCK-8,cell scratch,flow cytometry and reactive oxygen species detection showed that overexpression of COL1A1 gene significantly promoted the proliferation ability(P<0.01)and migration ability(P<0.05)of granulosa cells,changed the cell cycle process and reduced the level of ROS in cells(P<0.01).Down-regulation of COL1A1 gene expression significantly inhibited the proliferation(P<0.01)and migration(P<0.05)of granulosa cells,changed the cell cycle progression,and increased the intracellular ROS level(P<0.05).In addition,RT-q PCR results showed that up-regulation of COL1A1 gene expression could promote the expression levels of kidding-related genes GDF9,BMP15 and FSHβ in granulosa cells(P<0.01),while interfering with the expression of COL1A1 gene,the above genes showed significantly opposite expression results.The above results indicated that COL1A1 gene was positively correlated with the proliferation,migration and kidding gene expression of goat granulosa cells,and negatively correlated with ROS level in cells.4.COL1A1 gene affects the proliferation and apoptosis of granulosa cells by regulating the PI3 K / AKT / m TOR pathway.The study on the signaling pathway of COL1A1 gene affecting the proliferation and apoptosis of granulosa cells found that up-regulating the expression level of COL1A1 gene in granulosa cells promoted the ratio of p-PI3K/PI3 K,p-AKT/AKT,p-m TOR/m TOR and the expression level of cell proliferation related protein PCNA(P<0.05),and inhibited the expression of proteins associated with apoptosis BAX,Caspase-3 and Caspase-9(P<0.05).The down-regulation of COL1A1 gene expression,p-PI3K/PI3 K,p-AKT/AKT,p-m TOR/m TOR,BAX,Caspase-3 and Caspase-9proteins,has the opposite expression pattern.In summary,COL1A1 gene may regulate the proliferation,migration,cycle and ROS level of granulosa cells by activating the expression of PI3K/AKT/m TOR pathway proteins and regulating the expression of PCNA,BAX,Caspase-3 and Caspase-9 proteins,thereby affecting the biological function of granulosa cells,and indirectly affecting the development and maturation of goat follicles by changing the expression of GDF9,BMP15,BMPR-1B and FSHβ in granulosa cells,and regulating the kidding performance of Guizhou black goats.