| In order to explore the genetic mechanism of high fecundity of Qianbei Ma Goat,this study takes single and multiple lamb Qianbei Ma Goat as the research object,conducted reproductive hormone detection,transcriptome sequencing to screen differentially expressed genes,and detected their function at the tissue expression level and at the cellular level to verify and explore the effect of differentially expressed genes on the control of reproductive ability of Qianbei Ma Goat,and to reveal the mechanism of high-birth and multi-birth of Qianbei Ma Goat.In this study,ELISA method was used to detect the content of reproductive hormones during the estrous cycle.RNA-seq sequencing technology was used to sequence the ovary to screen candidate genes related to lamb traits,and the expression level was verified.Fluorescence quantitative PCR technology was used to detect the reproduction related gene BMP4,FSHR,Gn RHR,TIMP3 and differentially expressed genes in the gonadal axis tissues of Qianbei Ma Goat,using immunohistochemical techniques to express the differentially expressed genes in ovarian tissue protein,successfully isolated and cultured ovarian granulosa cells,constructed the difference recombinant plasmids expressing genes and transfected into ovarian granulosa cells,the effects of differential genes on reproduction-related genes,proliferation / apoptosis of ovarian granulosa cells,and production of estradiol / progesterone were detected at the cellular level.The research results are as follows:1.The results of circ RNA sequencing showed that there were 126 circ RNA items with significant differences,among which 97 items were down-regulated from the multi-lamb group and 29 items were up-regulated.Differential genes are mainly enriched in relaxin signaling pathway,prolactin signaling pathway,bone cell differentiation pathway,cell receptor signaling pathway,and mainly exist in cellular processes,biological regulation,metabolic processes,developmental processes,organelles,biofilms,intracellular,adhesion,catalytic activity,signal transduction,protein binding,etc.The multi-lamb group was screened from the TGF-β signaling pathway,oocyte meiosis,and oxytocin signaling pathway.The single-lamb group down-regulated entries chi＿circ＿0005390,chi＿circ＿0005005,and the up-regulated entries chi＿circ＿0000369,chi＿circ＿0002096 genes were verified by q PCR,which showed that the trend was consistent with the transcriptome sequencing results.2.Fluorescence quantitative PCR detected the expression levels of chi＿circ＿0005390(hereinafter referred to as SMAD1 gene),BMP4,FSHR,Gn RHR,TIMP3 genes in the uterus,fallopian tube,pituitary,hypothalamus,and ovary tissues of Qianbei Ma Goat.In the ovary and hypothalamus of Qianbei Ma Goat,the single lamb group was extremely highly expressed in the multi-lamb group(P<0.01),and the fallopian tube was significantly expressed in the single lamb group in the multi-lamb group(P<0.05);BMP4 and TIMP3 genes were in ovarian tissue the mid-lamb group was significantly overexpressed in the single-lamb group(P<0.05),and TIMP3 was significantly over-expressed in the single-lamb group in the fallopian tube,pituitary,and hypothalamus tissues(P<0.01);the FSHR gene was in the ovary and fallopian tube The expression of Gn RHR ovary and hypothalamus in the multi-lamb group was significantly higher than that in the single-lamb group(P<0.01),and in the pituitary tissue was significantly higher in the multi-lamb group.Expressed in the single lamb group(P<0.05).3.The SMAD1 gene was successfully cloned,and the p EGFP-N3-SMAD1 eukaryotic expression vector was successfully constructed.The results of bioinformatics analysis showed that SMAD1 protein was hydrophilic protein with no signal peptide region,conserved domain,or special domain.SMAD1 Protein 43.5% may exist in the cytoplasm,39.1% may exist in the nucleus,8.7% may exist in the mitochondria,and 8.7% may exist in the cell structure.4.The effect of peripheral blood reproductive hormones and SMAD1 gene on granulosa cells found that the content of estradiol and progesterone in the single lamb traits during natural estrus decreased faster than that in the multi-lamb group,and its content in the single-lamb group was higher than that in the multi-lamb group before estrus Group,but the multi-lamb group is higher than the single-lamb group during the estrus period;estradiol is significantly higher than the multi-lamb group 11 days before the first estrus(P<0.05),and progesterone is in the second natural estrus period The multi-lamb group was significantly higher than the single-lamb group(P<0.01);overexpression of SMAD1 gene found thatSMAD1 gene promoted the proliferation of ovarian granulosa cells,inhibited apoptosis,and also promoted the production of estradiol and progesterone.The expression of SMAD1 protein in the ovary was completed,and the goat ovarian granulosa cells were successfully isolated.The results showed that SMAD1 protein was expressed in oocytes and granulosa cells,but the expression level in primary oocytes was less,and the expression level in the corpus luteum most.Overexpression of SMAD1 gene was found to have an inhibitory effect on the m RNA expression of reproduction-related genes BMP4,FSHR,Gn RHR,TIMP3.In summary,SMAD1 gene can increase the production of E2 and P4,help maintain the estrus and pregnancy of Qianbei Ma Goat,promote cell proliferation,inhibit apoptosis,and also significantly inhibit the expression of reproduction-related gene m RNA,and these reproduction-related genes can regulate the number of follicles,follicular development and formation,and ovulation.Therefore,SMAD1 gene inhibits the expression of reproduction-related genes,which indirectly affects the development of follicles and the number of ovulations.Reducing the number of lambs,this result provides a theoretical basis for the study of genes related to lamb traits.It is initially believed that SMAD1 gene can be used as a candidate gene for exploring the traits of goat lamb traits. |
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