Study On The Function Of BMP7 Gene In The Puberty And Its Polymorphism Related To The Number Of Lambs In Dolang Sheep

Dolang sheep is a local breed in Xinjiang,with excellent reproductive performance.The puberty is a sign of animals obtaining reproductive ability,and the timing of puberty plays an important role in animal reproduction.This study focuses on the genetic mechanism of BMP7 gene activation during the puberty in Dolang sheep,using the BMP7 gene identified as associated with the puberty by previous transcriptome sequencing.The study conducted functional research on BMP7 gene at cellular level,explored its mechanism of action in sheep ovarian granulosa cells and its association with polymorphism and litter size.The research obtained the differentially expressed BMP7 CDS sequence through cloning technology and conducted bioinformatics analysis using the obtained sequence.Real-time fluorescent quantitative PCR technology was used to analyze the expression level of BMP7 gene in five different tissues of the hypothalamus,pituitary gland,ovary,oviduct and uterus of Dolang sheep.Sheep ovarian granulosa cells were isolated and BMP7 gene overexpression vector was constructed and transfected into sheep ovarian granulosa cells.CCK-8 was used to detect cell proliferation,and Elisa was used to detect the effect of BMP7 gene overexpression on reproductive hormone secretion and related gene m RNA expression.The polymorphic loci related to BMP7 gene and litter size were screened by PCR amplification.The research results are as follows:The CDS sequence of Dolang Sheep BMP7 gene was obtained 1296bp,encoding 431 amino acids.Using MEGA 7.0 to construct an evolutionary tree,the results show that the closest relative to the Manchurian wild sheep is the Ovis areis and then Bos taurus,followed by Homo sapiens,Macaca mulatta,Equus cacallus,with the furthest relative being Mus musculus and Canislupus familiaris.The amino acid sequence of BMP7 protein in the Manchurian wild sheep is similar to those of Homo sapiens,Bos taurus,Canislupus familiaris,Mus musculus,Ovis areis,Macaca mulatta,and Equus cacallus with similarity percentages of 97.4%,99.5%,46.9%,26.5%,99.3%,97.4%,and 96.8%,respectively.The bioinformatics analysis shows that the BMP7 protein molecule formula is C₂₂₀₀H₃₃₇₄N₆₃₂O₆₂₈S₁₇,theoretical isoelectric point is 8.17,total number of atoms is 6851,instability index is 53.18,lipid solubility coefficient is 76.96,and the overall average hydrophilicity is-0.419.The protein is an unstable hydrophilic alkaline protein with theα-helix and irregular curl being the main secondary structures,as predicted by tertiary structure Real-time fluorescence quantitative PCR detection shows that the BMP7 gene is expressed in five tissues of the Manchurian wild sheep before,during,and after the onset of estrus,with hypothalamus having the highest level of expression,followed by the uterus,oviduct,pituitary gland,and ovary,with the expression level of the hypothalamus being significantly higher than that of the other tissues（P<0.05）.By constructing BMP7 gene overexpression vector p EGFP-N1-BMP7 and transfecting it into sheep ovarian granulosa cells,a large amount of green fluorescence can be emitted in the cells,and the relative expression level of BMP7 gene after transfection is significantly higher than that of the blank group（P<0.01）.Western Blot result shows that the relative expressed protein level in BMP7 overexpression group is significantly higher than the other two groups（P<0.05）.CCK-8 results show that BMP7 overexpression can promote granulosa cell proliferation with increasing time.Elisa results show that BMP7 gene can significantly up-regulate the secretion of E2,Gn RH,P4,and LH.q RT-PCR detection shows that BMP7gene can increase the expression of BMPR1B,BMPR2,Gn RHr,and FSHR genes in the cells.The BMP7 gene was detected with 8 polymorphic loci in the Dolang sheep,including g.58172009C>T,g.58172131C>T,g.58172205C>T,g.58172226G>A,g.58171885G>A,g.58171886A>C,g.58171996G>A,and g.58172002A>G.The g.58172002A>G locus showed significant differences between multiple and single-birth lambs（P<0.05）.The genotype frequencies of the g.58172131C>T,g.58171885G>A,and g.58171886A>C loci were extremely significantly different between multiple and single-birth lambs（P<0.01）.The genotype frequencies of the four polymorphic loci,g.58172009C>T,g.58172205C>T,g.58172226G>A,and g.58171996G>A,showed no significant difference between multiple and single-birth lambs（P>0.05）.In conclusion,the CDS region of BMP7 gene in Dolang sheep was cloned in this study,and the expression rules of BMP7 gene in five tissues of Dolang sheep in different puberty stages were explored,and the BMP7 overexpression vector was successfully constructed.It was preliminarily confirmed that BMP7 gene was involved in the initiation of puberty in Dolang sheep,and the relationship between BMP7gene polymorphism and lambing was elucidated.