Genetic Effects Analysis And Functional Verification Of SH2B1 Gene On Growth Traits Of Large Body Lean Pigs

Pork is the main source of people’s meat consumption,and accelerating the genetic process of growth traits can increase the supply of pork.The existing lean pig breeds have good growth and meat production performance,but after 110 kg,their growth and meat production performance has turned point and gradually declined,and large-sized lean pigs（110 kg-140 kg）have gradually become the direction of pig production.Therefore,in this study,according to the differentially expressed SH2B1 gene in different Average Daily Gain（ADG）screened by the previous full transcriptome sequencing of our research group,its genetic effect in Duroc pig,landrace pig and large white pig population was analyzed.After that,the SH2B1 gene was verified and the mechanism was analyzed.The specific content and main test results are as follows:（1）In this study,direct sequencing was used to excavate the genetic variation of SH2B1gene in 722 pig populations of three lean pig breeds,namely Duroc,Landrace,and Yorkshire pig,and it was found that there was a SH2B1-g.7848 C>T mutation site on the second exon of SH2B1 gene.This site is a missense mutation.The amino acid encoded by this site is alanine（Ala）before the mutation and becomes valine（Val）after the mutation.The mutation site of the SH2B1 gene was significantly associated with DAY_{130kg BW} in Duroc pigs（P<0.05）,and with DAY_{130kg BW} and BF_{130kg BW} in Yorkshire pigs（P<0.01）.Among the significant association groups,genotype TT>CT>CC at DAY_{130kg BW}.Therefore,we identified SH2B1 as a candidate gene for growth traits in large body lean pigs.（2）The expression of the SH2B1 gene in 4 Duroc pig dorsal longus muscles with high mean daily gain（H-ADG）and 4 low mean daily gain（L-ADG）was detected.The results showed that the expression of the SH2B1 gene in the H-ADG group was significantly higher than that in the L-ADG group（P<0.05）.（3）Interference fragments and overexpression vectors of the SH2B1 gene were transfected into C2C12 cells to investigate their effects on cell proliferation and myoblast differentiation.（1）Effects on proliferation:The results showed that although interference could reduce the proliferative activity of cells and block cells more in the G0/G1 phase,overexpression could increase cell activity and increase the proportion of cells in the S phase,but the difference was not significant（P>0.05）.（2）Effects on cell differentiation:The expression of the SH2B1 gene showed an upward trend and was the highest on day 6;RT-q PCR and WB detection showed that at day 6 of cell differentiation,overexpression of the SH2B1 gene could significantly upregulate the expression level of m RNA and protein of myomarker genes（Myo D,Myo G,Myf5,and MEF2C）（P<0.05）,interfere with the m RNA and protein expression levels of myomarker genes after interfering with SH2B1 gene（P<0.05）,and overexpression of SH2B1 gene can significantly upregulate the expression of PI3K/AKT signaling pathway marker genes PIK3R1and AKT1 in C2C12 cells（P<0.05）.Interference with the SH2B1 gene inhibited the activity of the PI3K/AKT signaling pathway.（4）While transfecting SH2B1 overexpression vectors into C2C12 cells,the PI3K/AKT signaling pathway was treated with LY294002 inhibitor to further study the mechanism by which SH2B1 gene promotes C2C12 cell differentiation.The day 6 of differentiation of C2C12cells,RT-q PCR and WB showed that the expression levels of m RNA and protein of myogenic differentiation marker genes in the NC+pc DNA3.1-SH2B1 group were significantly higher than those in the LY+pc DNA3.1-SH2B1 group（P<0.05）.However,the expression levels of m RNA and protein of LY+pc DNA3.1 constituent muscle differentiation marker genes were not significantly different from those in the LY+pc DNA3.1-SH2B1 group.In summary,the polymorphisms of Hal gene and SH2B1 gene in this study were detected and found that both had a significant impact on the growth rate of pigs,and no halo recessive harmful genes were found in the detected population,and SH2B1-g.7848 C>T mutation sites were screened,and SH2B1 gene was used as a candidate gene for the growth traits of large body lean pigs.It was found that SH2B1 gene can promote the myogenic differentiation of C2C12cells through the PI3K/AKT signaling pathway.The above results finally provide effective molecular marker information for large body lean pigs,and provide a theoretical basis for further elucidation of the regulatory mechanism of SH2B1 gene in the process of myogenesis.