Polymorphism Of Chicken SLCO1B3 Gene And Its Interaction Protein Screening Analysis

Solute carrier organic anion transporter family member 1B3(SLCO1B3)is a major gene regulating eggshell color of green-shell eggs,and its encoded protein is OATP1B3.Chishui black bone chicken produces green shell eggs with good meat quality and rough feeding resistance.It is one of the typical representatives of green shell black bone chicken.In this study,the genetic variation sites in the exon of SLCO1B3 gene of Chishui black bone chicken hens were identified by direct sequencing,and the correlation with egg quality indexes was analyzed.At the same time,the eukaryotic expression vector p EGFP-C1-SLCO1B3 was constructed,the primary cells of eggshell gland epithelium of Chishui black bone chicken were cultured and identified,and the expression and subcellular localization of SLCO1B3 gene in eggshell gland epithelial cells were studied.Screening and identification of cellular proteins interacting with chicken SLCO1B3 using co-immunoprecipitation(Co-IP)combined with mass spectrometry(MS).The SLCO1B3 gene silencing expression vector was constructed,and the regulatory relationship between SLCO1B3 gene and FXR1,CTTN,RPL12,and RPS17 genes was studied,and the expression of FXR1,CTTN,RPL12,and RPS17 genes in different tissues of Chishui black bone chicken was studied.Tests were carried out to explore the effect of related genes on eggshell color.This study enriches the molecular theoretical research on egg green shell traits.The main findings are as follows:1 The functional mutation site EAV-HP of SLCO1B3 gene in 300 Chishui black bone chicken hens was genotyped,and 109 hens with green shell homozygous genotype(SL/SL)were screened.A total of 3 SNPs mutation sites were detected in all exons of the SLCO1B3 gene of these109 Chishui black bone chicken hens with homozygous genotype(SL/SL).These loci are located on exon 5 and exon 6,and they are all moderately polymorphic,and there is no strong linkage disequilibrium between the three mutation sites.The g.65235858G>A site mutation on exon 5 is a non-synonymous mutation,and the secondary structure of m RNA changes after mutation;The results of haplotype analysis showed that a total of 5 haplotypes and 6 diplotypes were found in 3SNP loci,Association analysis showed that the diplotypes constructed by these three SNP loci had significant effects on the eggshell color △L* value,eggshell color △b* value and egg shape index of Chishui black bone chicken(P < 0.05).In conclusion,the genetic variation of SLCO1B3 gene is significantly correlated with eggshell color and egg shape index.Western blotting showed that SLCO1B3 gene fusion protein could be successfully expressed in chicken eggshell glandular epithelial cells,and its subcellular localization was in the cytoplasm and nucleus.2.In this study,the pEGFP-C1-SLCO1B3 eukaryotic expression vector was constructed and transfected into the primary cells of the eggshell glandular epithelium of Chishui black bone chicken.The interacting cellular proteins of chicken SLCO1B3 protein were enriched by co-immunoprecipitation experiments,and the collected proteins were identified by mass spectrometry.The results showed that 37 interacting proteins of chicken SLCO1B3 gene were identified by mass spectrometry.Bioinformatics analysis showed that these proteins were mainly located in cytoplasm and nucleus,and were mainly involved in cellular processes,biological regulation and energy metabolism,with protein-binding ability and structural molecular activity.Combined with the functional analysis of genes,it was screened that FXR1,RPS17,CTTN and RPL12 proteins may be closely related to the green shell trait of eggs.3.The SLCO1B3 silencing expression vector was constructed,and the SLCO1B3 overexpression vector and the silencing vector were transfected into the eggshell glandular epithelial cells of Chishui black bone chicken,respectively,and the expression of FXR1,CTTN,RPL12 and RPS17 genes in the eggshell glandular epithelial cells was detected.The results showed that overexpression of SLCO1B3 could significantly reduce the expression of FXR1 gene in eggshell gland epithelial cells(P < 0.01),significantly increase the expression of RPL12 gene(P <0.05),and significantly increase the expression of RPS17 gene(P < 0.01),SLCO1B3 overexpression had no significant effect on the expression of related gene CTTN.When SLCO1B3 was silenced,the expression levels of FXR1 and CTTN genes in eggshell gland epithelial cells were significantly increased(P < 0.01),and the expression levels of RPL12 gene were significantly decreased(P < 0.05).4.According to the color of egg shell,Chishui black bone chicken was divided into three types: white shell,light green and dark green.The expression of FXR1,CTTN,RPL12 and RPS17 genes in three types of Chishui black bone egg shell gland,liver and hypothalamus were detected by qPCR technology.The results showed that the expression level of FXR1 gene was the highest in the hypothalamus(P < 0.01),and the expression level of FXR1 gene in the hypothalamus tissue of the white-shelled Chishui black-bone chicken was significantly higher than that of the Chishui black-bone chicken whose shell eggs were light green and dark green.(P < 0.05);The expression of RPL12 gene was the highest in the liver(P < 0.05).The expression of RPL12 gene in the liver and eggshell glands of the white-shelled Chishui black-bone chicken was significantly higher than that of the light green and dark green Chishui black-bone chickens(P < 0.05);The expression of RPS17 gene in the liver of white-shelled hens was significantly higher than that of dark green and light green hens(P < 0.05);The expression of CTTN gene was the highest in eggshell gland(P<0.05),There was no significant difference in the expression of CTTN gene in tissues of various types of Chishui black-bone chickens(P > 0.05).5.The correlation analysis between eggshell color and the expression levels of FXR1,CTTN,RPL12 and RPS17 genes was carried out.The results showed that the expression of FXR1 gene in the hypothalamus was significantly positively correlated with eggshell color △a* value(P < 0.01);the expression of RPL12 gene in eggshell glands and liver was extremely significantly correlated with eggshell color △a* value Positive correlation(P < 0.01);the expression of RPL12 gene in hypothalamus was significantly negatively correlated with eggshell color △a* value(P < 0.05).