Screening Of Functional Mutations Affecting Expression And Chicken Growth In Cholecystokinin A Receptor Gene

Cholecystokinin A receptor(CCKAR)is a receptor that mediates cholecystokinin(CCK)satiety signal.Therefore,down-regulation of CCKAR expression could reduce the threshold of satiety signal and increase chickens’ feed intake and growth rate o.However,the molecular basis of CCKAR down-regulation is not clear.In this study,the luciferase reporter gene system was used to study the expression regulation of CCKAR gene sequence variation,and to analyze the relationship between candidate mutations and chicken weight,in order to screen out functional regulatory mutations that affect CCKAR gene expression and chicken growth.Two upstream fragments(-1185 bp and-799 bp)of the CCKAR gene and one component containing conserved element variation(CSNP5)at downstream 5.6 Mb was inserted into the upstream of luciferase reporter gene of only p NL plasmid.A double luciferase reporter genotype system studied the expression regulation of the CCKAR sequence.The body weight at the ages of 6,10,18 and 22 weeks in the first generation of egg breeding core population of Lueyang black chicken(nasty 347),the body weight,feed intake and feed conversion rate of the third generation(nasty 129)at 13-16 weeks,and the feed intake,egg production and feed-egg ratio at 27-30 weeks in the fourth generation(nasty 150)were measured.The genotypes of candidate mutations(PSNP3 and CSNP5)screened by PCR-RFLP report gene experiment.The correlation of PSNP3 and CSNP5 with growth and feed conversion traits of Lueyang black chicken was tested by analysis of variance.The body weight of the first generation(nasty 40)at the age of 1-10 weeks was measured,and the early growth curve of Lueyang black chicken was fitted by Logistic and Gompertz model.The luciferase activity of fast-growth haplotype of plasmid p NL-1185/+183 was significantly higher than that of slow-growth haplotype.However,when the four mutations(PSNP3～PSNP6)at the 1835 ’end of the fragment-1185 were deleted,the difference in expressive activity disappeared.The above differences in luciferase activity were also detected on plasmid p NLCSNP5.These results suggest that PSNP3～PSNP6 and CSNP5 are candidate mutations affecting the expression of CCKAR gene.In order to further clarify the association between these candidate mutations and chicken growth traits,according to the prediction results of transcription factors,the study selected two mutation sites,PSNP3 and CSNP5,and analyzed their association with body weight,feed intake and feed conversion rate.All of them are significantly associated with those traits,describe them.The fitting results of early growth curve showed that the fitting effect of Gompertz model was better than that of Logistic model,and the first inflection point appeared at 7 weeks old.Through reporter gene experiment and association analysis,CCKAR gene promoter region mutation PSNP3 and downstream conservative element mutation CSNP5 were identified as functional mutations affecting the expression of CCKAR gene.G of PSNP3 is the dominant allele associated with low feed intake and high feed conversion rate.These results lay a foundation for the study of the growth regulation mechanism of CCKAR gene and provide a basis for the establishment of molecular breeding methods for feed utilization efficiency of Lueyang black chickens.