| As a member of the ubiquitin-dependent proteasome degradation pathway(UPP),UCHL1 involves in ubiquitin-dependent protein catabolism and protein deubiquitination,has cysteine-type endopeptidase and thiol-dependent ubiquitin-specific protease activity,and plays an key role in various biological processes of the organism,such as adult walking behavior,mitochondrial axon transport,diet behavior,muscle fiber development And ubiquitin-dependent protein catabolism processes.However,its function remains unclear in goose growth performance.In this study,the full-length coding sequence of 675 bp UCHL1 gene was firstly characterized in Yangzhou geese.According to the ORF prediction result,this CDS sequence encodes a protein of 224 amino acids with a molecular weight of 25038.43,and its Theoretical pI is 5.68.Its product was an unstable and hydrophilic protein,mainly distributed on the cytoplasm.The nucleotide sequence of the coding region of the goose UCHL1 was highly similar to that of other avian species such as duck(97.6%),chicken(94.%)and turkey(92.9%),but was relatively less similar to human(75.5%),rat(73.4%),mouse(74.1%),horse(76.3%),pig(74.8%),cattle(75.9%),sheep(76.2%),dog(75.7%)and zebrafish(64.0%).Through direct sequencing,we found that there is a SNP c.-652C>T in the 5’flanking region of UCHL1,which is significantly related to the 64day bodyweight of male Yangzhou geese.Genotype by Allele-specific PCR(AS-PCR)and correlation analysis with 64day bodyweight found that TT genotype individuals showed higher body weight compared with CC individuals(P<0.01).Given that the SNP is located UCHL1 gene promoter region,we further analyzed whether the mutation caused a change in the transcriptional activity of the promoter region of the UCHL1 gene.We constructed three UCHL1 gene-promoted luciferase reporter vectors with different lengths.Through fluorescence activity analysis,we found that pGL3-767 activity was significantly higher than pGL3-421 and pGL3-1173(P<0.01),confirming the interval-421 to-1173 contains the core promoter region of the UCHL1 gene.The luciferase reporter vector containing different alleles of c.-652C>T site was constructed by Site-directed mutagenesis.The results showed that the fluorescent activity of pGL3-T was significantly higher than that of pGL3-C(P<0.05).Q-PCR results showed that UCHL1 was Specific expressed in the brain and gonads.Though the detection of UCHL1 and some hypothalamic appetite-related genes mRNA level in goose brain tissue between two genotypes,the results showed that geese with CC genotype had lower mRNA expression level of UCHL1 in brain tissue than those of TT genotype(P<0.01),Compared with the TT groups,NPY and AdipoR1 mRNA level was significantly lower in the CC groups(P<0.05),while the FAS mRNA level was significantly higher in the TT groups(P<0.05).In order to verify whether changes in mRNA levels of hypothalamic appetite-related genes were affected by the expression level of UCHL1 gene,we used siRNA to specifically knock down UCHL1 gene in mouse hypothalamic N38 cell line,Q-PCR experiments showed that UCHL1 gene expression level was significantly reduced in knockdown group,Compared with the control groups,NPY,AdipoR1 and ADPN mRNA level was significantly decreased(P<0.05),while the FAS and AMPKA1 mRNA level was significantly increased in the UCHL1 knockdown groups treated with siRNA(P<0.01).This result is consistent with changes in appetite-related genes in goose brain tissue.Therefore,we believe that the UCHL1 gene c.-652C>T mutation can change the transcriptional activity of the promoter and affect the transcription level of the UCHL1 gene,resulting in changes in the expression level of hypothalamic related genes,which in turn affects the growth performance of Yangzhou goose.In this study,the UCHL1 gene of Yangzhou goose was cloned and characterized,and the molecular mechanism related to the bodyweight of Yangzhou goose SNP c.-652C>T in the promoter region of UCHL1 gene was found and discussed,which provided an effective molecular marker for goose breeding. |
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