| The conservation of the tRNA-modified gene structure in eukaryotes suggests similar functions and regulatory roles.Studies on model organisms such as zebrafish and mice showed that the state of tRNA modification was related to the rate of protein synthesis and affected the growth and development of animals.However,these genes have not been reported in large-scale livestock.In this study,four Chinese yellow cattle(Qinchuan cattle,Luxi cattle,Nanyang cattle and Jixian red cattle)were used as experimental materials,using biosignal analysis,sequencing technology,qPCR,Western Blot and dual luciferase reporter assays were used to systematically detect the genetic variation of tRNA-modified genes in different yellow cattle populations.The genotype distribution of TYW5,TRIT1 and TDRMT1 genetic variation in the population was analyzed.The TRDMT1 exon and promoter region were scanned to analyze the linkage structure of genetic variation sites.The mRNA and protein produced by the linkage structure were analyzed to explore different The expression pattern of haplotypes,transcriptional activity analysis of promoter mutations;analysis of genetic variation of tRNA modification genes in different yellow cattle populations and body size traits of yellow cattle(body height,Oblique length,chest circumference,chest depth,chest width,abdominal circumference)The correlation between lumbar horn width,sciatic width,hip length,waist height,cross height and body weight is intended to provide a theoretical basis for the study of the function of yellow cattle tRNA modification gene and provide reliable materials for yellow cattle molecular breeding.Research indicates:1.According to the Ensembl database published genetic variation data,the pooled sequencing method was used to identify the InDel locus larger than 10 bp.In the tRNA anti-codon loop modification gene,a TRDMT1 InDel(rs383375500)locus was found,and an TYW5 InDel(rs521024959)was found.Two InDel loci at the TRIT1 gene interval were found.The relationship between InDel locus and growth traits of different yellow cattle TYW5 genes was found: ID and DD genotypes were significantly higher than those of II genotypes(P <0.05),ID genotype Qinchuan cattle cow chest depth Significantly higher than the II genotype(P <0.05).The relationship between InDel locus and growth traits of different yellow cattle TRIT1 genes was found: there was no significant difference in the InDel-4 locus located upstream of the gene;InDel-5 locus in the 3’-UTR region was genotype Qinchuan cattle body.The Oblique length,height and cross height were significantly higher than the DD genotype(P <0.01).The ID genotype Qinchuan cattle cattle waist width was significantly higher than the II genotype(P <0.05),ID genotype Jixian Red cattle waist width was significantly higher than DD genotype(P <0.05).2.The differences in the distribution of the InDel locus genotypes and alleles of the different yellow cattle TRDMT1 genes were compared.The distribution frequencies of Qinchuan cattle and the other three yellow cattle alleles were significantly different(P <0.05).The chest width of the DD genotype Qinchuan cattle was significantly higher than that of the II genotype(P <0.05).The waist height of the II genotype Qinchuan cattle was significantly higher than that of the DD genotype(P <0.05).The Oblique length of the DD genotype of Jixian Red cattle was significantly higher than that of the II genotype.3.The eleven exons of TRDMT1 gene were sequenced and scanned.It was found for the first time that a fourth exon of the different yellow cattle showed a synonymous mutation locus 2(G>A,Q88>Q)and a missense mutation.locus 3(T>C,H101>Y);a missense mutation locus 9 in the sixth exon(A>G,T101>A);and six mutation loci in the third intron,included locus11(EX3+12,T>C),locus 12(EX3+13,A>G),locus 13(EX3+62,A>G),locus 4(EX3+1881,G>A),locus 5(EX3+1904,C>A),locus 6(EX3+2131,C>T);the second intron two mutation,included locus 1(EX4+23,C>T),locus 14(EX4+593,A>G);the fifth intron two mutation,included locus 7(EX5+559,T>C),locus 8(EX5+699,C>T);one mutation wes founded in the seventh,ninth and tenth introns respectively,included locus 15(EX7+15,C>T),locus 16(EX9+71,A>T),and locus 17(EX10+18,G>A).Genotyping statistics were performed on 570 individual cattle by PCR and sequencing techniques,and correlation analysis was performed on 9 genetic variation loci of Lcous 1 to 9 with yellow body measurements.The results showed that: TT gene of Lcous 1 the body height of the Qinchuan cattle was significantly higher than that of the CC genotype(P < 0.05).The height of the GG genotype of the Luxi cattle was significantly higher than that of the GA genotype(P <0.05).The body height of CC genotype Qinchuan cattle was significantly higher than that of TT genotype(P <0.05).The body height of AA genotype Qinchuan cattle at locus 4 was significantly higher than that of GG genotype(P <0.05).The body height of locus 7 TT genotypes of Nanyang cattle was significantly higher than that of CC genotype(P <0.05).The weight of CC genotype Nanyang cattle was significantly higher than that of TC genotype(P <0.05).The chest circumference and body weight of the Nanyang cattle were significantly higher than the CT genotype(P <0.05).The linkage analysis of SHEsis software showed that 9 mutations in the TRDMT1 gene and an InDel chain analysis showed that there were strong linkage disequilibrium at these 10 loci.According to the relative quantification of mRNA and protein in the muscle tissue of Qinchuan cattle according to the combination of three mutations of TRDMT1 exon,the gene expression difference between the three genomic mutations was significant(P <0.05).4.A G>C mutation was found at 1223 bp upstream of the translation initiator codon of the bovine TRDMT1 gene,which was predicted by AliBaba2.1 and Genomatix software.This site may cause differences in transcription factors such as the transcription factors sp1,PLAG1,ZNF35,MZF1 and other transcription factors.The results of double luciferase assay showed that the G>C mutation caused gene expression differences(P <0.05).In summary,the genetic variation of tRNA anti-codon loop modification gene indicates that TYW5,TRIT1 and TRDMT1 have medium and low abundance polymorphism,which can be used as Qinchuan cattle,Luxi cattle and Nanyang cattle in molecular breeding of Body height.These can become potential candidate DNA markers for growth traits such as waist height,chest depth for Qinchuan cattle and Oblique length,sciatic width,high cross for Jiaxian red cattle.The detection of genetic variation of TRDMT1 gene revealed that the 1223 bp G>C mutation upstream of the promoter translation initiator codon increased its transcriptional activity,and there was a strong linkage disequilibrium structure between sites 1 and 9,and different haplotypes have special expression patterns.The expression law provides a theoretical basis for the study of tRNA modification function of yellow cattle,and provides reliable molecular material for yellow cattle molecular breeding. |
