Clonging, Identification And Characterization Of Thedifferentially Expressed Genes In Longissimus Dorsi Of Yanbian Yellow Cattle

Intramuscular fat(IMF) is the material basis for beef marbling and is one of major determinants of the tenderness and taste of beef. Gene expression patterns of muscle tissue are various in cattles with different cotent of intramuscular fat. Compared with bulls, steers have a more slowly growth rate, faster lipid accumulation, more intramuscular fat, tenderer muscle. Further understanding on gene structure and function has indicated that these differences should be the result of different expression of genes. In the present study, Longissimus muscles Yanbian yellow cattle under the same age and same raising condition were used as materials. Gene Fishing was used to filter, clone and identify the differentially expressed genes of longissimus muscles in cattles with different cotent of intramuscular fat and steers, bulls. The findings are as follows:1. Longissimus muscle samples were collected from 30 cattles and evaluated for intramuscular fat content.30 Yanbian steers at the age of 28 months were selected and divided into two groups consisted of 3 samples with highest intramuscular fat content and 3 samples with lowest intramuscular fat content. The average intramuscular fat content of each group was 17.58%±0.14%and 5.16%±0.32%, and group difference was significant (P<0.01). GeneFishing was used to analyze the differentially expressed genes of two groups. Twelve Expressed Sequence ESTs were gained and sequenced. Eight out of twelve ESTs sequence were known genes. The differentially expressed genes included 5 categories: functional groups of myocinesimete(Titin, ACTA1), cytokine signal transduction(ASB12), transcription complex related genes (RPS14, RPS26 and RPLP1), cell energy metabolismand (COX4), protein of unknown function(Glutamyl tRNA Synthetase) gene and four unknown genes.2. From 30 steers of Yanbian cattle selected two groups which was 9 individuals each of the highest and the lowest IMF content, and IMF content was separately 13.26%±0.28%and 4.65%±0.18%(P<0.05). By RT-PCR and real-time quantitative PCR detected the qualitative and quantitative expression level of individual genes. The results of quantitative PCR showed that the correlation between ASB12, mRNA expression levels of Titin gene and IMF content was positively; the correlation between Glutamyl tRNA Synthetase, RPLP1, mRNA expression levels of ACTA1 gene and IMF was negatively.3. Obtained differentially expressed genes were performed in different tissues (heart, liver, spleen, lung, kidey, stomach, small intestine, muscle) by semi-quantitative RT-PCR analysis.4. Using ORF Finder, CLUSTAL W and some other related software, we analyzed ACTA1, RPLP1, ASB12and Glutamyl tRNA Synthetase structure, protein structure and conserved motifs of these genes. In addition, the corresponding phylogenetic trees were constructed.5. Partial genomic sequence of cattle ACTA1,Glutamyl tRNA Synthetase,ASB12 and Titin genes were obtained by Comparative genomics and PCR, and the polymorphism of the 4 genes was analyzed. The results were as follows:(1) Two SNPs were identified in ACTA1,1598bp, whose GenBank accession number is HQ608159. One is 193A→G mutation and the other is 288A→G. (2) Titin showed three SNPs. g. [45003A→G, 70541A→G,70553 C→A]. (3) ASB12 showed three SNPs. g. [712T→C, 886G→C,1C42 C→T]. (4) Glutamyl tRNA Synthetase showed three SNPs. g. [4734G→A,4858A→G]. and one is insertion mutation.6. Using PCR-RFLP and PCR-SSCP,4 SNPs in Yanbian yellow cattles were detected and production traits were analyzed. The results showed:(1) There are significant correlation between ACTA1-Avr II-RFLP genotypes and body length, heart girth, average daily gain, and body weight; (2) G4734A gene mutation of Glutamyl tRNA Synthetase was significantly associated with linoleic acid, palmitic acid, glutamic acid, histidine, glycine and alanine; (3) T712C gene mutation of ASB12 was significantly associated with palmitic acid, myristic acid, lysine, aspartic acid, praline, leucine, and arginine; (4) The Titin-MspI-RFLP were significantly correlated with L*24(Brightness), c*24 (Saturation) palmitic acid, palmitic acid, oleic acid and IMF.7. The gene expression were analyzed between steers and bulls by Gene Fishing. Ten expressed sequence ESTs were gained and sequenced. ACTA1 and NADH6 was confirmed as downregulation in steers, whereas MYH7, NDUFS8 and HSL was confirmed as upregulation in steers.8. Real-time PCR was used to analyse the change of mRNA expression level of HSL, ACTA1,MYH7, NDUFS8 and NADH dehydrogenase subunit 6 gene between steers and bulls. The mRNA levels showed these genes were differentially expressed genes, HSL, ACTA1, MYH7, NDUFS8 and NADH dehydrogenase subunit 6 which were considered as the most important genes involved in meat quality differences before and after Yanbian yellow cattles were castrated.9. ORF Finder, CLUSTALW and some other related software were used to analyze the gene structure the encoded protein structure, function, phylogenetic tree of HSL,MYH7 and NDUFS8, and phylogenetic trees were constructed.