Construct Subtractive CDNA Library Between Longissimus Muscle From Steer And Bull, Screening And Identification Of The Differentially Experssed Genes

Skeletal muscle comprises the most proportion of an animal's body mass. Hence there is much interest in understanding the development, physiology and metabolism of this tissue. Steers have a more slowly growth rate, faster lipid accumulation, more intramuscular fat, tenderer muscle as compared with bulls. Further understanding on gene structure and function has indicated that the trait difference is in fact the external exhibition of gene expression and regulation. For further studying on gene's expression rule between different types of individuals, we constructed subtracted cDNA libraries between Longissimus muscles from three Chinese Simmental steers and three Chinese Simmental bulls with same age and same raising condition by suppression subtractive hybridization technology, real-time PCR and bioinformatics method in order to clone and identify the differentially expressed genes responsible for the trait difference. The findings are as follows:1. Feeding and slaughter experiments indicated that, bulls after fattening had higher body measurement and carcass character than steers, in which the body steep length , body weight for sale, slaughter weight, carcass depth ,carcass weight, haunch weight, shoulder meat weight had significant differences(P<0.05); tenderloin weight, superiotentorial cerebral weight had extremely significant differences(P<0.01). Meat quality character analysis indicated that, bulls had significantly higher eye muscle area, marble score and Zn content than steer(sP<0.05); but steers had significantly higher fat colors, back fat thickness, water content, Ca content than bulls(P<0.05).2. A housekeeping gene,β-actin, was used to estimate the subtraction efficiency. In each subtracted cDNA library,β-actin was subtracted very efficiently at more than 25 folds, indicating that some differentially expressed genes were also enriched at the same folds and the subtracted cDNA libraries were very successful.3. More than 300 clones were randomly selected from each subtracted cDNA library. By PCR analysis, 223 positive clones were isolated from the subtracted cDNA libraries, by steers as Tester, bulls as Driver, respectively. Most of all plasmids in the clones contain 150～700bp inserts.4. By Longissimus muscles from steers and bulls cDNA as probes, high-throughput screening was carried out. We selected 84 differential expressed clones for further analysis, which showed that they represent 10 ESTs, all of them are known in cattle.5. Using real-time PCR and tissue expression character analysis, we validated four differential expressed genes. There is a difference homoplasy of expression in muscle, liver and adipose tissue, which may have correlation with its function in the skeletal muscle development of steers and bulls.6. Using CLUSTAL W and DNAman softwares, we analyzed the gene structure, protein structure and fuction of genes ACTG2,TPM2,IGF-1,HSL. In addition, the corresponding phylogenetic trees were constructed.