Study On The Expression Pattern Of Racl Gene In Pigs And The Construction Of Eukaryotic Expression Vector For PAcGFP-Rac1

The skeletal muscle fibers are not change after the birth of mammals, they are fixed number in embryonic period. The development of Muscle is not only related to cell proliferation but also associated with cell fusion. Ras-related C3 botulinum toxin substrate 1, Rac1,which belongs to the family of Rac, small G-protein are related to the fusion of myogenic cells. Racl plays an important role not only in the fusion of myogenic cells but in the enlargement of muscle fibers and the actual production during the developmental period. Racl regulate cell polarity, phagocytosis, migration and affects plasma membrane protrusion and vesicle traffic and the actin dynamics. The aim of this study was to investigate the expression profile of Racl gene in different tissues and the developmental expression of Racl mRNA and protein in 13 tissues including heart, liver, Spleen, Lung, Kidney, pancreas, tongue, Smallintestine, Stomach, Longissimus dorsi, Cerebellum, Hypothalamus, spinal cord of Mashen pig at birth and the developmental expression pattern of Racl at the level of mRNA and protein in longissimus dorsi at seven stages(1,30,60,90,120,150,180-day old)in Mashen and Large White pigs were studied by quantitative real-time PCR and wastern blot.By extractting the total RNA of the mouse muscle and reverse transcripted it into cDNA, amplificating the Racl CDS using the PCR, identification of single enzyme digestion and double enzyme digestion and sequencing, pAcGFP-Rac1 was successfully constracted laying the foundation for subsequent cellular level study.The main results were showed as follows:1.The Rac1 mRNA was expressed heart, liver, spleen, lung,kidney, pancreas, tongue,small intestine, Stomach, longissimus dorsi, cerebellum, hypothalamus, spinal cord of Mashen pigs at birth at a low expression level.The greatest mRNA expression of Racl gene was in the lung, significantly higher than other one comparing to the other tissues(P<0.01).The greater mRNA expression of Racl gene was in Hypothalamus (P<0.05) and the lowest was in liver. The Rac1 protein was expressed heart,liver,Spleen,Lung,Kidney, spinal cord,cerebellum, hypothalamus, stomach, longissimus dorsi, psoas major, biceep femoris, backfat. The greatest protein expression parerns of Racl was in the lung and backfat, significantly higher than other one comparing to the other tissues(P<0.01). The higher protein expression parerns of Rac1 was in the lung, Spleen and Kidney. Cerebellum and Stomach were not higher and Stomach was the lowest from all.2. From the developmental expression patterns of Racl mRNA in Large white and Mashen pigs’ longissimus dorsi,we found that the greatest mRNA expression of Racl gene in longissimus dorsi was at 1-old day,120-old day and 180-old day,significantly higher than other days. The expression of the other day kept low stable levels and 60-old day is the lowest. The higher mRNA expression of Racl gene in longissimus dorsi of Mashen pigs was 60-old day, significantly higher than other days(P<0.01).The expression was significantly decreased in 90-old day,followed by it, the expression of the Racl mRNA kept lower stable levels at other stages.The lowest expression was 150-old day.Compared between the two kinds of pigs at the same developmental stage, there were significantly differences between them except 30-day old(P<0.01).From the developmental expression patterns of Rac1 protein in Large white’ longissimus dorsi,we found that the greatest protein expression of Rac1 gene in longissimus dorsi was at 1-old day and the 120-old day was the lowest. The expression was significantly decline form 30-day old and 120-day old,then was on the rise; The protein expression parerns of Rac1 in Large white’ longissimus dorsi was always in a high level,and 60-day old was the greatest,significantly higher than other days(P<0.01),while,90-day old was the lowest. Compared between the two kinds of pigs at the same developmental stage, the protein expression parerns of Racl in Mashen’ longissimus dorsi was much higher than Large wight’s. There were significantly differences between 1-day old and 60-day old (P<0.01).3.The cloned sequence of Rac1 gene was connected with expression vector pAcGFP-N1.After identifying by restrictive enzyme,PCR and sequencing,the result showed that the mammalian vector o pAcGFP-Racl was successfully constracted.