Variation Of Bovine Myostatin Gene And Preliminary Study On Myostatin Gene Immunization

Muscle growth is one of the most interested traits in beef cattle breeding. Some of local cattle breeds have advantages of good beef quality and bearing the careless feed, while disadvantages of low growth speed and meat yield. Myostatin, a member of TGF-βsuperfamily, plays an essential role in regulating skeletal muscle growth and functions by inhibiting myoblast proliferation and differentiation. Animals with loss-of-function mutations in myostatin gene have excellent growth performance due to large fiber number and size. In this study, a total of 321 cattle, including Angus, Hereford, Simmental, Charolais, Limousin, Luxi, Qinchuan and Jinnan, were used to investigate genetic variations of bovine myostatin gene and the relationship between SNPs and meat quality traits; meanwhile, effects of myostatin gene immunization on mice were also investigated. The results were as the following:1. 5'flanking sequences of bovine myostatin gene from 16 samples (2 of each breed) were amplified and sequenced. No variation was detected in the amplified 5'flanking region. The g433 C>A and g2974 C>T sites were genotyped by PCR-RFLP. The allele and genotype frequencies were analyzed within and across breeds. The association between production traits and SNPs was analyzed and the results showed that: g433 C>A site was associated significantly with meat percentage (P<0.05), the mean of genotype AA was significantly higher than that of genotype CC (P<0.05); g433 C>A site was associated significantly with marbling score and LM area (P<0.01), the mean of genotype AA was significantly higher than that of genotype CC (P<0.01); g2974 C>T site was associated significantly with LM area (P<0.01), the mean of genotype TT was significantly higher than that of genotype CC and CT (P<0.01).2. The homology of myostatin propeptide and mature peptide from cattle, human, mouse, rat, pig, horse, dog, sheep and macaque were analyzed by DNAMAN software. The results showed that the homology of propeptide from these spacies was more than 96% and that the homology of mature peptide was more than 99%. The myostatin mature peptide sequence was analyzed by DNAStar, Antheprot and JaMBW software. The results showed that: the region of amino acids 1-17 was composed of random coil andβ-turn structure and was flexible to combine with antibodies; the region of amino acids 1-17 had high hydrophilicity, surface probability and antigenicity.3. Two DNA fragments encoding N-term of myostatin were incorporated into Hepatitis B virus S gene at C-term and site of amino acid sequence 112 and 113 to construct an expression plasmid of pVAX-S2M. Two DNA fragments encoding N-term of myostatin and N-term of inhibin were incorporated into Hepatitis B virus S gene at C-term and site of amino acid sequence 112 and 113 to construct an expression plasmid of pVAX-SMI. The plasmids were identified by restriction endonuclease digestion and sequencing. The plasmids were then transfected into HeLa cells. The transcripts and recombinant protein were detected 48 h after tranfection by RT-PCR and Western Blot, respectively. The results showed that: the plasmid pVAX-S2M could express recombined protein with antigencity of myostatin; the plasmid pVAX-SMI could express recombined protein with antigencity of both myostatin and inhibin.4. The plasmids were immunized to tibialis anterior of mice mediated by electric pules. The antibodies were detected by dot blot and the results showed that pVAX-S2M could induce myostatin antibodies in mice and that pVAX-SMI could induce myostatin and inhibin antibodies. The growth traits and reproductive traits were measured and analyzed. The results showed that: (1) the body weight (BW) of mice from pVAX-S2M, pVAX-SMI and control groups was compared. Male mice in control group had higher BW than that of pVAX-S2M, pVAX-SMI group at 2 weeks after immunization (P<0.05), while BW of male mice among three groups showed no significant difference on the other time; BW of female mice in pVAX-S2M, pVAX-SMI and control groups had no significant difference from 1 to 6 weeks after immunization. (2) The litter size (LS) of pVAX-S2M group was significantly less than control group (P<0.01), and average birth weight (ABW) of pVAX-S2M group was significantly higher than control group (P<0.01). The LS and ABW of pVAX-SMI showed no significant difference with those of pVAX-S2M and control groups. (3) The male offspring of female mice from pVAX-S2M group had significantly higher BW than that of control group from 4 to 8 week-age (P<0.05). The male offspring of female mice from pVAX-SMI group had significantly higher BW than that of control group from 5 to 7 week-age (P<0.05). There was no significant difference between the pVAX-S2M group and pVAX-SMI group. The female offspring of female mice from pVAX-S2M group had significantly higher BW than that of pVAX-SMI and control group from 5 to 8 week-age (P<0.05). There was no significant difference between the the control group and pVAX-SMI group. (4) The male offspring of female mice from pVAX-S2M group had significantly higher gastrocnemius weight (GW) than that of pVAX-SMI and control group (P<0.05). There was no significant difference between the pVAX-SMI and control group. Female offspring of female mice from pVAX-S2M, pVAX-SMI and control group had no significant GW. (5) Male and female offspring from pVAX-S2M group had significantly larger cross section area (CSA) than that of pVAX-SMI and control group (P<0.05). There was no significant difference between pVAX-SMI and control group.