【作者】 张路培；

【导师】 许尚忠；

【作者基本信息】 中国农业科学院 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 肌肉发育一直是肉牛育种领域中最受关注的问题之一。我国一些地方牛品种肉质好、耐粗饲,但是生长速度缓慢,产肉率低。属于TGF-β超家族的肌肉生成抑制素具有抑制肌肉增殖和分化的功能。肌肉生成抑制素功能缺失性突变的个体肌纤维数量和直径显著增大,导致肌肉产量显著增加。为了探索牛肌肉生成抑制素基因遗传变异与肉质性状的关系,本研究以8个品种(安格斯、海福特、利木赞、西门塔尔、夏洛来、鲁西牛、秦川牛和晋南牛)321个个体为研究对象,检测了牛肌肉生成抑制素基因的SNPs,并对其与肉质性状进行了关联分析;同时,本文对利用肌肉生成抑制素基因免疫提高动物生长性能做了初步研究。得到如下主要结果:1.对每个品种2个个体牛肌肉生成抑制素基因DNA 5’侧翼区进行扩增和测序,未发现扩增区间内的碱基存在变异。利用PCR-RFLP法对g433 C>A和g2974 C>T位点多态性进行了检测,并分析了各位点在不同品种中的基因频率和分布差异。对g433 C>A和g2974 C>T位点与牛生产性状进行关联分析,结果发现:g433 C>A位点与净肉率性状显著相关(P<0.05),AA型显著高于CC型(P<0.05);g433 C>A位点与大理石花纹等级和眼肌面积两个性状存在极显著相关(P<0.01),AA基因型个体的大理石花纹得分和眼肌面积极显著的高于AC和CC基因型个体(P<0.01)。g2974 C>T位点与眼肌面积存在极显著相关(P<0.01),TT型极显著高于CC和CT型(P<0.01)。2.通过DNAMAN软件分析发现,牛、人、小鼠、大鼠、猪、马、狗、绵羊和猕猴的肌肉生成抑制素的前体蛋白氨基酸序列的同源性在96%以上,成熟蛋白氨基酸序列的同源性在99%以上。利用DNAStar程序、Antheprot软件和JaMBW软件对肌肉生成抑制素成熟蛋白氨基酸序列分析,发现1-17位氨基酸由易发生形变和与抗体结合的无规则卷曲和β-转角构成;具有较强的亲水性,暴露在蛋白表面的可能性较强,抗原性较强。3.以乙肝表面抗原S基因为载体,将两拷贝肌肉生成抑制素N端基因片段分别插入S基因第112~113氨基酸密码子之间和C末端,构建肌肉生成抑制素重组表达质粒pVAX-S2M;将一拷贝肌肉生成抑制素N端基因片段和一拷贝抑制素N端片段分别插入S基因C末端和第112~113氨基酸密码子之间,构建重组表达质粒pVAX-SMI。酶切和测序鉴定表明序列正确。重组质粒转染Hela细胞,转染后48小时成功用RT-PCR和Western blot检测到重组质粒的转录产物和重组蛋白。重组质粒pVAX-S2M表达产物具有肌肉生成抑制素抗原性,pVAX-SMI质粒表达的重组蛋白同时具有肌肉生成抑制素和抑制素抗原性。4.利用电脉冲介导,将重组质粒免疫小鼠双侧胫骨前肌。经斑点免疫杂交检测,pVAX-S2M成功诱导机体产生肌肉生成抑制素抗体,pVAX-SMI成功诱导机体产生肌肉生成抑制素抗体和抑制素抗体。对免疫小鼠的生长和繁殖性状检测,结果发现(1)免疫后0-6周,对pVAX-S2M处理组、pVAX-SMI处理组和对照组体重比较发现,在免疫后2周时,对照组公鼠体重显著高于两个处理组(P<0.05),两个处理组之间差异不显著,其余时间组间差异不显著;母鼠在免疫后0-6周期间,三组间差异不显著。(2)pVAX-S2M免疫组母鼠产仔数极显著的低于对照组(P<0.01),仔鼠平均初生重极显著的高于对照组个体(P<0.01)。pVAX-SMI组和其他两组相比,产仔数和平均初生重差异均不显著。(3)对pVAX-S2M处理组、pVAX-SMI处理组和对照组仔鼠体重比较发现,pVAX-S2M处理组子代公鼠4-8周龄体重显著高于对照组(P<0.05),pVAX-SMI处理组子代公鼠5-7周龄体重显著高于对照组(P<0.05),pVAX-S2M处理组和pVAX-SMI处理组间子代公鼠体重差异不显著;pVAX-S2M处理组子代母鼠5-8周龄体重显著高于对照组和pVAX-SMI处理组(P<0.05),对照组和pVAX-SMI处理组间差异不显著。(4)pVAX-S2M处理组子代公鼠的腓肠肌重显著高于pVAX-SMI处理组和对照组(P<0.05),pVAX-SMI处理组和对照组之间差异不显著;pVAX-S2M处理组、pVAX-SMI处理组和对照组的子代母鼠之间腓肠肌重差异不显著。(5)pVAX-S2M处理组的子代公鼠和母鼠的肌纤维的截面积分别显著高于pVAX-SMI处理组和对照组子代公鼠和母鼠(P<0.05);pVAX-SMI处理组和对照组之间差异不显著。更多还原

【Abstract】 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.更多还原