浙江大学学报(农业与生命科学版)  2017, Vol. 43 Issue (3): 365-370
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福建黄兔肌纤维性状及Myh6基因表达水平分析[PDF全文]
穆琳, 王文洲, 赵博昊, 胡帅帅, 赵斌, 陈阳, 吴信生    
扬州大学动物科学与技术学院,江苏省动物繁育与分子设计重点实验室,江苏 扬州 225009
摘要: 选取我国地方品种福建黄兔为研究对象,利用冰冻切片技术对不同发育阶段福建黄兔背肌和腿肌的肌纤维性状进行研究,通过荧光定量聚合酶链式反应技术分析在肌肉发育过程中起重要作用的Myh6基因在福建黄兔不同发育阶段表达水平的差异,并与肌纤维性状进行相关性分析。结果显示:2、4、6、8、10和12周龄福建黄兔的白肌纤维密度均显著大于红肌纤维和中间型肌纤维,白肌纤维的面积显著高于红肌纤维,长径和短径的大小排列为白肌纤维>中间型肌纤维>红肌纤维,但差异无统计学意义;在6周龄前,背肌和腿肌中各类肌纤维长径、短径和面积呈显著增大的趋势,而6周龄后增大趋势逐渐变缓;Myh6基因在公母兔背部和腿部的表达均呈先上升后下降的趋势,其中母兔在4周龄表达量最高,公兔在6周龄表达量最高;Myh6基因的表达量与肌纤维面积、长径和短径的变化一致。表明Myh6基因与肌纤维类型和肌纤维的生长发育密切相关。
关键词: 福建黄兔    肌纤维    Myh6基因    表达水平    
Analysis of muscle fiber traits and Myh6 gene expression level in Fujian yellow rabbit
MU Lin, WANG Wenzhou, ZHAO Bohao, HU Shuaishuai, ZHAO Bin, CHEN Yang, WU Xinsheng    
Jiangsu Key Laboratory of Animal Genetics & Breeding and Molecular Design, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu, China
Summary: With growing demands for the diversity and quality of meat, rabbit meat is more and more popular with its special flavor. The traits of muscle fibers have a direct effect on the quality of meat, and the number, size, density, and type of muscle fibers are closely related to the meat flavor. However, recent studies on genes related to meat quality of rabbit are quite few, so the formation mechanism of the edibility characteristic of the muscle fibers is still unclear. Myh6, a member of the myosin heavy chain family, plays an important role in the development of muscles. What's more, Myh6 is a marker gene in cardiac development which promotes the expansion of ventricular myocardial cell.>With growing demands for the diversity and quality of meat, rabbit meat is more and more popular with its special >flavor. The traits of muscle fibers have a direct effect on the quality of meat, and the number, size, density, and type of muscle >fibers are closely related to the meat flavor. However, recent studies on genes related to meat quality of rabbit are quite few, so >the formation mechanism of the edibility characteristic of the muscle fibers is still unclear. Myh6, a member of the myosin heavy >chain family, plays an important role in the development of muscles. What's more, Myh6 is a marker gene in cardiac >development which promotes the expansion of ventricular myocardial cell. In order to clarify the relationship of Myh6 and muscle fiber traits, correlation analysis was used to study the expression level of Myh6 in rabbit muscle fibers of longissimus dorsi muscle and leg muscle with different ages, which could also provide theoretical basis for some improvement in rabbit meat. In this study, Fujian yellow rabbits were used as experimental animals, and their myofiber traits of longissimus dorsi muscle and leg muscles at different development stages were evaluated by a frozen section technique. Meanwhile, expression levels of Myh6 gene of Fujian yellow rabbit at different development stages were analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the density of white muscle fibers was significantly higher than that of red muscle fibers and intermediate muscle fibers at 2, 4, 6, 8, 10 and 12 weeks old; and the area of white muscle fibers was significantly greater than that of red muscle fibers. The descending order for the long and short diameters of muscle fibers of legs and backs was white muscle fibers, intermediate muscle fibers and red muscle fibers. The inverse relationship between the density and area of muscle fibers was that the density of muscle fibers decreased with the increase of muscle fiber area. The density of muscle fibers had an obviously decreasing trend from 2 to 6 weeks old. In the whole, with the age increasing, the density decreased but not significant. There was no significant difference between long and short diameters of muscle fibers, and both long and short diameters of the different muscle fibers increased gradually with the age increasing, although the trend was very obvious before 6 weeks old, but insignificant after 6 weeks old The results also showed that the expression level of Myh6 first increased and then decreased in longissimus dorsi muscles and leg muscles of bucks and does, which showed the highest expression level at 4 weeks old in does and 6 weeks old in bucks. Furthermore, there is no difference in the change tendency between the expression level of Myh6 gene and the variation of area, long and short diameters of muscle fibers. Above all, it can be concluded that the development of muscle fibers is related to Myh6 gene.
Key words: Fujian yellow rabbit    muscle fiber    Myh6 gene    expression level    

肌球蛋白由多个高度保守的基因编码,含有多种同工蛋白家族,在骨骼肌运动中主要起收缩作用。肌球蛋白分子由2条肌球蛋白重链(myosin heavy chain, MyHC)和2对起调节作用的肌球蛋白轻链(myosin light chain, MyLC)组成。Myh6基因属于肌球蛋白重链家族。研究发现,敲除Myh6基因的小鼠患有心肌衰竭与异常肥大症[1]Myh6基因表达下调可能引起心房间隔缺陷[2]。斑马鱼wea突变体由于干扰了斑马鱼Myh6基因的正常表达导致心房收缩缺陷[3]。Mef2转录因子缺失的果蝇在Myh6基因表达下调时出现肌球蛋白重链缺失的性状[4]。通过蛋白质组学技术筛选北京油鸡肌肉发育相关基因,发现Myh6基因可能参与肌肉细胞骨架结构[5]。在前期研究中,本实验室也利用表达谱芯片技术从新西兰白兔和福建黄兔不同部位肌肉中筛选出差异表达的Myh6基因[6],因此,推测其与家兔的骨骼肌发育密切相关。

福建黄兔是我国地方家兔优良品种[7],具有耐粗饲、繁殖性能好、抗病力强、肉质与口感好、药用价值高等优点[8],拥有“药膳之兔”的美誉,被农业部列入《国家级畜禽遗传资源保护名录》 [9]。但目前关于福建黄兔肌肉发育的研究较少,其肌纤维性状形成的分子遗传学基础更是鲜有报道。因此,本研究通过对比福建黄兔不同发育时期的肌纤维类型及Myh6基因的表达水平差异,分析Myh6基因与肌纤维性状的相关性,旨在为福建黄兔肌肉发育遗传学机制研究提供理论依据。

1 材料与方法 1.1 材料 1.1.1 实验动物

本实验动物来自饲养于江苏省金陵种兔场的福建黄兔。随机选取2、4、6、8、10、12周龄的公母兔各15只,取背最长肌(以下简称背肌)和后腿内侧肌(即股四头肌,以下简称腿肌)适当大小各2块:一块装入10 mL管中,编号后迅速投入冰盒,带回实验室立即制作冰冻切片;另一块放入1.5 mL指形管中,并迅速投入到液氮里,最后置于-70 ℃冰箱中保存,用于总RNA提取。

1.1.2 引物设计及合成

根据GenBank公布的家兔基因的mRNA序列,利用NCBI网站Primer-BLAST在线引物设计程序设计得到Myh6基因和内参基因GAPDH的荧光定量引物序列,由生工生物工程(上海)股份有限公司合成。引物信息见表 1

表1 目的基因引物序列 Table 1 Primer sequences of the target genes
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1.1.3 主要试剂

磷酸缓冲液、琥珀酸盐缓冲液、0.1%硝基四氮盐和Trizol均购买自美国Invitrogen公司;SuperRT cDNA第一链合成试剂盒、荧光定量试剂盒和无RNA酶水(RNase-free water)均购自北京康为世纪生物科技有限公司。

1.2 方法 1.2.1 组织学测定

在恒温冷冻切片机内进行冰冻切片制作:将修好的样品固定在涂抹了包埋剂的样品托上,并保证肌纤维走向与样品托垂直,同时做好标记,切片厚度一般为5~10 μm。利用琥珀酸脱氢酶(succinate dehydrogenase, SDH)染色法染色,封片后置于显微镜下选取合适的视野观察测量,利用励扬万能视频成像系统装置和ScopePhoto软件在10倍物镜下拍照,每个样品选取2个视野。

1.2.2 总RNA提取和cDNA合成

肌肉总RNA提取按Trizol提取试剂说明书进行,总RNA提取完成后用DNA酶(DNase)水处理以去除基因组DNA的污染,并用分光光度法检测RNA的质量,置于-20 ℃冰箱中保存。cDNA合成根据SuperRT cDNA试剂盒说明书配制反应体系,其中RNA模板2 μL,然后利用反转录试剂盒(RevertAidTM first strand cDNA synthesis kit)合成单链cDNA。

1.2.3 荧光定量聚合酶链式反应

荧光定量聚合酶链式反应(polymerase chain reaction, PCR)采用SYBR Green I法进行。将每个待测样品的反转录(reverse transcription,RT)产物等体积混合,用混合样(cDNA mix)进行反应条件优化,包括最佳退火温度、引物浓度、模板浓度等,确定最佳反应条件。以GAPDH基因为内参基因,每个样品设置3个重复,利用ABI 7500荧光定量PCR仪(Applied Biosystems,美国)分析在PCR过程中荧光信号值的变化,并获取各样品基因的CT值,采用2-ΔΔCT法计算基因的相对表达水平。

1.2.4 数据统计分析

利用SPSS 21.0软件对实验数据进行方差分析及多重比较,结果以平均值±标准差表示,显著性检验采用独立样本t检验;利用IPP专业图像分析软件分析冰冻切片的结果,统计所有图片中各类肌纤维的组织学特性。

2 结果与分析 2.1 福建黄兔背肌和腿肌纤维类型

利用冰冻切片技术分析2、4、6、8、10和12周龄福建公母黄兔背肌和腿肌的肌纤维类型,根据染色的深浅可判断不同的肌纤维类型。以8周龄为例,染色最深的为红肌纤维,染色最浅的为白肌纤维,染色中等的为中间型肌纤维(图 1)。

A:8周龄公兔背肌组织;B:8周龄公兔腿肌组织;C:8周龄母兔背肌组织;D:8周龄母兔腿肌组织。 A: Longissimus dorsi muscle of buck at 8 weeks old; B: Leg muscle of buck at 8 weeks old; C: Longissimus dorsi muscle of doe at 8 weeks old; D: Leg muscle of doe at 8 weeks old. 图1 8周龄公母兔背肌和腿肌的冰冻切片 Fig. 1 Frozen sections of the longissimus dorsi muscles and leg muscles of buck and doe at 8 weeks old
2.2 不同周龄福建黄兔肌纤维密度和面积变化趋势

图 2所示:随着福建黄兔周龄的增加,肌纤维面积逐渐增加,肌纤维密度逐渐减小;在2~6周龄内,肌纤维密度的下降趋势明显,但8~12周龄内,下降趋势越来越小,几乎保持不变。因此,从整体来看,随着周龄数的增加,肌纤维密度逐渐下降,但减小的趋势越来越不明显。

图2 不同周龄福建黄兔肌纤维密度和面积变化趋势 Fig. 2 Changes of the density and area of muscle fiber in Fujian yellow rabbits at different week-ages
2.3 不同周龄福建黄兔肌纤维长径和短径变化趋势

图 3所示,不同周龄福建黄兔背部和腿部肌纤维的长径和短径差异均无统计学意义(P>0.05),但随着周龄数的增大,各类肌纤维的长径和短径逐渐变大,且在6周龄前变大的趋势更明显,6周龄之后增长缓慢。

图3 不同周龄福建黄兔肌纤维长径和短径变化趋势 Fig. 3 Changes of the long and short diameters of muscle fibers in Fujian yellow rabbits at different week-ages
2.4 Myh6基因在福建黄兔背肌和腿肌中的表达水平

图 4可知:随着周龄的增大,Myh6基因在福建黄兔背肌和腿肌中的表达量都呈现出先增大再变小最后趋于稳定的趋势;且Myh6基因在母兔背部和腿部中的表达量在4周龄时达到最大,而在公兔背部和腿部中的表达量在6周龄时最大,显著高于其他几个周龄(P<0.05);同时,Myh6基因在公兔腿肌中的表达量大于在母兔腿肌中的表达量。

*表示同一性别同一部位在不同周龄差异有统计学意义(P<0.05)。 Single asterisk (*) shows statistically significant difference at different week-ages in the same part and gender at the 0.05 probability level. 图4 福建黄兔背肌和腿肌中Myh6基因表达水平 Fig. 4 Myh6 expression level in the longissimus dorsi muscle and leg muscle of Fujian yellow rabbits

结合肌纤维性状的表型数据,即背肌和腿肌中各类肌纤维长径、短径和面积在6周龄前增大趋势明显,6周龄后趋势减缓,恰好与Myh6基因随着周龄的变化规律一致。这表明Myh6基因可能在肌纤维发育过程中发挥重要作用。

3 讨论

肌纤维作为构成肌肉的基本单位,其密度、直径、面积是影响肌肉组织学特性和评定肉质品质的重要指标。本研究以福建黄兔为实验对象,利用冰冻切片技术对不同周龄的福建黄兔背部和腿部的肌纤维密度、面积、长径和短径等性状进行了组织学测定与统计分析。陈宽维等[10]对我国7种不同遗传背景肉鸡进行研究发现,白肌纤维直径大于红肌纤维和中间型纤维直径,且其所占比例与肉的嫩度呈负相关。本研究在福建黄兔中测得的结果与此基本一致。此外,本研究还发现,在6周龄前福建黄兔背肌和腿肌中各类肌纤维长径、短径和面积增大的趋势明显,而6周龄后趋势缓慢;这与俞荣林等[11]在新西兰白兔骨骼肌中的研究结果一致。而福建黄兔的肌纤维密度随着日龄数的增加而逐渐减小,这与陈岩锋等[12]在闽西南黑兔中的研究结果类似。

本实验室前期利用表达谱芯片技术筛选出了与家兔发育有关的肌球蛋白重链家族基因Myh6[6]Myh6基因对肌肉发育的影响至关重要,其表达下调可能会引起心房间隔缺陷[2],出现肌球蛋白重链缺失[4]。目前,在小鼠、果蝇、斑马鱼等动物中已对Myh6基因的功能进行了研究,但在家兔或其他动物组织肌肉中对该基因的研究尚未见报道。本研究通过荧光定量PCR技术对不同发育阶段的福建黄兔Myh6基因的表达量进行了分析,发现随着周龄数的增加,Myh6基因的表达量先变大后变小,最后趋于稳定。将该定量结果与肌纤维组织学特性进行关联分析可以看出,当肌肉的生长速度较快时,Myh6基因的表达水平也较高。表明Myh6基因的表达量与肌纤维的类型即生长发育密切相关,但其具体的作用机制仍有待进一步研究。

4 结论

随着周龄的增加,福建黄兔背肌和腿肌中各类肌纤维的密度逐渐变小,而肌纤维面积、长径和短径逐渐变大,且6周龄前变化趋势明显,之后变化趋缓。Myh6基因在福建黄兔背肌和腿肌中的表达量都呈现出先增大后变小,最后趋于稳定,与肌纤维面积、长径和短径的变化趋势相一致,说明Myh6基因与肌纤维类型及生长发育密切相关,推测其在肌纤维的发育过程中发挥了重要作用。

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