【作者】 杨飞云；

【导师】 陈代文； 张克英；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2008， 博士

【摘要】 本论文研究共设3个试验,旨在研究猪骨骼肌肌纤维类型分布及转化的分子机理。1.品种、体重及营养对猪肌纤维类型分布与转化的影响:选用荣昌猪和杜×长×大(DLY)杂交猪为试验动物,测定10～120kg体重时背最长肌中肌纤维直径和密度及I、2a、2x和2b 4种MyHC的基因表达,以探讨猪背最长肌纤维生长及类型的发育性变化,并分析品种及营养影响特点。结果表明:(1)背最长肌纤维直径均随体重增加而加粗,其中在10～35 kg阶段的增加幅度最大(＞50%);而肌纤维密度则随体重增加逐渐下降,尤其是在10～35 kg阶段下降幅度超过了1倍多,至80 kg后变化不大。肌纤维密度与直径存在显著负相关;(2)从10～20 kg,2个品种的背最长肌肌纤维类型的百分组成发生显著改变,MyHC I和2x型纤维比例显著降低,而2b型纤维比例显著提高;(3)从20～120 kg,背最长肌肌纤维的变化规律因品种和纤维类型而异,除MyHC2b外,2个品种的MynC I、2a和2x型肌纤维的发育规律存在一定差异;(4)背最长肌纤维大小和类型的百分组成在10～50kg阶段未见品种间显著差异,但在80 kg,荣昌猪的肌纤维大小和MynC 2b型纤维比例显著低于DLY,而2a型纤维比例则正好相反:(5)饲粮营养水平对2个品种猪背最长肌纤维直径、密度及类型的百分组成均无显著影响。以上结果提示,2个品种的背最长肌纤维类型的发育性规律及组成存在差异,且纤维组成差异主要表现在80kg,荣昌猪的MyHC 2b型纤维比例显著低于DLY猪,可能与其优良肉质相关。2.品种与体重对猪肌纤维生长相关功能基因表达的影响:选用荣昌猪和DLY杂交猪为试验动物,测定10～120 kg体重时背最长肌中与肌纤维生长相关功能基因MyoD、CaN和PGC-1的表达,以探讨猪背最长肌中这些相关功能基因的发育性变化,并分析品种特点。结果表明:(1)荣昌猪背最长肌MyoD mRNA水平先随体重增加显著增加,至20 kg达最高,之后均有不同程度下降;而DLY猪在10～120 kg阶段未见显著变化。而且,20 kg时荣昌猪的背最长肌MyoD mRNA水平显著高于DLY猪;(2)荣昌猪的背最长肌CaN mRNA水平在前80 kg未见显著变化,之后显著降低;DLY猪在50 kg前也未见显著变化,但50 kg后显著降低;(3)荣昌猪的背最长肌PGC-1 mRNA在80 kg前保持在较高水平,至100 kg显著降低,显著低于80 kg前;而DLY猪在20 kg前保持在较高水平,而后显著降低。而且,50和80 kg时荣昌猪的PGC-1 mRNA水平显著高于DLY猪。以上结果提示,猪肌纤维生长相关功能基因表达的发育性变化因品种和指标而异,MyoD、CaN和PGC-1可能在MyHC基因表达和肌纤维类型转化中发挥重要作用,但具体机制仍需进一步研究。3.共轭亚油酸对体外培养的猪肌纤维类型分布与转化的影响:试验以培养的原代猪骨骼肌卫星细胞为材料,添加不同水平CLA(0、50、100、150、200μg/mL),通过观测处理后4、8和12 d的骨骼肌细胞中I、2a、2b和2x 4种MynC的基因表达,研究CLA对骨骼肌细胞纤维类型分布与转化的影响及随时间的变化规律。结果表明:(1)肌纤维类型的分布随培养时间的延长发生显著变化。D8与D4相比,MyHC 2x型肌纤维比例未发生显著变化,但MyHC I和2a型肌纤维比例显著下降,而MyHC 2b型肌纤维比例显著上升。在D12,除MyHC 2b型肌纤维比例显著上升外,其余3种类型的肌纤维比例均显著下降。(2)添加50μg/mL CLA对D 4～12的4种类型肌纤维的百分组成没有显著影响。(3)添加100μg/mL CLA对肌纤维类型百分组成的影响主要体现在D12,显著提高MyHC I和2a型肌纤维比例,而显著降低MyHC 2x和2b型肌纤维比例。(4)添加150～200μg/ml CLA则可显著影响D4～12的4种肌纤维类型的百分组成,显著提高MyHC I和2a型肌纤维比例,而显著降低MyHC 2b型肌纤维比例。以上结果提示,添加CLA可使肌纤维类型的组成发生变化,且该作用与添加水平和处理时间密切相关。CLA对肌纤维类型组成的影响主要表现为提高MyHC I和2a型肌纤维的比例,而降低MyHC 2b和2x型肌纤维的比例,这在一定程度上解释CLA提高猪肉品质的原因。更多还原

【Abstract】 Three experiments for this thesis were conducted to study the molecular mechanism of myofiber type distribution and transformation in skeletal muscle of pigs.1.Effects of breed,body weight,and nutrition on myofiber type distribution and transformation in pigs:This experiment was conducted to study the developmental changes of fiber size and myofiber types in longissimus dorsi(LD) muscle of Rongchang and Duroc×Landrace×Yorkshire(DLY) pigs under different dietary nutritioal condition by determining fiber diameter,density,and the ratios of mRNA abundance of four isoforms of myosin heavy chain(MyHC I,2a,2b and 2x) with semi-quantitative RT-PCR.The results showed that:(1) Fiber diameter increased with BW,especially from 10 kg to 35 kg BW,it increased by more than 50%.Fiber density decreased with BW.It decreased by more than 100%from 10 kg to 35 kg BW,and aider 80 kg BW,no significant change was observed.Fiber density had a significant negative correlation with fiber diameter.(2) From 10 kg to 20 kg BW,the composition of myofiber types in LD muscle of both breeds changed dramatically with significant decrease of MyHCⅠand 2x fibers but ramarkabl increase of MyHC 2a fiber.(3) From 20 kg to 120 kg BW,the developmental changes of myofibers in LD muscle varied with breed and myofiber type.Except MyHC 2b fiber,the developmental patterns of the other three myofiber types were different between the two breeds(4)There was no difference in fiber size and the proportion of four myofiber types in LD muscle during 10～50 kg BW between the two breeds,but at 80 kg BW,Rongchang pigs showed significantly smaller fiber size,lower proportion of MyHC 2b fiber and higher ratio of MyHC 2a fiber compared with DLY pigs.(5) Dietary nutitional levels had no effect on fiber diameter,density,and the composition of myofiber types in LD muscle of both breeds.The results suggested that the developmental changes and composition of myofiber types in LD muscle varied with pig breed.The breed difference of the composition appeared at 80 kg,and RC pigs showed significantly lower proportion of MyHC 2b fiber, which might explain for their better meat quality. 2.Effects of breed,and body weight on some gene expressions related to fiber growth in pigs:The second experiment was conducted to study the developmental changes of MyoD,CaN and PGC-1 mRNA in LD muscle of Rongchang and DLY pigs. The results showed that:(1) MyoD mRNA levels in LD muscle of Rongchang pigs increased with BW,reaching the highest at 20 kg BW,and then decreased.However,in DLY pigs,there were no significant changes in MyoD mRNA levels during the period of 10～120 kg BW.At 20 kg BW,MyoD mRNA level in Rongchang pigs was markedly higher than that in DLY pigs.(2) There was no difference in CaN mRNA of LD muscle in Rongchang pigs untill 80 kg BW,and then dramatical decrease.While in DLY pigs,no significant changes were observed before 50 kg BW,and there was then marked decrease. (3) PGC-1 mRNA levels in LD muscle of Rongchang pigs were kept relatively high before 80 kg BW,following by a marked decline.However,in DLY pigs,PGC-1 mRNA levels were kept relatively high before 20 kg BW,and then decreased significantly.Rongchang pigs showed higher levels of PGC-1 mRNA at 50 kg and 80 kg BW compared to DLY pigs. These results suggested that developmental patterns of these gene expressions observed in this experiment varied with breed and parameter.MyoD,CaN and PGC-1 might play an important role in the expression of MyHC genes and the transformation of muscle fiber types,but the mechanisms need further studies.3.Effects of conjugated linoleic acids(CLA) on the distribution and transformation of myofiber types of pigs in vitro:The third experiment was conducted in vitro to study the effects of different CLA levels(0,50,100,150,200μg/mL) on the distribution and transformation of myofiber types in skeletal muscle cells of pigs after 4,8 and 12 d of culture time by determining the ratios of mRNA abundance of four isoforms of myosin heavy chain(MyHC I,2a,2b and 2x) with semi-quantitative RT-PCR.The results showed that:(1) The distribution of myofiber types in skeletal muscle cells changed markedly with culture time.On d 8, MyHC I and 2a fibers decreased clearly,while MyHC 2b fibers increased dramatically. On d 12,MyHC 2b fiber was increased markedly,hut the other three types of muscle fibers were decreased significantly.(2) 50μg/mL CLA had no effect on the composition of myofiber types from d 4 to d 12.(3) The effects of 100μg/mL CLA on the composition of myofiber types appeared mainly on d 12,with an up-regulation of MyHC I and 2a fibers, and a down-regulation of MyHC 2x and 2b fibers.(4) The compositions of myofiber types from d 4 to d 12 were significantly changed by 150～200μg/mL CLA with increase of the proportions of MyHC I and 2a fibers and decrease of the ratio of MyHC 2b fiber.These results suggested that the compositions of myofiber types in skeletal muscle cells were influenced by the addition of CLA,which depended on the CLA dose and culture time.The effects of CLA on the composition of myofiber mainly were represented by increasing proportions of MyHC I and 2a fibers and decreasing the ratios of MyH 2b and 2x fibers, which could explain partially why CLA improved pork quality.更多还原