为了筛选秦川牛公牛和阉牛肌肉组织差异表达的基因, 探讨二者肉质差异的分子生物学机理, 文章利用Affymetrix公司生产的牛基因组芯片技术, 分别检测了3头36月龄秦川牛公牛与阉牛背最长肌肌肉组织的mRNA 表达水平变化; 运用Significance Analysis of Microarrays (SAM) 法对秦川牛公牛和阉牛基因表达谱进行了差异分析; 并通过分子注释系统平台(MAS 2.0)对差异表达基因进行了功能富集类分析和调控通路分析; 最后应用实时定量PCR 技术对部分差异表达基因进行了验证。基因表达谱分析结果显示, 在36月龄秦川牛的肌肉组织中, 共检测到约11 000个探针, 代表大约10 000个基因。共筛选出差异表达基因143条, 主要涉及胶原纤维的组织和合成、细胞粘附、细胞生长调控、泛素介导的蛋白分解代谢和横纹肌收缩等生物学过程; 在分子注释系统数据库中注释到的显著调控通路为细胞外基质受体反应、细胞通讯、焦点粘连和紧密连接等; 所验证的差异表达基因的荧光定量 PCR结果与芯片结果基本一致。结合已有的文献报道, 文章初步认为ECM受体反应、细胞通讯、焦点粘连、紧密接头等调控通路及COL3A1、COL1A1、COL1A2、SPP1、FBN1、MMP2、ECM1、MYH3、MYH8、S100A4、ASPN、CFD等基因可能是参与调控秦川牛阉割前后肉质性状差异的重要调控通路和基因。此外, 还筛选出一些尚未在GenBank上登陆的序列, 推测可能是未知的新基因, 它们在牛肉质代谢过程中的作用还需进一步的研究证明。
Bovine Genome Array was used to construct gene expression profile to screen differentially expressed genes of the Longuissimus dorsi muscle (LDM) tissue between intact male and castrated Qinchuan cattle and investigate the molecular mechanism related to meat quality differences between the two groups. Significance Analysis of Microarray (SAM) methods was used to identify the differentially expressed genes. Go (Gene Ontology) and the pathway analyses were con-ducted on differentially expressed genes using a free web-based Molecular Annotation System 2.0 (MAS 2.0). About 11 000 probe sets represented about 10 000 genes were detected in LDM of 36 months old Qinchuan cattle. A total of 143 genes were identified to be differentially expressed genes. They were mainly involved in collagen fibril organization and synthesis, regulation of cell growth and development, ubiquitin-dependent protein catabolism, and striated muscle contrac-tion etc. The enriched pathways mainly included ECM-receptor interaction, cell communication, and focal adhesion etc. Subsequently, real-time PCR was performed to validate eight differentially expressed genes screened out by the microarray approach and sufficient consistency was observed between the two methods. According to our study and published papers, the regulated pathways including ECM-receptor interaction, cell communication, focal adhesion, tight junction and genes including COL3A1, COL1A1, COL1A2, SPP1, FBN1, MMP2, ECM1, MYH3, MYH8, S100A4, ASPN, CFD etc were con-sidered as the most important parthways and genes involved in meat quality differences between males and castrated Qin-chuan cattle. Moreover, some genes had no annotation in GenBank were screened out, which were presumed to be unknown new genes. The roles that they may play in muscle metabolism need to be clarified in future.
[1] 符金录, 刘汉卿. 秦川牛肉用性能开发前景及对策. 黄牛杂志, 2005, 31(5): 39-41.
[2] GB 5797-2003, 秦川牛. 北京: 中国标准出版社, 2003.
[3] Li C, Wong WH. Model-based analysis of oligonucleotide arrays: Expression index computation and outlier detec-tion. Proc Natl Acad Sci USA, 2001, 98(1): 31-36.
[4] Li C, Wong WH. Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error ap-plication. Genome Biol, 2001, 2(8): research0032.1-0032.11.
[5] Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA, 2001, 98(9): 5116-5121.
[6] Qin LX, Beyer RP, Hudson FN, Linford NJ, Morris DE, Kerr KF. Evaluation of methods for oligonucleotide array data via quantitative real-time PCR. BMC Bioinformatics, 2006, 7: 23-34.
[7] Field, RA. Effect of castration on meat quality and quan-tity. J Anim Sci, 1971, 32(5): 849-858.
[8] Ende C, Gebhardt R. Inhibition of matrix metallopro-teinase-2 and -9 activities by selected flavonoids. Planta Med, 2004, 70(10): 1006-1008.
[9] Dietz HC, Pyeritz RE. Mutations in the human gene for fi-brillin-1 (FBN1) in the Marfan syndrome and related disorders. Hum Mol Genet, 1995, 4(Review 1): 1799-1809.
[10] Okamoto O, Suzuki Y, Kimura, S, Shinkai H. Extracellular matrix 22-kDa protein interacts with decorin core protein and is expressed in cutaneous fibrosis. J Biochem (Tokyo), 1996, 119(1): 106-114.
[11] Forbes EG, Cronshaw AD, MacBeath JRE, Hulmes DJS. Tyrosine-rich acidic matrix protein (TRAMP) is a tyro-sine-sulphated and widely distributed protein of the extracel-lular matrix. FEBS Lett, 1994, 351(3): 433-436.
[12] MacBeath JR, Shackleton DR, Hulmes DJ. Tyrosine-rich acidic matrix protein (TRAMP) accelerates collagen fibril formation in vitro. J Biol Chem, 1993, 268(26): 19826-19832.
[13] Oberhauser AF, Marszalek PE, Erickson HP, Fernandez JM. The molecular elasticity of the extracellular matrix protein tenascin. Nature, 1998, 393(6681): 181-185.
[14] Riley RR, Savell JW, Murphey CE, Smith GC, Stiffler DM, Cross HR. Palatability of beef from steer and young bull car-casses as influenced by electrical stimulation, subcutaneous fat thickness and marbling. J Anim Sci, 1983, 56(3): 592-597.
[15] Vanderwert W, McKeith FK, Bechtel PJ, Berger LL. Influ-ence of zeranol implants and electrical stimulation on the pal-atability traits of five muscles in Angus and Limousin bulls and steers. J Anim Sci, 1986, 63(1): 114-120.
[16] Lepetit J. Collagen contribution to meat toughness: Theo-retical aspects. Meat Sci, 2008, 80(4): 960-967.
[17] Lepetit J. A theoretical approach of the relationships be-tween collagen content, collagen cross-links and meat tenderness. Meat Sci, 2007, 76(1): 147-159.
[18] Cross HR, Schanbacher BD Crouse JD. Sex, age and breed related changes in bovine testosterone and intramuscular collagen. Meat Sci, 1984, 10(3): 187-195.
[19] McCormick RJ. The flexibility of the collagen compart-ment of muscle. Meat Sci, 1994, 36(1-2): 79-91.
[20] Crouse JD, Cross HR, Seideman SC. Effects of sex condi-tion, genotype, diet and carcass electrical stimulation on the collagen content and palatability of two bovine mus-cles. J Anim Sci, 1985, 60(5): 1228?1234. [21] Gerrard DE, Jones SJ, Aberle ED, Lemenager RP, Diekman MA, Judge MD. Collagen stability, testosterone secretion and meat tenderness in growing bulls and steers. J Anim Sci, 1987, 65(5): 1236–1242.
[22] Fritsche S, Steinhart H. Differences in natural steroid hormone patterns of beef from bulls and steers. J Anim Sci, 1998, 76 (6): 1621-1625.
[23] Boccard RL, Naudé RT, Cronje DE, Smit MC, Venter HJ, Rossouw EJ. The influence of age, sex and breed of cattle on their muscle characteristics. Meat Sci, 1979, 3(4): 261-280.
[24] Warrick HM, Spudich JA. Myosin structure and func
