摘要
肌内脂肪(Intramuscular fat, IMF)是形成牛肉大理石花纹的物质基础,是影响牛肉嫩度和风味的一个主要因素。不同肌内脂肪含量的个体肌肉组织基因的表达模式存在差异；阉牛与公牛相比,肌内脂肪增加,肉的嫩度增加,提高了牛肉等级。随着人们对基因结构和功能的深入研究,这些差异的产生应是基因的差异表达的结果。因此,本试验以相同月龄、同一饲养条件下的延边黄牛背最长肌为材料,利用GeneFishing差异显示技术对肌内脂肪含量差异极显著的个体间以及阉牛和公牛背最长肌进行了差异表达基因的筛选、克隆和鉴定,取得了如下结果：
1、试验选取30头28月龄延边黄牛阉牛分别选取肌内脂肪含量最高和最低各3个个体组成两组,其平均肌内脂肪含量分别为17.58%±0.14%和5.16%±0.32%,组间差异极显著(P<0.01)。采用GeneFishing技术,分析了两组个体背最长肌组织差异表达基因。利用40对随机引物差异显示扩增下,共获得12条ESTs,通过NCBI比对发现其中8个为已知的ESTs,这些差异基因主要包括5’类：细胞骨架形成相关基因(Titin, ACTAl),细胞因子信号转导相关基因(ASB12),转录复合体相关基因(RPS14、RPS26和RPLPl),细胞能量代谢密切相关基因(COX4),未知功能蛋白Glutamyl tRNA Synthetase基因和4个未知基因。
2、从30头延边黄牛阉牛中选取肌内脂肪含量最高和最低的9个个体组成两组,肌内脂肪含量分别为13.26%±0.28%和4.65%±0.18%(P<0.05)‘。通过RT-PCR和实时定量PCR定性和定量检测个体基因表达水平。荧光定量PCR结果显示ASB12和Titin基因mRNA表达水平与肌内脂肪含量呈正相关关系；Glutamyl tRNA Synthetase, RPLPl和ACTAl基因mRNA表达水平与肌内脂肪含量呈负相关关系。
3、利用半定量RT-PCR技术对所获得的差异表达基因进行了不同组织(背最长肌、心脏、肝脏、脾脏、肺脏、肾脏、小肠和瘤胃)间的表达分析。
4、利用ORF Finder、CLUSTALW等软件对牛ACTA1、ASB12和Glutamyl tRNA Synthetase基因结构、所编码的蛋白质结构、功能、系统进化树等特征进行了预测和分析,并进行了系统进化树的构建。
5、利用比较基因组学和PCR技术,克隆了ACTA1、Titin、ASB12和Glutamyl tRNA Synthetase基因的部分基因组序列,并分析了这4个基因在延边黄牛群体中的多态性：(1)ACTA1,1598bp,GenBank登录号为HQ608159,在其上发现了2个SNPs位点,第193位A→G突变和第288位A→G突变。(2)Titin发现了3个SNP位点,分别为第45003位A→G突变,第70541位A→G突变和第70553位C→A突变。(3)ASBl 2发现了3个SNPs位点,分别为第712位T→C突变,第886位G→C突变和第1042位C→T突变。(4)Glutamyl tRNA Synthetase发现了3个SNP位点,第4734位G→A突变,第4845位A→G突变,1处插入突变。
6、利用PCR-RFLP和PCR-SSCP技术,对其中4个SNP位点在延边黄牛群体中进行了基因分型,并与生产性状进行了关联分析,结果表明：(1)牛ACTA1一Avr II-RFLP的不同基因型主要与体长、胸围、体重和平均日增重显著相关；(2)Glutamyl tRNA Synthetase基因G4734A突变在脂肪酸方面与亚油酸和棕榈油酸存在相关性,在氨基酸含量方面与甘氨酸、丙氨酸、谷氨酸和组氨酸存在显著相关(3)牛ASB12基因T712C突变在脂肪酸方面与棕榈油酸和肉豆蔻酸存在相关性,在氨基酸含量方面与赖氨酸、天冬氨酸、脯氨酸、亮氨酸和谷氨酸值存在显著相关；(4)牛Titin-Msp I-RFLP的不同基因型主要与L*24(亮度)、c*24(彩度)、棕榈酸、硬脂酸、油酸和肌内脂肪含量显著相关。
7、利用GeneFishing差异显示技术,对延边黄牛阉牛和公牛背最长肌差异表达基因进行了筛选。共获得10条ESTs,通过NCBI比对发现其中8条序列与已知序列高度同源,MYH7、NDUFS8和HSL基因在阉牛肌肉中表达上调,NADH脱氢酶亚单位6和ACTA1基因在阉牛肌肉中表达下调,2条为未知新基因。
8、选取其中5个差异表达基因NADH脱氢酶亚单位6、MYH7、HSL、NDUFS8和ACTA1基因进行荧光定量PCR的验证,结果均为差异表达,提示这些基因可能参与延边黄牛阉割前后肉质性状差异的重要调控基因。
9、利用ORF Finder、CLUSTALW等软件对牛MYH7、HSL、NDUFS8基因结构、所编码的蛋白质结构、功能、系统进化树等特征进行了预测和分析,并进行了系统进化树的构建。
Intramuscular fat(IMF) is the material basis for beef marbling and is one of major determinants of the tenderness and taste of beef. Gene expression patterns of muscle tissue are various in cattles with different cotent of intramuscular fat. Compared with bulls, steers have a more slowly growth rate, faster lipid accumulation, more intramuscular fat, tenderer muscle. Further understanding on gene structure and function has indicated that these differences should be the result of different expression of genes. In the present study, Longissimus muscles Yanbian yellow cattle under the same age and same raising condition were used as materials. Gene Fishing was used to filter, clone and identify the differentially expressed genes of longissimus muscles in cattles with different cotent of intramuscular fat and steers, bulls. The findings are as follows:
1. Longissimus muscle samples were collected from 30 cattles and evaluated for intramuscular fat content.30 Yanbian steers at the age of 28 months were selected and divided into two groups consisted of 3 samples with highest intramuscular fat content and 3 samples with lowest intramuscular fat content. The average intramuscular fat content of each group was 17.58%±0.14%and 5.16%±0.32%, and group difference was significant (P<0.01). GeneFishing was used to analyze the differentially expressed genes of two groups. Twelve Expressed Sequence ESTs were gained and sequenced. Eight out of twelve ESTs sequence were known genes. The differentially expressed genes included 5 categories: functional groups of myocinesimete(Titin, ACTA1), cytokine signal transduction(ASB12), transcription complex related genes (RPS14, RPS26 and RPLP1), cell energy metabolismand (COX4), protein of unknown function(Glutamyl tRNA Synthetase) gene and four unknown genes.
2. From 30 steers of Yanbian cattle selected two groups which was 9 individuals each of the highest and the lowest IMF content, and IMF content was separately 13.26%±0.28%and 4.65%±0.18%(P<0.05). By RT-PCR and real-time quantitative PCR detected the qualitative and quantitative expression level of individual genes. The results of quantitative PCR showed that the correlation between ASB12, mRNA expression levels of Titin gene and IMF content was positively; the correlation between Glutamyl tRNA Synthetase, RPLP1, mRNA expression levels of ACTA1 gene and IMF was negatively.
3. Obtained differentially expressed genes were performed in different tissues (heart, liver, spleen, lung, kidey, stomach, small intestine, muscle) by semi-quantitative RT-PCR analysis.
4. Using ORF Finder, CLUSTAL W and some other related software, we analyzed ACTA1, RPLP1, ASB12and Glutamyl tRNA Synthetase structure, protein structure and conserved motifs of these genes. In addition, the corresponding phylogenetic trees were constructed.
5. Partial genomic sequence of cattle ACTA1、Glutamyl tRNA Synthetase、ASB12 and Titin genes were obtained by Comparative genomics and PCR, and the polymorphism of the 4 genes was analyzed. The results were as follows:(1) Two SNPs were identified in ACTA1,1598bp, whose GenBank accession number is HQ608159. One is 193A→G mutation and the other is 288A→G. (2) Titin showed three SNPs. g. [45003A→G, 70541A→G,70553 C→A]. (3) ASB12 showed three SNPs. g. [712T→C, 886G→C,1C42 C→T]. (4) Glutamyl tRNA Synthetase showed three SNPs. g. [4734G→A,4858A→G]. and one is insertion mutation.
6. Using PCR-RFLP and PCR-SSCP,4 SNPs in Yanbian yellow cattles were detected and production traits were analyzed. The results showed:(1) There are significant correlation between ACTA1-Avr II-RFLP genotypes and body length, heart girth, average daily gain, and body weight; (2) G4734A gene mutation of Glutamyl tRNA Synthetase was significantly associated with linoleic acid, palmitic acid, glutamic acid, histidine, glycine and alanine; (3) T712C gene mutation of ASB12 was significantly associated with palmitic acid, myristic acid, lysine, aspartic acid, praline, leucine, and arginine; (4) The Titin-MspI-RFLP were significantly correlated with L*24(Brightness), c*24 (Saturation) palmitic acid, palmitic acid, oleic acid and IMF.
7. The gene expression were analyzed between steers and bulls by Gene Fishing. Ten expressed sequence ESTs were gained and sequenced. ACTA1 and NADH6 was confirmed as downregulation in steers, whereas MYH7, NDUFS8 and HSL was confirmed as upregulation in steers.
8. Real-time PCR was used to analyse the change of mRNA expression level of HSL, ACTA1,MYH7, NDUFS8 and NADH dehydrogenase subunit 6 gene between steers and bulls. The mRNA levels showed these genes were differentially expressed genes, HSL, ACTA1, MYH7, NDUFS8 and NADH dehydrogenase subunit 6 which were considered as the most important genes involved in meat quality differences before and after Yanbian yellow cattles were castrated.
9. ORF Finder, CLUSTALW and some other related software were used to analyze the gene structure the encoded protein structure, function, phylogenetic tree of HSL,MYH7 and NDUFS8, and phylogenetic trees were constructed.
引文
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