鸡脂肪组织和骨骼肌miRNA的筛选和表达研究
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摘要
microRNA(miRNA)是一类非编码的22 nt左右的小RNA,它广泛存在于各种生物体中,通过对靶mRNA的翻译抑制和降解对基因的表达起负性调节作用。miRNA参与了包括增殖、分化、凋亡、发育等很多生物过程。
本研究构建了4周龄肉鸡脂肪和骨骼肌中小分子RNA的cDNA文库,脂肪文库中筛选出44个已公布鸡miRNA,共16种,39个与其他物种同源miRNA,共5种;骨骼肌文库中筛选出81个已公布鸡miRNA,共27种,31个与其他物种同源miRNA,共4种。通过进一步分析,预测出11个脂肪中潜在新miRNA,共9种,7个骨骼肌中潜在新miRNA,共4种。通过已测出的各种miRNA序列在鸡基因组中的定位,找到5个miRNA基因簇,其中4个基因簇各含有2个miRNA基因,1个基因簇含有4个miRNA基因。在测出的miRNA序列中,通过对相同miRNA序列分析,发现其中一些相同miRNA含有不同的序列,即miRNA序列存在多样性。
采用生物信息学方法,运用miRNA靶基因预测软件miRanda和TargetScan对脂肪和骨骼肌中miRNA的靶基因进行预测,在脂肪的250个表达基因中预测出192个潜在靶基因,在骨骼肌的250个表达基因中预测出216个潜在靶基因。并对其中每个miRNA的潜在靶基因数和与每个潜在靶基因相互作用的miRNA数进行了统计。这些结果为进一步研究鸡脂肪和骨骼肌中miRNA的靶基因奠定了基础。
运用TaqMan实时荧光定量PCR技术,结合stem-loop法对文库中的10个miRNA进行鸡12个组织表达量的检测。结果显示,gga-miR-133a和gga-miR-1a在肌肉中特异表达,提示它们在鸡肌肉的生长发育中可能起重要作用;gga-miR-122在肝中表达量最高,在脂肪中有少量表达,提示它可能参与鸡脂肪酸代谢和脂肪生长发育;gga-miR-22、gga-miR-199、miR-23a和miR-145在心肌和肌胃中表达量高,而在骨骼肌中表达量却很少,提示它们在不同的肌肉类型中可能起不同的作用;gga-miR-126、gga-miR-200b和miR-191在骨骼肌和脂肪中表达量都很低,提示它们可能在这两个组织中并不发挥功能。
microRNAs(miRNAs) are a class of～22 nt small noncoding RNAs. They exist extensively in various organisms, negatively regulate gene expression by translational repression and degradation of target mRNAs. miRNAs are involved in many bioprocesses, including proliferation, differentiation, apoptosis and development.
In this study, we constructed the cDNA libraries of small RNAs from 4 week chicken’s adipose tissue and skeletal muscle. In the adipose library 44(16 varieties) were chicken miRNAs published, 39(5 varieties) were miRNAs homolog to other species. In the skeletal muscle library 81(27 varieties) were chicken miRNAs published, 31(4 varieties) were miRNAs homolog to other species. By further analysis, 11(9 varieties) potential new miRNAs were predicted in adipose, 7(4 varieties) potential new miRNAs were predicted in skeletal muscle. By location of various sequenced miRNAs in chicken genome, 5 miRNA gene clusters were found. Among the 5 miRNA gene clusters, 4 clusters have 2 miRNA genes respectively, 1 cluster has 4 miRNA genes. The sequence polymorphism was revealed by sequence alignment of the obtained miRNAs.
Based on the bioinformatics method, 192 potential target genes were predicted in 250 expression genes in adipose, 216 potential target genes were predicted in 250 expression genes in skeletal muscle by using miRNA target prediction software miRanda and TargetScan. The interaction between miRNAs and their target genes was analyzed. These results establish a basis for further research on miRNA target genes in chicken adipose and skeletal muscle.
The expression level of 10 miRNAs in 12 chicken tissues were detected using stem-loop RT and TaqMan real-time fluorescence quantitative PCR. gga-miR-133a and gga-miR-1a are expressed specifically in muscle. They may play an important role in growth and development of chicken muscle. gga-miR-122 has the highest expression level in liver and a low expression level in adipose. It may be involved in fatty acids metabolism and growth and development of chicken adipose. gga-miR-22, gga-miR-199, miR-23a and miR-145 has a high expression level in heart and muscular stomach and a very low expression level in skeletal muscle. They may play different roles in different type of muscle. gga-miR-126, gga-miR-200b and miR-191 has very low expression level in skeletal muscle and adipose. They may play no roles in the two tissues.
本研究构建了4周龄肉鸡脂肪和骨骼肌中小分子RNA的cDNA文库,脂肪文库中筛选出44个已公布鸡miRNA,共16种,39个与其他物种同源miRNA,共5种;骨骼肌文库中筛选出81个已公布鸡miRNA,共27种,31个与其他物种同源miRNA,共4种。通过进一步分析,预测出11个脂肪中潜在新miRNA,共9种,7个骨骼肌中潜在新miRNA,共4种。通过已测出的各种miRNA序列在鸡基因组中的定位,找到5个miRNA基因簇,其中4个基因簇各含有2个miRNA基因,1个基因簇含有4个miRNA基因。在测出的miRNA序列中,通过对相同miRNA序列分析,发现其中一些相同miRNA含有不同的序列,即miRNA序列存在多样性。
采用生物信息学方法,运用miRNA靶基因预测软件miRanda和TargetScan对脂肪和骨骼肌中miRNA的靶基因进行预测,在脂肪的250个表达基因中预测出192个潜在靶基因,在骨骼肌的250个表达基因中预测出216个潜在靶基因。并对其中每个miRNA的潜在靶基因数和与每个潜在靶基因相互作用的miRNA数进行了统计。这些结果为进一步研究鸡脂肪和骨骼肌中miRNA的靶基因奠定了基础。
运用TaqMan实时荧光定量PCR技术,结合stem-loop法对文库中的10个miRNA进行鸡12个组织表达量的检测。结果显示,gga-miR-133a和gga-miR-1a在肌肉中特异表达,提示它们在鸡肌肉的生长发育中可能起重要作用;gga-miR-122在肝中表达量最高,在脂肪中有少量表达,提示它可能参与鸡脂肪酸代谢和脂肪生长发育;gga-miR-22、gga-miR-199、miR-23a和miR-145在心肌和肌胃中表达量高,而在骨骼肌中表达量却很少,提示它们在不同的肌肉类型中可能起不同的作用;gga-miR-126、gga-miR-200b和miR-191在骨骼肌和脂肪中表达量都很低,提示它们可能在这两个组织中并不发挥功能。
microRNAs(miRNAs) are a class of～22 nt small noncoding RNAs. They exist extensively in various organisms, negatively regulate gene expression by translational repression and degradation of target mRNAs. miRNAs are involved in many bioprocesses, including proliferation, differentiation, apoptosis and development.
In this study, we constructed the cDNA libraries of small RNAs from 4 week chicken’s adipose tissue and skeletal muscle. In the adipose library 44(16 varieties) were chicken miRNAs published, 39(5 varieties) were miRNAs homolog to other species. In the skeletal muscle library 81(27 varieties) were chicken miRNAs published, 31(4 varieties) were miRNAs homolog to other species. By further analysis, 11(9 varieties) potential new miRNAs were predicted in adipose, 7(4 varieties) potential new miRNAs were predicted in skeletal muscle. By location of various sequenced miRNAs in chicken genome, 5 miRNA gene clusters were found. Among the 5 miRNA gene clusters, 4 clusters have 2 miRNA genes respectively, 1 cluster has 4 miRNA genes. The sequence polymorphism was revealed by sequence alignment of the obtained miRNAs.
Based on the bioinformatics method, 192 potential target genes were predicted in 250 expression genes in adipose, 216 potential target genes were predicted in 250 expression genes in skeletal muscle by using miRNA target prediction software miRanda and TargetScan. The interaction between miRNAs and their target genes was analyzed. These results establish a basis for further research on miRNA target genes in chicken adipose and skeletal muscle.
The expression level of 10 miRNAs in 12 chicken tissues were detected using stem-loop RT and TaqMan real-time fluorescence quantitative PCR. gga-miR-133a and gga-miR-1a are expressed specifically in muscle. They may play an important role in growth and development of chicken muscle. gga-miR-122 has the highest expression level in liver and a low expression level in adipose. It may be involved in fatty acids metabolism and growth and development of chicken adipose. gga-miR-22, gga-miR-199, miR-23a and miR-145 has a high expression level in heart and muscular stomach and a very low expression level in skeletal muscle. They may play different roles in different type of muscle. gga-miR-126, gga-miR-200b and miR-191 has very low expression level in skeletal muscle and adipose. They may play no roles in the two tissues.
引文
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