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为了探求水牛泌乳和非泌乳期关键调控差异表达miRNAs及其表达模式,试验结合Solexa高通量测序和生物信息学分析结果筛选出18个水牛乳腺组织在泌乳期和非泌乳期差异表达的miRNAs,设计、筛选可行的miRNAs反转录及实时荧光定量PCR引物,进行实时荧光定量PCR验证差异表达miRNAs的表达模式。结果发现,miRNA的差异表达模式与测序结果基本一致。泌乳期miRNA-103、miRNA-125a、miRNA-30a-5p和miRNA-148a的表达量均显著高于非泌乳期(P<0.05);miRNA-29a在非泌乳期表达量显著高于泌乳期(P<0.05);miRNA-141、miRNA-125b和miRNA-497在泌乳期表达量均高于非泌乳期,但差异不显著(P>0.05);低丰度的miRNA-490和miRNA-592在泌乳期表达量均显著高于非泌乳期(P<0.05)。Novel-123b在泌乳期和非泌乳期差异不显著(P>0.05),Novel-57在泌乳期比非泌乳期高29.79倍(P<0.01)。本试验结果为后续水牛乳腺研究提供了16个miRNA的可用特异性反转录引物和实时荧光定量PCR引物,miRNA-103、miRNA-125a、miRNA-30a-5p、miRNA-148a、miRNA-29a和Novel-57 6个水牛差异表达miRNAs值得进一步研究。
In order to explore differentially expressed miRNAs and their expression patterns in buffalos and non-lactating buffaloes, the miRNAs differentially expressed in lactating and non-lactating stages of 18 buffaloes were screened by Solexa high-throughput sequencing and bioinformatics analysis. Design and screening of feasible miRNAs reverse transcription and real-time PCR primers, real-time fluorescence quantitative PCR validation differentially expressed miRNAs expression patterns. The results showed that miRNA expression patterns and sequencing results are basically the same. The expression of miRNA-103, miRNA-125a, miRNA-30a-5p and miRNA-148a during lactation were significantly higher than those during non-lactation (P <0.05). The expression of miRNA-29a during lactation was significantly higher than that during lactation P <0.05). The expression of miRNA-141, miRNA-125b and miRNA-497 in lactation was higher than that in non-lactation, but the difference was insignificant (P> 0.05) The expression of lactation was significantly higher than non-lactation (P <0.05). Novel-123b had no significant difference between lactation and non-lactation (P> 0.05), Novel-57 was 29.79 times higher than non-lactation during lactation (P <0.01). The results of the present study provided 16 specific miRNA reverse transcription primers and real-time PCR primers for subsequent buffalo mammary research. The miRNA-103, miRNA-125a, miRNA-30a-5p, miRNA-148a, miRNA- Novel-57 Six buffalo differentially expressed miRNAs deserve further study.