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【目的】研究龙眼miR172家族的进化特性及其在龙眼不同组织部位中的表达模式。【方法】采用生物信息学分析及实时荧光定量PCR的方法,对龙眼miR172家族5条前体(pre-miR172)序列及1条成熟体序列进行分析,并预测其前体二级结构,构建系统发育进化树,预测靶基因及分析不同组织部位的定量表达情况。【结果】对龙眼miR172家族5条前体序列进行多序列比对,发现miR172家族两端相对保守,中间区域则形成各自的特异性区域。对miR172家族5条前体及1条成熟体进行多序列比对,发现6条序列间存在1个约20个碱基的保守区域,推测该区域可能为miR172家族成熟体所在区域。Mfold软件预测结果显示,pre-miR172家族成员均能形成典型的茎环二级结构,其最小折叠自由能为-35.80~-54.45 kal·mol-1,较为稳定。系统发育进化树结果显示,龙眼pre-miR172家族成员与甜橙、毛果杨等植物的miR172亲缘关系较近。靶基因预测结果显示,龙眼miR172a的靶基因包括AP2、蝶呤型钼辅因子(molybdopterin cofactor)、谷氨酰-t RNA(glutamyl-t RNA)、假定蛋白(hypothetical protein)等。实时荧光定量PCR结果显示,miR172家族各成员在龙眼不同组织部位中的表达模式总体相似,均在叶芽和生殖生长阶段中大量表达,在营养生长阶段及果实中表达量则较低,但dlo-miR172-scaffold4293特异地在叶片中高表达。【结论】龙眼miR172家族在进化过程中保守性与特异性并存,且可能参与了龙眼部分器官的形成及发育过程。
【Objective】 The objective of this study was to investigate the evolutionary characteristics of longan miR172 family and its expression pattern in different tissues of longan. 【Method】 Bioinformatics analysis and real-time fluorescence quantitative PCR were used to analyze the sequence of pre-miR172 and one mature sequence of the miR172 family of longan, and to predict the secondary structure of precursor and construct the system Development of evolutionary tree, prediction of target genes and analysis of quantitative analysis of different parts of the organization. 【Result】 Multiple sequence alignment of the 5 precursor sequences of the miR172 family of longan showed that the miR172 family was relatively conserved at both ends and the middle region formed their specific regions. Multiple sequence alignment of the 5 precursors and one mature body of the miR172 family revealed one conserved region of about 20 bases between the six sequences, suggesting that this region may be the region where the mature miR172 family is located. The predicted results of Mfold software showed that the members of pre-miR172 could form typical secondary structure of stem-loop, and the minimum free energy of folding was -35.80 ~ -54.45 kal · mol-1, which was more stable. Phylogenetic tree showed that the members of longan pre-miR172 family were closely related to miR172 in plants such as sweet orange and Populus trichocarpa. The target gene prediction results showed that the target genes of longan miR172a include AP2, molybdopterin cofactor, glutamyl-t RNA, hypothetical protein and the like. Real-time PCR results showed that the expression patterns of miR172 family members in different tissues of longan were similar, both of them were highly expressed in leaf buds and reproductive growth stages, but lower in vegetative growth stages and fruits. However, dlo- miR172-scaffold4293 is highly expressed specifically in leaves. 【Conclusion】 The longan miR172 family coexist in both evolutionary and evolutionary stages, and may be involved in the formation and development of some longan organs.