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【目的】填补绒毛状烟草(Nicotiana tomentosiformis)和林烟草(Nicotiana sylvestris)在miRNA相关领域的研究空白,揭示普通烟草(Nicotiana tabacum)的生长发育调控机理。【方法】在绒毛状烟草和林烟草全基因组中预测并分析miRNA及其靶基因,通过同源比对及miRNA前体二级结构特征进行预测:参考序列在绒毛状烟草和林烟草基因组的比对中,允许最多1—2个错配;miRNA二级结构为经典茎环结构,其中MEF最大值为-25,MEFI最小值为0.85,预测的miRNA与已知的同一家族的miRNA位于发夹结构的同一条臂上;去除E值小于等于1e-6的编码蛋白的序列。【结果】在绒毛状烟草中得到39个家族的162条miRNA,包括14对正/反义miRNA和5个基因簇。在林烟草中得到40个家族的169条miRNA,包括13对正/反义miRNA和3个基因簇。2个野生烟草在保守度高的miRNA家族中,其成员分布相似,且成员数相近。在保守度相对较低的家族中,2个野生烟草其成员分布差异较为明显,其中,miR5021、miR5203等9个家族仅在绒毛状烟草中有成员,miR1446、miR1509等10个家族仅在林烟草中有成员。2种野生烟草的正义miRNA与反义miRNA都有着1—4个碱基差异,这些差异位点在不同的家族中呈现出偏好性,而且在2种野生烟草中偏好性相似:miR164家族的9、12、13个碱基处,miR172家族的1、21个碱基处,miR396家族的2、17个碱基处,miR399家族的15、20个碱基处。2种野生烟草的基因簇主要是由miR156、miR169家族组成,其前体的间距小于350 nt,同时在绒毛状烟草中首次发现miR6019/miR6020基因簇。以普通烟草的unigene数据库作为靶基因集进行预测与分析,在绒毛状烟草122条miRNA中得到749个靶基因,去掉重复基因得到非冗余靶基因206条,其中89条(43%)得到GO功能注释;在林烟草中117条miRNA得到650个靶基因,去掉重复基因得到非冗余靶基因169条,其中78条(46%)得到GO功能注释。在分子功能方面,大多数靶基因具有结合等活性。在生物学过程中,靶基因主要参与了发育过程、生殖过程、多细胞器官发育过程、胁迫应答过程等。【结论】控制发育和多细胞器官发育过程的靶基因数方面以林烟草居多,而胁迫应答的靶基因数以绒毛状烟草较多。
【Objective】 The objective of this study is to fill the gaps in the field of miRNA related to Nicotiana tomentosiformis and Nicotiana sylvestris, and to reveal the regulatory mechanism of growth and development of Nicotiana tabacum. 【Method】 The miRNAs and their target genes were predicted and analyzed in the whole genome of V. villosa and N. tabacum and predicted by homology alignment and miRNA precursor secondary structure features. Which allows for a maximum of 1-2 mismatches. The miRNA secondary structure is a classical stem-loop structure with a maximum of MEF of -25 and a minimum of MEFI of 0.85. The predicted miRNA is associated with a known miRNA of the same family in the hairpin Structure of the same arm; remove E value less than or equal to 1e-6 encoded protein sequence. 【Result】 162 miRNAs from 39 families were obtained in villous tobacco, including 14 pairs of positive / negative sense miRNAs and 5 gene clusters. 169 miRNAs from 40 families were obtained from N. tabacum, including 13 pairs of positive / negative miRNAs and 3 gene clusters. Two wild tobacco miRNA families with high conservatism, its members are similar in distribution, and the number of members is similar. Among the families with relatively low conservatism, the distributions of the two wild tobacco species were more obvious. Among them, 9 families such as miR5021 and miR5203 only had members in the villus, while 10 families such as miR1446 and miR1509 were only found in the forest tobacco There are members. The two miRNAs showed 1-4 bases difference between sense miRNA and antisense miRNA, which showed preference in different families, and had similar preference in two wild tobaccos: miR164 family 9 , 12,13 bases, 1,21 bases of the miR172 family, 2,17 bases of the miR396 family, and 15,20 bases of the miR399 family. The genomes of two wild tobacco species were mainly composed of miR156 and miR169 families with a distance of less than 350 nt. The miR6019 / miR6020 gene cluster was first found in the villus. In order to predict and analyze the unigene database of tobacco, 749 target genes were obtained from 122 miRNAs of villus, 206 repeats were obtained without repeats, of which 89 (43%) got GO Functional annotation. Among 117 tobacco miRNAs, 650 target genes were obtained, and 169 repeats of the target genes were deleted, of which 78 (46%) were GO functional annotations. In molecular function, most of the target genes have binding activity. In the biological process, the target gene is mainly involved in the developmental process, the reproductive process, the development of multicellular organs, the stress response process and so on. 【Conclusion】 Most of the target genes controlling development and the development of multicellular organism are mostly tobacco, while the number of target genes responding to stress is more in the form of villous tobacco.