基于基因组的绒毛状烟草和林烟草microRNA及其靶基因分析

【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。

The Genome-Wide Analysis of MicroRNAs and Their Target Genes in Nicotiana tomentosiformis and Nicotiana sylvestris

【Objective】The objective of this study is to fill the gaps in miRNA-related fields of Nicotiana tomentosiformis and N. sylvestris research as quickly as possible, and to reveal the growth and development regulation mechanism in N. tobacum, 【Method】the microRNAs and their target genes of N. tomentosiformis and Nicotiana sylvestris were genome-wide predicted and analyzed. miRNAs were predicted by the method of homologous alignment and secondary structure characteristics of pre-miRNA: reference sequence in sequence alignment of N. tomentosiformis and N. sylvestris were allowed 1-2 mispairings; secondary structure of miRNA was classic stem loop structure, maximum value of MEF was -25, minimum value of MEFI was 0.85, the predicted miRNA and the same family miRNA located on the same arm of hairpin structure; E-values of encoding protein sequence less than or equal to 1e-6 were eliminated. 【Result】 A total 162 miRNAs belonging to 39 families were identified in N. tomentosiformis, including 14 pairs of sense and antisense strand miRNAs and 5 gene clusters. A total 169 miRNAs belonging to 40 families were identified in N. sylvestris, including 13 pairs of sense and antisense strand miRNAs and 3 gene clusters. In high degree of miRNA conservative families, members of the distribution and membership were near in 2 wild tobaccos. While in a relatively low degree of conservative families, members of 2 wild tobaccos differed obviously. Nine families like miR5021, miR5203 and so on, got members in N. tomentosiformis. Ten families like miR1446, miR1509 and so on, got members in N. sylvestris. Antisense miRNA and their sense partners from 2 wild tobaccos differed from 1 to 4 bases, these differences location presented preferences in different families, and the preferences were similar in 2 wild tobaccos: 9th, 12th, 13th base in miR164 family, 1st, 21st base in miR172 family, 2nd, 17th base in miR396 family, 15th, 20th base in miR399 family. Gene cluster of 2 wild tobaccos consisted of miR156 family and miR169 family, distance of pre-clusters was less than 350nt. miR6019/miR6020 gene clusters were found in N. tomentosiformis for the first time. Unigene of N. tabacum was used as target genes. In N. tomentosiformis, 749 target genes of 122 miRNAs were identified. With duplicate genes eliminated, 206 non-redundant target genes were identified, in which, 89 target genes (43%) got GO annotations. In N. sylvestris, 117 target genes of 650 miRNAs were identified. With duplicate genes eliminated, 169 non-redundant target genes were identified, in which, 78 target genes (46%) got GO annotations. In terms of molecular function, most of the target genes have binding activity. In the process of biology, target genes mainly involved in the development process, reproductive process, multicellular organ development process, stress response and so on. 【Conclusion】 In N. sylvestris, there are more target genes control development and multicellular development, while in N. tomentosiformis, there are more target genes control stimulus and press reply.