【摘要】 为了解Cd胁迫下马蔺〔Iris lactea var.chinensis(Fisch.)Koidz.〕根系miRNA的表达模式,采用高通量测序法对100μmol·L-1Cd胁迫0(CK)和24 h(Cd)后马蔺根系的sRNA文库(分别为CK和Cd文库)进行分析,筛选出显著差异表达的miRNA,并对这些miRNA的靶基因功能进行预测;在此基础上,采用qRT-PCR技术对部分miRNA及其靶基因的表达模式进行验证。结果表明:在CK和Cd文库中,未注释的sRNA序列较多,分别占各自sRNA特异序列总数的86.4%和80.5%;在已注释的sRNA序列中,miRNA所占比例最低(分别为0.3%和0.5%),而rRNA所占比例最高(分别为9.4%和11.8%);2个文库中的sRNA长度主要为21~24 nt,且均以21 nt为最多。从Cd胁迫下马蔺根系sRNA中共筛选出32个显著差异表达的miRNA,其中20个miRNA表达量下调(分别属于miR165、miR166、miR167、miR168、miR390和miR396家族),12个miRNA表达量上调。功能预测结果表明:这些miRNA靶基因的功能主要集中在生物学过程、细胞组分和分子功能3个方面;而从KEGG通路富集分析看,富集在核糖体、氨基酸生物合成和碳代谢3个通路上的差异表达miRNA的靶基因数分别为122、88和82个。qRT-PCR验证结果表明:在CK和Cd文库间,11个差异表达miRNA及8个靶基因的相对表达量均有显著差异(P<0.05);其中,11个miRNA相对表达量的上调和下调趋势与上述筛选结果一致,并且miRNA相对表达量上调时,其靶基因的相对表达量下调,反之亦然,说明Cd胁迫条件下马蔺根系的miRNA负调控其靶基因的表达,并且这些靶基因主要参与编码转录因子、HD-ZIP蛋白和信号蛋白等过程。更多还原

【Abstract】 In order to understand the expression pattern of miRNA in root of Iris lactea var. chinensis( Fisch.) Koidz. under Cd stress,sRNA libraries( which is CK and Cd libraries,respectively) from root of I. lactea var. chinensis after stressed by 100 μmol·L- 1Cd for 0( CK) and 24 h( Cd) were analyzed by method of high throughput sequencing analysis. Also,miRNA with significantly differential expression was screened,and functions of target genes of these miRNA were predicted. On this basis,expression pattern of some miRNA and their target genes were verified by qRT-PCR technology. The results show that in CK and Cd libraries,there are more unannotation sRNA sequences,accounting for 86. 4% and80. 5% of total number of their own sRNA unique reads, respectively. Among annotation sRNA sequences,percentage of miRNA is the lowest with a value of 0. 3% and 0. 5%,respectively,while that of rRNA is the highest with a value of 9. 4% and 11. 8%,respectively. Length of sRNA in the two libraries is mainly 21- 24 nt,and the most of them is 21 nt. Thirty-two miRNA with significantly differential expression are screened from sRNA in root of I. lactea var. chinensis under Cd stress,inwhich,expression of twenty miRNA is down-regulated( belonging to miR165, miR166, miR167,miR168,miR390 and miR396 families,respectively),that of twelve miRNA is up-regulated. The result of function prediction indicates that functions of target genes of these miRNA are mainly concentrated in three aspects,i. e. biological process,cellular component and molecular function,while from KEGG pathway enrichment analysis,number of target gene of differential expression miRNA enriching in three pathways of ribosome, biosynthesis of amino acids and carbon metabolism is 122, 88 and 82,respectively. Verification result of qRT-PCR shows that between CK and Cd libaries, there are significantly differences in relative expression of eleven miRNA with differential expression and eight target genes( P < 0. 05). In which,up-regulated and down-regulated trends of relative expression of eleven miRNA are consistent with above screening result,and when relative expression of miRNA is upregulated,that of its target gene is down-regulated,and vice versa,meaning that under Cd stress condition,miRNA from root of I. lactea var. chinensis negatively regulates its target gene expression,and these target genes mainly are involved in processes of coding transcription factor,HD-ZIP protein and signaling protein,etc.更多还原