2024年3月29日 星期五
马蔺根系响应 Cd 胁迫的 miRNA 高通量测序分析
High throughput sequencing analysis on miRNA in root of Iris lactea var. chinensis response to Cd stress
2016年 第25卷 第3期 页码[1-11]    下载全文[1.2MB]  
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 100  μmol· L-1 Cd 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。

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% and 80. 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, in which, 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 up- regulated, 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.

关键词马蔺; Cd 胁迫; miRNA; 靶基因; 高通量测序; 差异表达
Key wordsIris lactea var. chinensis (Fisch.) Koidz.; Cd stress; miRNA; target gene; high throughput sequencing; differential expression
作者刘凉琴1, 宋爱萍1, 张永侠2, 原海燕2, 黄苏珍2, 刘兆磊1, 顾春笋2
所在单位1. 南京农业大学园艺学院, 江苏 南京 210095; 2. 江苏省中国科学院植物研究所(南京中山植物园), 江苏 南京 210014
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基金项目国家自然科学基金资助项目(31301807; 31300436); 江苏省自然科学基金资助项目(BK20130734)