基于硝态氮或铵态氮条件下杨树根尖miRNAs特征分析

miRNAs Analysis of Poplar Root Tips Treated with Nitrate- or Ammonium-Nitrogen

目的通过小RNAs高通量测序技术,在硝态氮或铵态氮处理下,对杨树根尖差异表达miRNAs及其靶基因调控机制进行了研究,分析和描述了杨树根尖生长和形态特征的规律,为进一步筛选和鉴定硝态氮或铵态氮吸收利用效率高的林木新品种提供依据。方法以灰杨水培苗根尖为研究对象,应用高通量测序技术进行小RNA文库构建,获得miRNAs表达谱,鉴定差异表达miRNAs,同时利用降解组测序技术鉴定miRNAs靶基因,并对差异表达靶基因进行功能注释以及代谢通路富集等分析。最后通过联合分析,阐明不同氮形态处理下,灰杨根尖miRNA-靶基因调控网络。结果从2种不同氮形态处理杨树根尖miRNAs文库中鉴定出523个已知miRNAs和42个新miRNAs,其中,有96个miRNAs显著差异表达。与硝态氮处理相比,铵态氮处理下有44个miRNAs上调表达,52个下调表达,其中,vvi-MIR399d-p3_1ss13GA上调倍数最大,新的miRNA PC-5p-35885_222下调最显著。对miRNAs靶基因进行KEGG通路富集分析表明,部分显著差异表达靶基因参与杨树根尖氮响应,从而影响杨树根尖生长发育和形态特征。利用RT-qPCR对7组随机挑选的miRNA-靶基因进行验证,结果与测序数据一致,说明测序结果可靠。结论miRNAs及其靶基因在不同氮形态处理后对杨树根尖氮响应起着重要的调控作用,从而使硝态氮处理下的灰杨根长比铵态氮处理的几乎长1倍。

Objective To screen the differentially expressed miRNAs from root tips of poplar through small RNAs high-throughput sequencing technology,to study the regulation mechanism of miRNAs and their target genes related to nitrate or ammonium responses and try to identify and explain the morphological characteristics of poplar root tips growth and development.Method Using root tips of poplar as the research object,the high-throughput sequencing technology for small RNA library construction was applied to reveal the genetic differences of nitrogen forms in miRNA level,and to identify the differentially expressed miRNAs.Meanwhile,degradation sequencing technology was used to identify the miRNA target genes,and the functional annotation,clustering and metabolic pathway enrichment of the differentially expressed target genes were analyzed.Through conjoint analysis,the regulation network of miRNA-target pairs in the root tips of poplar was clarified under different nitrogen forms treatments.Result 523 known and 42 novel miRNAs were identified from miRNA libraries.A total of 96 miRNAs with significantly different expression were identified.Under ammonium treatment,44 miRNAs were upregulated and 52 downregulated.Among them,the upregulation of vvi-MIR399d-p3_1ss13GA and the downregulation of novel miRNA PC-5p-35885_222 were the most significant.The KEGG pathway analysis of differentially expressed target genes of miRNAs in the libraries showed that some significantly different expression target genes were involved in nitrogen response pathway.Real time fluorescence quantitative PCR confirmed 7 randomly selected miRNA-target pairs,and the results were consistent with the sequencing data.Conclusion miRNAs and their target genes may play important roles in nitrogen response of poplar root tips after treatments with different forms of nitrogen,thus the root length under nitrate-nitrogen treatment is almost twice that under ammonium-nitrogen treatment.