A combined small RNA and transcriptome sequencing analysis illuminates the response of resistance to brown planthopper in rice [miRNA-seq]

Here, a combined small RNA and transcriptome sequencing analysis was performed on the BPH6-transgenic and Nipponbare plants sheath samples at the different feeding stage by BPH, and differentially expressed microRNAs (DEMs) and genes (DEGs) were identified and further analyzed for a possible involvement in BPH6 mediated resistance mechanism. A total of 217 DEMs and 7,874 DEGs were identified in the BPH6-transgenic and Nipponbare plants after BPH feeding. At the miRNA level, 29 miRNAs, including the members of miR160, miR166, miR169, miR1861, miR319 and miR390 family and other miRNAs, appeared opposite expression at early or late feeding stages between the two varieties, and 9 miRNAs specially expressed in the BPH6-transgenic plans, suggesting these miRNAs might play an important role in BPH6-mediated resistance to BPH. For the transcriptome, 949 DEGs appeared opposite expression at early or late feeding stages of two rice genotypes were identified and enriched in metabolic process, cellular development, cell wall organization, cellular component movement and hormone transport, and in involved in certain kinds of primary and secondary metabolite synthesis by GO and KEGG enrichment analysis. 24 genes were further selected to be considered as BPH resistance related gene candidates. Finally, integrated analysis of DEMs and DEGs showed that 34 miRNAs corresponding to 42 target genes were selected as BPH resistance related miRNA-mRNA interaction candidates. Overall design: 18 libraries were constructed, amplified and sequenced with the BPH6-transgenic and Nipponbare sheath before and after BPH feeding.

本研究对转BPH6基因水稻与日本晴（Nipponbare）植株的叶鞘样本，在褐飞虱（Brown Planthopper, BPH）不同取食时段开展小RNA与转录组联合测序分析，鉴定出差异表达microRNA（DEMs）与差异表达基因（DEGs），并进一步分析其是否参与BPH6介导的抗虫机制。经褐飞虱取食后，在转BPH6基因水稻与日本晴中共鉴定出217个DEMs与7,874个DEGs。在miRNA层面，两类水稻品种在取食早期或晚期共存在29个miRNA呈现相反的表达模式，其中包含miR160、miR166、miR169、miR1861、miR319及miR390家族成员在内的多个miRNA；另有9个miRNA仅在转BPH6基因水稻中特异性表达，提示这些miRNA可能在BPH6介导的抗褐飞虱过程中发挥重要作用。针对转录组数据，经基因本体（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析后，鉴定出两类水稻基因型在取食早、晚期共949个DEGs呈现相反表达模式，这些DEGs显著富集于代谢过程、细胞发育、细胞壁组织、细胞组分运动及激素转运等生物学过程，同时参与多种初级与次级代谢产物的合成。最终筛选出24个基因作为褐飞虱抗性相关候选基因。最后，通过DEMs与DEGs的联合分析，筛选出34个靶向42个靶基因的miRNA，作为褐飞虱抗性相关的miRNA-mRNA互作候选对。实验整体设计：针对褐飞虱取食前与取食后的转BPH6基因水稻与日本晴的叶鞘样本，共构建18个文库并进行扩增与测序。