角瓜与南方根结线虫非亲和互作相关MicroRNAs的鉴定

Identification of miRNAs Associated with Incompatible Interaction Between Cucumis metuliferus and Meloidogyne incognita

为鉴定角瓜与南方根结线虫非亲和互作中的植物源MicroRNAs(miRNAs),预测miRNAs的靶基因,解析miRNAs的表达模式。构建角瓜与南方根结线虫亲和、非亲和互作的小RNA(sRNA)文库,通过Solexa测序和生物信息学分析筛选鉴定角瓜与南方根结线虫非亲和互作相关的miRNAs,利用PsRNATarget软件预测miRNAs的靶基因,通过实时荧光定量PCR(qRT-PCR)对测序获得的miRNAs进行表达验证。结果表明,18份样品测序获得的Unique reads数量在808262~3926308条,长度24 nt的sRNA丰度最高,miRNAs占sRNA的0.3%~0.5%,包含565个已知miRNAs,归属75个miRNAs家族,其中miR156包含的家族成员数目最多。26条差异表达的miRNAs响应南方根结线虫胁迫的表达模式分为4种类型,其中10条显著差异表达的角瓜miRNAs与南方根结线虫非亲和互作密切相关。miRNAs靶基因预测显示,一个miRNA可调控多个靶基因,同一miRNA的靶基因同源性较高,miRNAs靶基因功能主要涉及转录激活,参与植物生长发育和调控,以及编码信号传导和细胞代谢相关蛋白等。这些研究结果为探明角瓜与根结线虫非亲和互作的miRNAs调控机理奠定了基础。

The objective of the present study was to identify the plant-derived MicroRNAs(miRNAs),predict the potential target genes and analyze the expression patterns of miRNAs.Small RNA(sRNA)libraries with compatible and incompatible interactions were constructed,sRNAs libraries were then subjected to Solexa sequencing followed by bioinformatics analysis,and miRNAs related to incompatible interaction between C.metuliferus and M.incognita were screened and identified.The target genes of miRNAs were predicted by PsRNATarget software,and the expression of miRNAs was verified by Real-time quantitative expression(qRT-PCR).The results showed that the number of unique reads obtained from 18 samples ranged from 808262 to 3926308,the highest abundance was observed in 24 nt sRNA in length.The miRNAs accounted for only 0.3%-0.5% of all sRNA,including 565 known miRNAs,belonging to 75 miRNAs families,of which miR156 contained the largest number of family members.The expression patterns of 26 differentially expressed miRNAs in response to M.incognita infection can be divided into four types,of which 10 miRNAs were significantly differentially expressed and were closely related to incompatible interaction between C.metuliferus with M.incognita.The prediction of miRNAs target genes showed that one miRNA could regulate multiple target genes,and the target genes of the same miRNA had high homology.The functions of miRNAs target genes are mainly involved in transcriptional activation,participating in plant growth and development and regulation,and encoding proteins related to signal transduction and cell metabolism.These results laid a foundation for exploring the regulatory mechanism of miRNAs in the incompatible interaction between Cucumis metuliferus and Meloidogyne incognita.