lncRNA调控下冬小麦PPP途径抗寒应答的分子机制

Dongnongdongmai 1(dn 1) was the first freezing-tolerant(resistant to low temperature of -30℃)winter wheat that can live through the winter in extreme frigid region in Heilongjiang Province. miRNAs targets was regulated by miRNA, then initiated the cold resistant response. lncRNA (long noncoding RNA) competitivly binded miRNA and coregulated the expression level of miRNA targets.Our previous study indicated that the key enzyme genes in pentose phosphate pathway could response to cold stress and up-expressed. The expression level of miRNA was also affected by the expression level of some noncoding RNA, such as lncRNA. So the cold resistance of winter wheat was affected, but the mechanism was not cleared. dn1 was used as materials, the rhizomes were sampled under natural cooling condition in fields. Using high throughput sequencing, bioinformatics analysis method and modern molecular biology technology，the relationship between miRNA and mRNA was verified from DNA and protein level, the members of ceRNA (lncRNA-miRNA-target mRNA) were determined and their function was identified, the study results were onjointly analyzed and control network diagram of ceRNA was drawn. After The mutual regulation mechanism under cold stress of lncRNA-miRAN-target mRNA is comprehensively analyzed, in winter wheat, it will be revealed that the molecular regulation mechanism of cold resistense response of pentose phosphate pathway by lncRNA. Our project is featured by researching on winter wheat in extreme frigid region. The results could supply new ideas for cold-resistance molecular breeding and cultivation, and rich cold-resistance genetic resources.

东农冬麦1号(dn1)是我国首例黑龙江省高寒区越冬强抗寒(可耐-30℃低温)冬小麦(返青率＞85%),是耐寒研究珍贵材料。miRNA调控其靶基因启动抗寒应答；lncRNA可竞争性结合miRNA共同调控其靶基因表达。我们前期研究发现,抗寒应答miRNA靶基因中PPP途径关键酶基因上调表达,miRNA还受到lncRNA调控进而影响冬小麦抗寒性,但机制尚不清楚。本项目以dn1为材料,大田自然降温下取样根茎,用高通量测序、生物信息学和现代分子生物学技术,从DNA和蛋白水平验证miRNA-靶mRNA关联；确定ceRNA成员（lncRNA-miRNA-靶mRNA）并鉴定功能；联合分析和绘制ceRNA调控网络图。分析寒胁迫下lncRNA-miRAN-靶mRNA调控模式,揭示lncRNA调控下冬小麦PPP途径抗寒应答的分子机制。本项目有高寒区冬小麦研究特色,结果可为抗寒分子育种及栽培调控提供新思路和基因。

以耐寒珍贵品种东农冬麦1号(Dn1)为实验材料，大田自然降温下取样根茎,用高通量测序构建了3个lncRNA库；应用生物信息软件分析等技术预测了几十条lncRNA-miRNA-靶mRNA关联关系；应用5'RACE和RAP技术验证了5条lncRNA-miRNA-靶mRNA关联，其中2条（lncR0310/lncR0150- miR5049-3P-Ta6PGL）调控PPP途径关键酶基因表达；应用基因克隆、农杆菌转化技术等分别获得过表达miRNAs和lncRNAs的转基因拟南芥植株若干和过表达lncRNA的转基因二穗短柄草植株；应用real-time PCR技术验证转基因植株中lncRNAs、miRNAs及其靶mRNA寒胁迫下表达模式变化。以上结果证实Dn1 中寒胁迫相关lncRNAs行驶ceRNA功能；lncRNA和miRNA共同调控PPP途径关键酶基因上调表达，应答寒胁迫。本项目有高寒区冬小麦研究特色,结果可为抗寒分子育种及栽培调控提供新思路和基因。