miR159介导的PndMYB转录调控在欧美杨-落叶松-杨栅锈菌互作中的分子机制研究

MiR159 is common to all types of plants, and involved in plant biotic and abiotic stress responses by targeting MYB or MYB-like transcription factor. Based on the previous studies about the poplar- Melampsora larici-populina interaction systems, we have found that PndMYB is involved in anti-rust infection, and four PndMYB is regulated by 6 miR159 family genes. To explore the role of miR159 family genes in gene expression regulation mechanism under pathogen stress, we will conduct studies as fellows: expression patterns of miR159 family genes and its target gene; cleavage site of miR159 family genes; regulation mechanism of miR159 family genes by targeting PndMYB. The miR159 family genes and its target genes expression patterns is validated by Q-PCR. RLM-RACE is employed in studying the cleavage site of miR159 family genes. The miR159s genes are transformed into Arabidopsis thaliana and poplar to generating the overexpression transgenic plant, respectively. And the phenotypes and disease resistance of the overexpression transgenic plant is assessed. These results may be contributed in revealing the gene expression regulation mechanism of miR159 family genes in poplar-pathogen interaction systerm, and provide a scientific basis for poplar breeding for disease resistance.

miR159普遍存在于各类植物中，其靶基因大多是MYB和MYB-like转录因子，参与植物生物及非生物胁迫应答。前期研究发现在欧美杨-落叶松-杨栅锈菌E4小种互作系统中，转录因子PndMYB与欧美杨抗锈菌侵染相关，有4个PndMYB受6个miR159家族miRNA转录后调控。为探索杨树miR159家族miRNA在病原菌胁迫下参与的基因表达精确调控机制，本项目拟进一步研究：miR159家族miRNA（miR159s）及其靶基因的表达模式；miR159s对PndMYB的剪切位点；miR159s对PndMYB的调控机理。应用Q-PCR验证miR159s及其靶基因表达模式，RLM-RACE鉴定剪切位点，将miR159s基因转入拟南芥/欧美杨中，验证miR159s基因过量表达植株的表型变化及抗病性，系统揭示杨树-病原菌互作体系中miR159s对PndMYB的调控机理，为杨树抗病育种提供科学依据。

欧美杨抗落叶松-杨栅锈菌E1~E3小种，但不抗强致病E4小种侵染。前期研究发现转录调控因子PndMYB与欧美杨抗性相关，E4小种侵染后有多个PndMYB受miR159家族miRNA转录后调控。为揭示欧美杨对不同锈菌抗性差异的分子机理，本项目在鉴定锈菌侵染欧美杨后miR159对PndMYB调控的基础上，对E4侵染后欧美杨miR159对PndMYB调控机理进行三个层次的研究：miR159家族miRNA及PndMYB的克隆、表达和转基因验证；miR159对PndMYB剪切位点验证和PndMYB候选靶基因研究；以及miR159对PndMYB调控机理研究。. 为系统研究E4锈菌侵染后杨树microRNA对抗/感病基因的转录后调控，本课题组构建了small RNA (sRNA)文库（2个）、降解组文库（1个）和数字表达谱文库（2个），并对所构建的5个文库进行了高通量测序，获得了miRNA数据、PndMYB数据和miRNA靶向PndMYB的数据。E4锈菌侵染后共发现40条miR159家族同源microRNA（包括前体），其中5条microRNA对10余条MYB基因存在调控关系。鉴定得到217个R2R3-MYB基因。综合分析miRNA组和降解组数据，鉴定到18个R2R3-MYBs与22个miRNA共组成86对转录后调控关系。R2R3-MYB基因表达量有差异的基因75个（40个锈菌接种后表达量上调，35个表达量下调），105个表达量未发生显著变化。其中有38条PdtMYB基因参与调控9条促细胞分裂原活化蛋白激酶（MAPK1，MAPK10，MAPK16，MAPK3，MAPK4，MAPK5，MAPK6，MAPK7和MAPK homolog 2）基因的表达。锈菌侵染不但可以影响R2R3-MYBs的表达量，还可以启动或关闭R2R3-MYBs的表达。欧美杨在E4强致病锈菌胁迫条件下，R2R3-MYBs转录后主要受miR159和miR858家族的miRNA调控。已验证R2R3-MYB与miRNA存在负向转录后调控关系。亚细胞定位表明候选基因在细胞核表达。转基因拟南芥对致病疫霉抗病性提高。R2R3-MYBs靶基因涉及多个与免疫、植物激素以及抗逆相关的生物过程。