青杄转录因子PwHAP5及其同源蛋白AtHAP5下游靶基因的鉴定

Identifying Target Genes of PwHAP5 in Picea wilsonii and Its Homologous Protein AtHAP5

利用分子生物学手段,对青杄PwHAP5及其拟南芥同源基因AtHAP5的下游调控基因进行探究,旨在丰富青杄以及拟南芥中HAP5转录因子的调控网络。通过转录激活活性分析,发现PwHAP5和AtHAP5均无转录激活活性。两个酵母单杂交体系(pHis2和pAbAi体系酵母单杂交实验)显示A37、HSFA2启动子全长、只含有CCAAT box的区域均不能与PwHAP5和AtHAP5直接结合。EMSA试验结果表明,AtHAP5与A37和HSFA2启动子在体外不能直接结合;单(双)荧光素酶试验结果表明,AtHAP5对HSFA2具有负调控作用,但对A37表达无影响。通过RT-qPCR试验检测在异源过表达OE-PwHAP5植株中预测靶基因的表达量,结果显示A37与HSFA2相对表达量均无显著变化,而在同源过表达植株OE-AtHAP5中,HSFA2的相对表达量下降。这些结果显示AtHAP5在体内负调控HSFA2表达,而PwHAP5在进化上与AtHAP5的功能存在差异。

The downstream regulatory genes of PwHAP5 and its Arabidopsis homologous protein AtHAP5 were studied by molecular biology to enrich the regulatory network of HAP5 transcription factors in both Picea wilsonii and the model plant Arabidopsis.By transcriptional activation assay,PwHAP5 and AtHAP5 subunits alone have no transcriptional activation activity.Yeast one hybrid experiment has been carried out with two systems included pHis2 and pAbAi.However,it was found that both the A37 and HSFA2 promoter or only the CCAAT box region could not be directly combined with PwHAP5 and AtHAP5.EMSA experiment showed that AtHAP5 could not directly combine to the promoters of A37 and HSFA2 in vitro.Single fluorescein and double fluorescein experiment showed that AtHAP5 had a negative regulation on the expression of HSFA2 but no effect on that of A37.RT-qPCR showed that the relative expression of A37 and HSFA2 did not change significantly in heterologous over-expressed OE-PwHAP5 plants,but decreased in homologous over-expressed OE-AtHAP5 plants,which further confirmed that AtHAP5 had a negative regulatory effect on the expression of HSFA2,but PwHAP5 had no regulatory effect on the possible downstream target genes.In summary,there is no transcriptional activation activity between PwHAP5 and AtHAP5.AtHAP5 inhibited the expression of HSFA2 in vivo and PwHAP5 had no significant effect on HSFA2 expression.These results suggest that AtHAP5 negatively regulates the expression of HSFA2 in vivo,which may be the downstream target gene.PwHAP5 differs from AtHAP5 in the evolutionary function.