沉默大豆GmWRKY33B基因导致大豆抗病性降低

Silencing GmWRKY33B genes leads to reduced disease resistance in soybean

WRKY转录因子基因家族是植物特有的转录因子,在防御中起着重要作用。通过生物信息学分析,本研究在古四倍体大豆(Glycine max)基因组中找到一对同源性高达93%的WRKY33同源基因,并将其命名为GmWRKY33B。从GmWRKY33B的两个同源基因保守区域选取一个315bp片段构建至菜豆豆荚斑驳病毒(bean pod mosaic virus,BPMV)沉默载体(BPMV-VIGS)上,以期同时沉默上述2个GmWRKY33B基因。结果表明,同时沉默2个GmWRKY33B基因并不显著改变沉默植株的表型,但却显著降低了大豆对大豆斑点病菌以及大豆花叶病毒的抗性,说明GmWRKY33B在大豆免疫反应中起正调控作用。激酶分析表明,GmWRKY33B沉默植株中flg22诱导的GmMPK6的磷酸化水平较空载体BPMV-0植株显著降低,说明GmWRKY33B可以通过调控GmMPK6的激酶活性而参与大豆的免疫反应。抗毒素为大豆中主要起防御作用的植保素,而大豆异黄酮类特异性异戊烯基转移酶(prenyltransferase,PT)基因家族是参与大豆抗毒素生物合成的主要基因,许多PT基因启动子区含有与WRKY特异性结合的W-box序列。在丁香假单胞菌pv.甘氨酸(Pseudomonas syringae pv.glycinea,Psg)侵染条件下,4个PT基因的表达水平在沉默株系中显著降低,说明GmWRKY33B参与PT基因的转录激活。综上所述,GmWRKY33B通过调控GmMPK6的激活以及调控大豆抗毒素生物合成途径中关键酶编码基因的表达而参与免疫反应。

The WRKYs are a group of plant-specific transcription factors that play important roles in defense responses.In this study,we silenced 2 GmWRKY33B homologous genes using a bean pod mosaic virus(BPMV)vector carrying a single fragment from the conserved region of the GmWRKY33B genes.Silencing GmWRKY33B did not result in morphological changes.However,significantly reduced resistances to Pseudomonas syringae pv.glycinea(Psg)and soybean mosaic virus(SMV)were observed in the GmWRKY33B-silenced plants,indicating a positive role of the GmWRKY33B genes in disease resistance.Kinase assay showed that silencing the GmWRKY33B genes significantly reduced the activation of GmMPK6,but not GmMPK3,in response to flg22 treatment.Reverse transcriptase PCR(RT-PCR)analysis of the genes encoding prenyltransferases(PTs),which are the key enzymes in the biosynthesis of glyceollin,showed that the Psg-induced expression of these genes was significantly reduced in the GmWRKY33B-silenced plants compared with the BPMV-0 empty vector plants,which correlated with the presence of the W-boxes in the promoter regions of these genes.Taken together,our results suggest that GmWRKY33Bs are involved in soybean immunity through regulating the activation of the kinase activity of GmMPK6 as well as through regulating the expression of the key genes encoding the biosynthesis of glyceollins.