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植物WRKY类转录因子的IIa亚类广泛参与调控植物的生物胁迫和非生物胁迫过程。本研究根据拟南芥和普通烟中具有抗病和抗胁迫功能的IIa亚类WRKY40转录因子的序列,利用基因同源克隆法,获得本氏烟中Nb WRKY40基因。序列分析表明在本氏烟中有5个属于IIa亚类的基因,其序列之间具有高度相似性。qRT-PCR检测发现Nb WRKY40基因是受寄生疫霉侵染诱导上调表达的基因,通过VIGS技术研究该类基因在本氏烟中的抗病相关功能,用半活体营养型寄生疫霉进行抗病性试验,结果表明Nb WRKY40基因的沉默降低了本氏烟对寄生疫霉的抗性,且在侵染点的细胞坏死程度增强,过氧化氢积累和胼胝质沉积都明显减少。Nb WRKY40基因沉默同样降低了本氏烟对死体营养型灰霉菌的抗性。检测Nb WRKY40基因沉默后4个不同抗病信号通路下游基因的表达,发现基因沉默导致参与抗病和SA途径的PR1b、PR2b基因受疫霉侵染诱导表达的水平明显下降。综上,推测Nb WRKY40基因可能依靠SA介导的信号通路参与调控本氏烟抗病性。
The IIa subgroup of plant WRKY transcription factors is widely involved in the regulation of plant biotic and abiotic stress processes. In this study, based on the sequence of class IIa WRKY40 transcription factor with resistance and stress in Arabidopsis and general tobacco, the gene of Nb WRKY40 in N. benthamiana was obtained by gene homology cloning method. Sequence analysis revealed five genes belonging to the IIa subclass in N. benthamiana, with highly similar sequences. The qRT-PCR assay showed that the gene of WRKY40 was up-regulated by Phytophthora infestans. The VIGS technique was used to study the resistance-related function of this gene in N. benthamiana. The results showed that the silencing of Nb WRKY40 gene decreased the resistance of N. benthamiana to Phytophthora parasitica, and increased the degree of cell necrosis at the infection site, and significantly reduced the accumulation of hydrogen peroxide and callose deposition. Nb WRKY40 gene silencing also reduced the resistance of N. benthamiana to dead vegetative Botrytis cinerea. The expression of genes downstream of four different resistance signaling pathways after the silencing of Nb WRKY40 gene was detected. It was found that PR1b and PR2b genes involved in disease resistance and SA pathway induced by Phytophthora infestans significantly decreased after gene silencing. In conclusion, it is speculated that Nb WRKY40 gene may be involved in the regulation of N. benthamiana disease resistance by SA-mediated signaling pathway.