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以‘丰香’草莓(Fragaria×ananassa‘Toyonaka’)为材料,采用病毒诱导的基因沉默技术(Virus-induced Gene Silencing,VIGS)对果实中类黄酮化合物代谢相关转录因子基因FaMYB5进行沉默,并对沉默后果实类黄酮化合物代谢途径相关的结构基因和其他转录因子基因表达量进行半定量RT-PCR及RT-qPCR分析。并分别采用HPLC和香草醛—硫酸比色法对沉默后果实中花青素苷和原花青素含量进行测定。成功构建了pTRV2-FaMYB5的VIGS沉默载体,并建立了草莓果实VIGS沉默体系,FaMYB5基因沉默效率达60%;FaMYB5基因沉默处理与阴性对照相比,草莓类黄酮化合物代谢途径相关结构基因和转录因子表达量均发生改变,其中FaMYB9、FaLAR、FaDFR、FaANR表达量显著增加,从而导致果实中原花青素含量增加;而花青素合成相关基因FaANS的表达量减少1/2,FaMYB10表达量变化并不显著。此外,bHLH家族的转录因子基因FabHLH3和FabHLH3Δ随着FaMYB5的沉默表达量显著下降,而FaGL3显著升高。结果进一步证明FaMYB5转录因子对草莓类黄酮化合物代谢途径中的原花青素合成途径起负调控作用。
Fragaria × ananassa’Toyonaka ’was used to silence FaMYB5, a transcriptional factor related to flavonoid metabolism in fruits, by using Virus-induced Gene Silencing (VIGS) Semiquantitative RT-PCR and RT-qPCR analysis of the gene expression levels of flavonoid-related metabolic pathway-related structural genes and other transcription factors were carried out after silencing. The content of anthocyanins and proanthocyanidins in the fruits after the silencing were determined by HPLC and vanillin - sulfuric acid colorimetry respectively. The VIGS silencing vector of pTRV2-FaMYB5 was successfully constructed and the strawberry VIGS silencing system was established. The silencing efficiency of FaMYB5 gene was up to 60%. Compared with the negative control, FaMYB5 gene silenced the related structural genes of the flavonoid pathway and the transcription factor The expression levels of FaMYB9, FaLAR, FaDFR and FaANR were significantly increased, which led to the increase of proanthocyanidins content in fruit. However, the expression of FaANS in anthocyanidin decreased by 1/2, while the expression of FaMYB10 did not change significantly . In addition, the bHLH family of transcription factor genes, FabHLH3 and FabHLH3Δ, decreased significantly as FaMYB5 silenced, whereas FaGL3 increased significantly. The results further demonstrate that the FaMYB5 transcription factor negatively regulates the proanthocyanidin biosynthetic pathway in the strawberry flavonoid metabolic pathway.