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园艺学报 ›› 2017, Vol. 44 ›› Issue (1): 33-42.doi: 10.16420/j.issn.0513-353x.2016-0346

• 研究论文 • 上一篇    下一篇

利用VIGS技术研究草莓FaMYB5的功能

王 玲,汤浩茹*,王小蓉,陈 清,江雷雨,林源秀   

  1. 四川农业大学园艺学院,成都 611130
  • 出版日期:2017-01-25 发布日期:2017-01-25
  • 基金资助:

    教育部高等学校博士学科点专项科研基金项目(20125103110005)

Virus-induced Gene Silencing as a Tool for FaMYB5 Gene Functional Studies in Strawberry

WANG Ling,TANG Haoru*,WANG Xiaorong,CHEN Qing,JIANG Leiyu,and LIN Yuanxiu   

  1. College of HorticultureSichuan Agricultural UniversityChengdu 611130China
  • Online:2017-01-25 Published:2017-01-25

摘要:

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FaMYB9FaLARFaDFRFaANR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FabHLH3FabHLH3Δ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。

关键词: 草莓, VIGS, FaMYB5, 负调控类黄酮代谢

Abstract:

In the present study,using strawberry cultivar‘Toyonaka’(Fragaria × ananassa) as plant materials,we silenced the gene encoding FaMYB5 transcription factor,which was imparted in flavonoid metabolism in fruits of strawberry by using Virus-induced gene silencing(VIGS)technology. After silencing of FaMYB5,the expression level of other important genes involved in flavonoid metabolic pathway was detected by semi-quantatitive PCR and RT-qPCR. Meanwhile,in order to fully investigate the role of FaMYB5 in regulating the flavonoid metabolic pathway,we determined the anthocyanins and proanthocyanidins content using HPLC and DMACA chemical methods respectively. The results showed that,the recombined pTRV2-FaMYB5 vector and the VIGS system were successfully constructed. By determining of the phenotype and detecting the expression of FaMYB5 after its silencing,the efficiency of gene silencing was about 60%. In constract with the negative control group,the expression of FaMYB9FaLARFaDFR,and FaANR increased,leading to an increasing of proanthocyanidins level. On the contrary,half of the expression level was observed for FaANS,accompanied with the anthocyanins decreasing. While the expression of FaMYB10 had no radical change. In addition,the expression of FabHLH3 and bHLH3Δ decreased after silencing of FaMYB5. However,the expression of FaGL3 had an obvious increasing phase. All the results showed that FaMYB5 transcription factor could negatively regulate the proanthocyanidins metabolism pathway.

Key words: strawberry, VIGS, FaMYB5, negatively regulation, flavonoids metabolism

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