草莓FaWRKY44和FaWRKY46调控花青素苷代谢的机理研究

Strawberry is abundant of anthocyanin, which is a final product of flavonoid biosynthesis branch. The anthocyanin metabolism is regulated by MYB-bHLH-WD ternary complex, which has been extensively well characterized. However, the roles of other transcription factors on regulating anthocyanin pathway have not been well studied yet, especially in strawberry. Recently, a few researches have suggested that WRKYs transcription factors could participate in regulating of anthocyanin pathway, while the mechanisms are not clear and need to be further investigated. Based on previous transcriptome analysis, virus-induced gene silencing and agrobacterium-based transient overexpression experiments, we found two new WRKYs transcription factors FaWRKY44 and FaWRKY46, which could positively influence anthocyanin levels, as well as the expression of key genes for anthocyanin biosynthesis in strawberry fruits, indicating they are involved in anthocyanin regulation. However, more details about how FaWRKY44 and FaWRKY46 affect anthocyanin content need to be deeply explored. Therefore, in order to investigate the roles of FaWRKY44 and FaWRKY46 in regulating anthocyanin, we intend to build WRKY44 and WRKY46 knocked-down and overexpressed strawberry mutants by using CRISPR/Cas9 and overexpression methods, and further validate by overexpression in arabidopsis wrkys mutants. What's more, yeast one- and two- hybrid, bimolecular fluorescence complementation (BiFC) and chromatin immunoprecipitation (ChIP) assay will be employed to identify the upstream regulatory genes, interact proteins and target genes of WRKY44 and WRKY46. All these efforts are done for unraveling the regulatory mechanism and constructing the anthocyanin regulatory network. The results from this project would provide novel knowledge of anthocyanin regulation in strawberry, and might improve the existing regulatory network of anthocyanin.

花青素苷是类黄酮代谢途径中一个重要分支的终产物，其代谢受MYB-bHLH-WD40三元复合体调控并已有广泛研究。近来有研究表明，WRKYs转录因子也参与了花青素苷的调控，但不同的WRKYs行使着不同的功能。申请人通过转录组分析、瞬时沉默与过表达等实验发现了两个新的WRKYs—FaWRKY44和FaWRKY46，且能调控草莓果实中花青素苷的含量以及花青素苷代谢关键基因的表达量，但其调控机理尚不明确。本项目拟采用CRISPR/Cas9和过表达技术构建草莓WRKY44和WRKY46敲除与过表达植株，结合在拟南芥突变体中过表达验证其功能，并通过酵母单/双杂交、BiFC和ChIP等技术，对草莓WRKY44和WRKY46调控花青素苷代谢的靶基因、互作蛋白及其上游调控基因进行筛选鉴定，以探明FaWRKY44和FaWRKY46对草莓果实花青素苷代谢的调控机理，籍以丰富对花青素苷途径调控网络的认知。

花青素苷是草莓果实品质的重要组成部分，其代谢的调控机理一直是草莓研究中的重点和热点。本项目研究发现，除MYB-bHLH-WD40三元复合体（MBW）外，WRKY转录因子FaWRKY44和FaWRKY46也参与了草莓果实中花青素苷的代谢调控。亚细胞定位分析发现，FaWRKY44和FaWRKY46均定位在细胞核中。qRT-PCR定量分析显示，FaWRKY44在草莓花和功能叶中表达量较高，而FaWRKY46在草莓各个组织中均高表达。此外，FaWRKY44和FaWRKY46均受低温和红/蓝光诱导。在活体草莓果实中，抑制FaWRKY44和FaWRKY46的表达均导致草莓果实花青素苷积累的下降；过表达FaWRKY46提高了草莓果实花青素苷的含量，而过表达FaWRKY44对草莓果实花青素苷的积累无明显的促进作用。qRT-PCR和转录组分析显示，FaWRKY44和FaWRKY46不是直接影响草莓果实花青素苷合成通路相关基因和FaMYB10的表达，而是通过影响Oxidative phosphorylation代谢通路间接调控草莓果实中花青素苷的积累。FaWRKY44和FaWRKY46均与MBW中的EGL3存在蛋白互作；FaWRKY44与TTG1、FaWRKY46与LWD1也存在互作；FaWRKY44和FaWRKY46与‘MBW’复合体的互作增强了草莓花青素苷的储存和运输。FaBT2与FaWRKY46通过蛋白互作后诱导了FaWRKY46蛋白的降解。FaWRKY44和FaWRKY46基因的转录水平不受上游信号传导通路中PP2C、NCED1、MAPK3、MAPK4-2、MAPK16、MAPK17、NCED1、SnRK2.2和SnRK2.6的影响。此外，采用DAP-seq结合ChiP技术还挖掘到了83个FaWRKY46的靶基因。本项目的研究结果部分已在《Plant Biotech. J.》、《Int. J. Mol. Sci.》和《园艺学报》等发表，这些结果不仅丰富了对草莓WRKY家族和类黄酮代谢调控网络的认知，而且也为WRKY基因的利用提供了理论基础。

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