β-葡萄糖苷酶基因调控西瓜果实成熟及品质进化的作用机制

Watermelon（Citrullus lanatus (Thunb.) Matsum. & Nakai）belongs to non-climacteric fruit. Until now, the mechanism of watermelon fruit ripening and evolution is not well known. Our previous study revealed that ABA involved in the regulation of watermelon fruit ripening and quality evolution; genetic variation analysis of the 130 resequenced watermelon accessions representing different evolution states showed that the β-glucosidase (converting the conjugated ABA-GE into free ABA) gene ClBG1 exhibited a significant genetic variance between the ripen and non-ripen accessions; ClBG1 expression, increasing with fruit development and ripening, was significantly higher in the cultivated watermelon 97103 than that in the wild watermelon PI296341-FR, which was consistent with ABA content variation. Above all, it turned out that ClBG1 could be the key ABA metabolism gene involved in the regulation of watermelon fruit ripening and quality evolution. Based on the above results, to systematically understand the mechanism of ClBG1 in regulating watermelon fruit ripening and quality evolution, the project proposed here firstly intends to clarify the effects of sequence variation in the promoter region of ClBG1 on gene expression, ABA content, fruit ripening and quality evolution; secondly, the biological function of ClBG1 during watermelon fruit development and ripening will be verified by gene transformation, and the gene network affected by ClBG1 will be identified. This study helps to establish the molecular mechanism of non-climacteric fruit ripening and provide technical support for molecular improvement of watermelon fruit quality, which will be of great importance in both theory and industry.

西瓜属于呼吸非跃变型果实，其成熟与进化机制尚不十分清楚。本项目前期研究发现：脱落酸（ABA）参与西瓜果实成熟和品质进化的调控；130份重测序西瓜材料群体遗传差异分析表明β-葡萄糖苷酶（水解结合态ABA产生游离ABA）基因ClBG1在不成熟和成熟材料间存在显著变异和进化痕迹；其在栽培西瓜果肉表达量随果实发育而升高，且显著高于野生西瓜，与ABA含量变化趋势一致。因此ClBG1可能是ABA路径中控制西瓜果实成熟和品质进化的关键基因。在此基础上，本项目首先明确ClBG1基因启动子区序列变异与基因表达、ABA含量、果实成熟和品质的关系，其次通过转基因验证ClBG1在西瓜果实成熟过程中的功能，鉴定其影响的基因网络，最终系统明确ClBG1调控西瓜果实成熟及品质进化的作用机制。本研究将进一步完善呼吸非跃变型果实成熟调控分子机制，为西瓜品质分子改良与生产提供技术指导，具有重要的理论意义和潜在的产业价值。

西瓜属于呼吸非跃变型果实，其成熟与进化机制尚不十分清楚。本项目前期研究发现：脱落酸（ABA）参与西瓜果实成熟的调控；130份重测序西瓜材料群体遗传差异分析表明β-葡萄糖苷酶（水解结合态ABA产生游离ABA）基因ClBG1在不成熟和成熟材料间存在显著变异和进化痕迹；其在栽培西瓜果肉表达量随果实发育而升高，且显著高于野生西瓜，与ABA含量变化趋势一致。因此ClBG1可能是ABA路径中控制西瓜果实成熟的关键基因。在此基础上，利用Crispr/Cas9技术，我们对ClBG1进行了敲除。在Clbg1敲除突变体中，果实转色稍有延迟，但是并没有最终抑制果实成熟。值得注意的是，由于ABA含量的降低，引起了细胞数目的减少，最终导致了种子大小和重量显著降低。转录组分析显示，在授粉后10和18天的敲除突变体中，分别有1015和1429个基因的表达发生了显著变化，这些基因主要富集在细胞骨架和细胞循环路径。而且，参与种子大小调控的主要信号路径中的基因表达也发生了改变。此外，由于ABA含量的降低，Clbg1敲除突变体中种子萌发提前。这些结果表明，ClBG1主要通过调控ABA的含量从而调控了细胞骨架、细胞循环和信号相关基因的表达，从而对西瓜种子大小和萌发的调控发挥了重要作用。我们的结果为西瓜种子大小调控的分子机制研究奠定了基础。

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