NAC转录因子介导ABA代谢途径调控西瓜果实成熟及品质进化的分子机制

The modern cultivated watermelon is the result of years of cultivation and selection for fruits with desirable qualities. The ABA content in cultivated watermelon fruit increased during fruit development and ripening, and was significantly higher than in wild watermelon. The expressions of NCEDs and CYP707As which encoded the key enzymes in the ABA metabolism were significantly different in cultivated and wild watermelon. However, these genes were not selected during domestication. In order to reveal the molecular mechanism of the difference of ABA content between cultivated and wild watermelon, we analyzed the promoters of NCEDs and CYP707As. NAC core recognition cites were found in the promoters of NCEDs and CYP707As, and NAC transcription factors were selected during the domestication. We hypothesized that through evolution, NAC transcription factors regulated watermelon fruit ripening via ABA metabolism. We analyzed the haplotypes and found SNPs in the CDS and promoters of candidate NAC transcription factors. We will detect the transcriptional ability of NAC transcription factors to NCED and CYP707As in cultivated and wild watermelon to reflect the effects of SNPs in NAC transcription factors to the transcriptional ability. We will knockout the candidate NAC transcription factors by CRISPR-Cas9 based on these SNPs. After we obtain the homozygous generations, we will analysis the quality traits and transcriptome of the transgenic lines and control lines to illustrate the molecular mechanism of NAC transcription factors regulating watermelon fruit ripening via ABA metabolism. These results will provide technical guidance to improve watermelon quality, and possess important theoretical significance and industrial value.

现代栽培西瓜果实成熟的优良品质是从野生种不成熟性状进化而来，本实验室前期研究表明：栽培西瓜果肉中ABA含量随着果实发育成熟逐渐升高，且显著高于野生西瓜。ABA代谢途径关键节点基因NCED和CYP707As的表达在栽培和野生西瓜果实间存在显著差异,但在全基因组驯化中未受到选择。NCED和CYP707As的启动子上包含NAC转录因子核心结合元件，且NAC在驯化中受选择。就此提出本项目科学假设：NAC转录因子通过进化，并介导ABA代谢途径，进而调控西瓜果实成熟。本项目利用西瓜野生与栽培材料重测序信息开展单倍型分析，明确NAC及其差异位点对ABA代谢关键节点基因调控差异；对NAC关键差异位点进行基因编辑，获得功能性状互补的转基因后代，进行品质性状与转录组分析，明确NAC介导ABA代谢途径调控果实成熟的分子网络。这必将丰富果实成熟调控机制，并为高品质西瓜改良提供技术指导，具有重要理论意义和产业价值。

现代栽培西瓜果实成熟的优良品质是从野生种不成熟性状进化而来，本实验室前期研究表明：栽培西瓜果肉中ABA含量随着果实发育成熟逐渐升高，且显著高于野生西瓜。ABA代谢途径关键节点基因NCED和CYP707As的表达在栽培和野生西瓜果实间存在显著差异,但在全基因组驯化中未受到选择。NCED和CYP707As的启动子上包含NAC转录因子核心结合元件，且NAC在驯化中受选择。就此提出本项目科学假设：NAC转录因子通过进化，并介导ABA代谢途径，进而调控西瓜果实成熟。NAC（NAM，ATAF1/2，CUC2）转录因子在果实成熟和品质中起重要作用。本研究发现，一个与SlNOR高度同源的NAC转录因子ClNOR在西瓜驯化过程中受到选择，并位于含糖量QTL上。敲除ClNOR基因西瓜果实不能正常成熟，clnor突变体中的ABA含量、番茄红素含量和糖含量均显著低于野生型果实。利用转录组和DNA亲和纯化测序（DAPseq）分析进一步证明，ClNOR不能直接结合ABA、类胡萝卜素和糖的关键生物合成基因，而是介导ClABI5、ClMYB44和ClNF-YC2分别调控成熟相关基因ClNCED1、ClPSY1和ClAGA2的表达。这说明ClNOR通过间接级联调控非呼吸跃变型果实成熟，不同于SlNOR通过直接靶向乙烯和番茄红素的生物合成调控呼吸跃变型果实成熟的分子机制。生物层干涉分析（BLI）和瞬时荧光素酶分析实验表明，驯化位点 SNP（564，T/C）提高了ClNOR与这些靶基因的结合稳定性和转录激活活性。这些发现拓宽了对NOR转录因子参与非呼吸跃变型果实成熟调控和驯化的普遍功能和独特机制的认识。ClNAC68是一个转录抑制子，在西瓜果肉中特异高表达，且在栽培西瓜果实中的表达显著高于野生西瓜。在西瓜中敲除ClNAC68显著降低了突变体果实中的ABA含量和含糖量。转录组分析发现，在clnac68突变体中，蔗糖代谢关键转化酶基因的表达显著提高。EMSA和瞬时荧光酶分析ClNAC68直接结合ClINV启动子并抑制其表达。本研究将进一步完善呼吸非跃变型果实成熟及品质进化调控分子机制，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。

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