转录因子LcHd-Zip2 调控荔枝落果的分子机制研究

The substantial production of Litchi (Litchi chinensis Sonn.), an important economic fruit crop widely grown in southern China and other southeast Asian areas, has been challenged by excessive fruit drop. Homeodomain-leucine zipper proteins(HD-ZIP) are transcription factors unique to plants, classified in four sub-families. Subfamily I members have been mainly associated to abiotic stress responses. The relationship between HD-ZIP and organ shedding has not so far been documented both in model plants and cultivated crops. Based on the previous solid research foundation and the latest findings in our group, in combination with related literature analysis, we speculate that LcHd-zip2, belonging to the HD-ZIP subfamily I member, might be as key regulators in the process of litchi fruit abscission by regulating the key target genes involved in ethylene (ACS, ACO), and ABA (NCED) biosynthesis. In this present project, to reveal the function and molecular mechanism of LcHd-zip2 in controlling the fruit abscission, firstly, we should further uncover the relationships among the LcHd-zip2 transcriptional factor and its candidate target genes, and litchi fruit abscission by analyzing the abundance of transcripts of genes, important physiological indexes (the content of ACC, ethylene, ABA and the activity of PG and EG) and field fruit abscission rate change under different independent experiments including different varieties with sever and minor fruit drop characteristics, treatments between promoting and inhibiting fruit abscission. Then, the downstream target genes regulated by LcHd-zip2 transcription factor would be further determined by using DNA - protein interactions analysis technology such as ChIP- qPCR, yeast hybrid and EMSA. Finally, the function of LcHd-zip2 in regulating organ shedding will be further confirmed by ectopic ove-rexpression and silence of LcHd-zip2 in tomato by transgenic and the CRISPR/Cas9 knockout technology respectively. The results obtained in this project will provide a new findings for understanding of the molecular mechanism for regulation of plant organ shedding and improving practices for alleviating litchi fruit drop.

针对我国特色果树荔枝生产中存在的严重落果问题，基于课题组前期研究和最新发现，结合相关文献分析，本课题提出LcHd-zip2可能通过对ABA和乙烯合成途径中关键靶基因的调控而参与荔枝果实脱落的假说。先通过比较分析落果程度不同品种间、同一品种不同促进和抑制落果处理间等对比样品中LcHd-zip 2基因及其候选靶基因表达情况、重要生理指标（ACC、乙烯、ABA含量及PG和EG酶活性）和田间落果率变化，进一步验证转录因子LcHd-zip2 及其候选靶基因与荔枝落果关系；后采用ChIP-qPCR和EMSA等 DNA-蛋白质互作分析技术，确定荔枝LcHd-zip2 转录因子调控的下游靶基因，并通过异源转基因技术和CRISPR/Cas9基因敲除技术确证 LcHd-zip 2转录因子在调控器官脱落中作用。研究结果为理解植物器官脱落的分子调节机制和改进荔枝防落措施提供新的依据。

荔枝果实鲜美，营养丰富，具有很高的经济价值，然而荔枝花多果少，落果现象非常严重，严重制约着荔枝的优产和丰产，影响着荔枝产业的整体效益。同源异型-亮氨酸拉链（HD-ZIP）蛋白是植物所特有的一类转录因子，但该转录因子是否参与植物器官，特别是果实脱落过程，尚无文献报道。本研究发现HD-ZIP I亚家族转录因子LcHB2直接正调控细胞壁水解酶基因LcCEL2/8的表达而促进荔枝幼果脱落；以及LcHB2/3 可正调控乙烯和脱落酸的生物合成而促进荔枝幼果的脱落的分子机制。主要研究结果如下：.1、花后25 d对‘妃子笑’荔枝进行碳水化合物胁迫（环剥去叶，GPD）和外源乙烯利（ET）处理，结果显示GPD和ET处理5 d后，累积落果率分别达到100%和80.63%，而对照处理的累积落果率仅为9.31%。GPD和ET处理可显著提高果实内乙烯的释放，处理后第4 d果实内乙烯的释放量分别达到60.27 µL kg-1 h-1和73.1 µL kg-1 h-1；GPD处理还促进果实内脱落酸（ABA）的含量显著上升，处理后第4 d果实内ABA含量达到4208.94 ng g-1 FW；.2、筛选鉴定到LcHB2/3是参与荔枝幼果脱落的关键基因，其在GPD和ET促进荔枝落果的处理中较对照有显著上调的表达，且其在荔枝自然落果动态中的表达趋势与生理落果曲线较为相似；.3、细胞壁水解酶在细胞壁降解中发挥着重要作用，本研究筛选到纤维素酶基因LcCEL2/8和多聚半乳糖醛酸酶基因LcPG1/2在荔枝幼果脱落中发挥着主要作用；且将LcCEL2/8超表达于野生型拟南芥中可促进花器官的提前脱落。此外，鉴定出乙烯和ABA生物合成基因LcACS1/4/7、LcACO2/3和LcNCED3在荔枝幼果脱落中显著被激活；.4、EMSA和双荧光素酶分析实验表明LcHB2直接正调控LcCEL2/8的表达而促进离区细胞壁水解；且LcHB2/3均可直接结合并正调控其表达而促进乙烯和ABA的生成，而促进荔枝幼果的脱落。此外，LcHB2/3的表达可受到植物激素乙烯和ABA的反馈调节而加速荔枝果实脱落。.综上，本研究很好地将学科前沿的应用基础研究与解决荔枝产业实际问题紧密结合起来，既为理解植物器官脱落的分子调节机制提供新的依据，也为生产上改进荔枝落果管理措施及后期的分子改良提供理论基础，具有重要的理论和实践意义。