细胞分裂素对荔枝果实成熟衰老的调控作用及其分子机制探索

Litchi industry is suffering from heavy burden of marketing the fruit as a result of a very short marketing period due to concentrated harvesting season and rapid senescence of ripe fruit. The current project is targeted to solving this problem through advancing or postponing ripening process and extending hangling life of litchi based on in-depth understanding of the regulation mechanisms of litchi fruit ripening and senescence. The study is to be carried out from the angle of cytokinins' (CTK) regulation. Cultivars of litchi differing in ripening period and litchi with very short hanging life and grape with long hanging life will be compared for their endogenous CTKs dynamics as well as changes in various ripening and senescence related physiological parameters and endogenous ABA concentration. Their correlations will be analyzed. Effect of exogenous CTK (CPPU) on ripening and senescencing processes will be examined and the interactions between CTKs and ABA in regulation of litchi ripening will be explored. Comparative transcriptomical analysis will be carried out between CPPU-treated fruit and control fruit. Effect of CTK on expression profiling and co-expression network will be analyzed. At the same time, transcription factors will be clustered based on the expression trend during fruit development and responses to CPPU and subject to pricipal component analysis. Based on the above analyses, key CTK-responding regulator genes will be screened. The functions of the target genes will be examined by virus induced gene silencing (VIGS) and their expression pattern will be studied. Throught this study, the molecular mechanisms of CTK actions on litchi fruit ripening and senescene will be unravelled.

荔枝果实成熟期集中、衰老快、留树时间短，导致产期短，销售压力大，成为产业重要制约因素。本项目针对此重大产业问题，深入揭示荔枝成熟衰老进程的调控机制，为成熟促成、延后、延长挂树期和产期的技术创新提供理论依据。项目从细胞分裂素（CTK)调控着眼，分析成熟期不同的荔枝品种（早、中、迟熟品种）及衰老进程不同的种间（荔枝vs葡萄）内源CTKs动态，及其与成熟衰老生理指标和内源ABA的动态关联；探索外源CTK（CPPU)对成熟和衰老各生理过程的影响，分析外源CTK与ABA的在成熟调控上的交互效应，揭示CTK与ABA的互作方式；展开CPPU处理与对照果实的比较转录组学分析，探索其对基因表达趋势和共表达网络格局的影响，对转录因子展开聚类和主成分分析，筛选响应CTK的荔枝成熟衰老调节关键基因，分析其表达调控规律，并以VIGS对其功能验证，解析CTK调控荔枝成熟衰老的分子机制。

荔枝果实成熟期集中、衰老快、留树时间短，导致产期短，销售压力大，成为产业重要制约因素。本项目针对此重大产业问题，深入揭示荔枝成熟衰老进程的调控机制，为成熟促成、延后、延长挂树期和产期的技术创新提供理论依据。本项目探索外源CTK（CPPU)对成熟和衰老各生理过程的影响，分析CTK与ABA的在成熟调控上的交互；展开CPPU处理与对照果实的比较转录组学分析，筛选响应CTK的荔枝成熟衰老调节关键基因，以VIGS对其功能验证，解析CTK调控荔枝成熟衰老的分子机制。研究结果表明：（1）CPPU在果实发育各阶段处理均抑制落果，但无明显增大果实的效应，仅增厚果皮；在花后8周以2.5-20ppm浓度处理也无显著提高果重效应，仅提高果皮重；花后6-9周处理，可以显著推迟果实着色，但不影响糖积累和酸含量；花后8周处理10ppm以上的CPPU，可以显著延缓成熟荔枝果肉的退糖和无氧呼吸发生，具有延长“留树保鲜期”的效应。CPPU通过抑制无氧呼吸关键酶-丙酮酸脱羧酶和乙醇脱氢酶活性而抑制果肉无氧呼吸发生。（2）葡萄成熟果内源活性CTK含量远高于荔枝，可能是葡萄留树期长的生理原因。（3）ABA不能逆转CPPU对荔枝着色的抑制效应，活性氧（ROS）发生剂MV显著促进荔枝着色，ABA可强化ROS的着色效应；但CPPU可完全抑制ROS的效应，CPPU显著抑制了ABA信号转导途径基因LcABF1、 LcABF2、LcSnRK表达。（4）通过比较转录组分析发现了大量响应CTK的基因，从类黄酮合成路径结构基因表达谱解析了CPPU抑制花青苷积累而不影响非花青苷类黄酮含量的机理；首次发现响应CTK的WRKY基因参与果实着色调控，其中，LcWRKY33正响应CPPU处理，在果实发育中期以VIGS处理沉默该基因，可促进果皮着色；而负响应CTK的LcWRKY75-2被VIGS沉默后，果皮着色受到抑制，通过共表达和单周分析，初步锁定其下游靶基因。（5）筛选出1个可同时调控绿色降解关键基因（LcSGR1）和花青苷合成关键基因（LcMYB1）表达的转录因子LcNAC2, 但CPPU并不能持续抑制该基因表达，但持续抑制LcSGR1和LcMYB1表达，不排除CTK可能通过蛋白修饰影响LcNAC2的转录调节。本研究结果具有理论创新，应用性也强。