miRNA经SnRK信号通路介导荔枝果实能量水平的机制

Fruit ripening and senescence are active processes initiated by internal and environmental factors. Considerable evidence suggests that the cellular energy supply is the key factor to control ripening and senescence events. However, mechanism of energy deficit is still unclear due to the diversity of the energy regulatory elements and the complexity of the energy control system. Energy is generated by respiration, and energy level is closely related to the activity, transcription and translation of the regulatory elements relating to the synthesis, dissipating, transport and regulation of ATP. Furthermore, microRNAs are the most abundant regulatory genes of the genome which may be involved in the inhibition of nearly 70% of the mRNA expression. Based on the above knowledge, the project will conduct the following researches: (1) to clone and identificate the miRNAs related to energy regulation in litchi fruit tissue; (2)to study the role of SnRK (sucrose non-fermenting-1-related protein kinase) and energy signals in deterioration and senescence of harvested litchi fruit given the energy signal was blocked by exogenous application of SnRK inhibitor and by treatment of exogeneous ATP and its nonhydrolytic analogues; (3) to study the role of SnRK in energy regulation, the transcription of its target genes and translation of its phosphorylated target protein by over-expression of its specific miRNA or artificial miRNA (amiRNA) in tomato. This study could provide a new strategy to postpone the aging process of harvested horticultural products by regulating the their development though energy control.

能量亏缺可能是启动园艺产品采后衰老劣变的关键因素。由于能量形成调控元件的多样性和调控体系的复杂性，造成植物组织能量亏缺的关键机制仍不清楚。能量由呼吸作用产生，并受到其合成、利用和转运相关酶基因表达水平的影响；而miRNA是最丰富的调节基因，可能参与抑制近70%mRNA的表达。项目将克隆和鉴定荔枝果实组织能量调控相关的miRNA；以我们前期克隆的能量调节器基因蔗糖非酵解型蛋白激酶SnRK为中心，通过外源施用ATP及其非水解类似物和SnRK专一性抑制剂米酵菌酸阻断能量信号，研究SnRK和能量信号在荔枝采后衰老劣变中的作用；在番茄中过表达特定miRNA或人工小分子RNA（amiRNA）抑制SnRK基因表达，研究SnRK及其磷酸化靶蛋白转录和翻译水平的变化及其对能量水平的影响。项目从miRNA和SnRK信号通路阐明荔枝果实采后衰老劣变机制，对于从能量角度提出园艺产品采后品质调控新策略具有重要意义。

采后荔枝果实的衰老与能量状态密切相关，蔗糖非酵解型蛋白激酶（SnRK）蛋白家族作为植物体内的能量调节器，在能量调节过程中发挥关键作用，是整个能量代谢调节网络的中心；而microRNAs 作为一种重要的负调控因子几乎参与植物的每一个生理过程。本项目从microRNAs和SnRK信号通路角度研究荔枝果实采后衰老劣变及其调控机制。主要结果如下：.（1）构建了常温贮藏0、4 天以及低温贮藏14 天常温货架期0、24和48 小时不同贮藏时期的小RNA文库和一个混合样品的降解组文库，并对它们进行高通量测序，首次从荔枝果实中鉴定出了296条保守miRNAs，它们分属49个miRNA家族。同时，找到11条荔枝特有的miRNAs。.（2）应用多种PCR技术验证荔枝miRNA对其靶基因的作用，鉴定出14对miRNA-靶基因参与包括花色素苷代谢、SnRK信号、激素信号传递以及病原菌侵染的防御等荔枝衰老相关过程。最终提出了miRNA-靶基因参与荔枝衰老过程的调控网络。.（3）采用同源克隆并结合荔枝转录组数据，获得5个LcSnRKs全长，并对其进行生物信息学分析、亚细胞定位和时空表达分析，结果证明：LcSnRK1a定位于细胞核中，而LcSnRK2.1则定位于细胞质和细胞核中；LcSnRK1a在荔枝果实采后衰老过程中可能起重要调控作用。.（4）通过酵母双杂交证明LcSnRK2.1与LcbZIP存在互作，荧光双分子互补（BiFC）结果显示LcSnRK2.1和LcbZIP的互作区域主要位于细胞核，并且二者存在相似的时空表达特性。.（5）通过酵母双杂交技术筛选到与LcSnRK1a存在互作关系的LcFBC、LcWRKY28、LcBRIS基因，回转酵母实验初步验证它们的互作关系。.（6）项目执行期间共发表论文11篇（其中SCI收录10篇）；获授权国家发明专利3件，申请国家发明专利1件，申请PCT国际发明专利1件；培养博士生1名、硕士生4名；获得广东省科学技术一等奖等科技奖励3项。