猕猴桃果实损伤诱导的木栓质合成及其脂肪ω-羟基酸/脂肪醇苯丙基转移酶（FHT）的调控机制

Fresh fruits incur mechanical wounds inevitably during harvest, storage and transportation, hastening deterioration and loss. Considering wound-healing ability in plant tissue, this project uses kiwifruit (Actinidia Chinensis) as model material and ‘kiwifruit--wound stress --phytohormone signals--functional protein/gene (FHT) expression--wound-induced suberization--wound-healing’ as research approach, to study wound-induced suberinzation of kiwifruit. The advanced methods and techniques including autofluorescence microscopy and ultrastructural observation, physiological and biochemical assay, transcriptome sequencing, yeast hybridization, luciferase assay, transient silencing and over-expression are used in this project. In the process of wound-healing, kiwifruit tissue structure, cell morphology, and suberin metabolic regulation are analyzed. The temporal and spatial expression of AchnFHT, the homologous gene of StFHT, is determined in the process of kiwifruit growth and development. AchnFHT cDNA is fused with green fluorescent protein (GFP) to construct a plant expression vector and localize the GFP-AchnFHT fusion protein. AchnFHT and protein responding to wound-induced suberin biosynthesis is identified. Cis-acting element of AchnFHT promoter and AchnFHT promoter-binding activity of transcription factor AchnMYB41 are analyzed, and then targeted regulation of phytohormone on AchnFHT is identified. This project will elucidate biosynthesis and regulation mechanism of wound-induced suberin in kiwifruit, and provide a theoretical basis for postharvest wound-healing and preservation technologies of fruits and vegetables.

本项目针对水果采摘和贮运过程不可避免遭受损伤，基于植物组织具有愈伤的能力，以猕猴桃果实为模式材料，采用转录组测序、酵母杂交、荧光素酶活性验证、瞬态沉默和过表达等方法，以“猕猴桃果实--损伤胁迫--激素信号--功能蛋白/基因（FHT）表达--愈伤细胞木栓化--伤口愈合”作为研究思路；观察猕猴桃果实伤口愈合过程的特点；测定木栓化过程的木栓质代谢规律；测定StFHT的同源基因AchnFHT在猕猴桃果实中的时空表达；构建AchnFHT cDNA与GFP融合基因，对AchnFHT蛋白表达进行亚细胞定位；确定AchnFHT基因和蛋白对猕猴桃果实愈伤木栓质合成代谢的响应；分析AchnFHT启动子的顺势作用元件，验证转录因子AchnMYB41与AchnFHT基因启动；确定植物激素对AchnFHT基因的靶标调控因子，揭示猕猴桃果实损伤诱导的木栓质合成及其调控机制，为果蔬采后愈伤保鲜技术开发提供理论基础。

木栓质是由脂肪类和芳香类物质聚合而成的生物多聚物，其定位在损伤组织的细胞壁和细胞膜之间防止水分损失和致病菌侵染。ω-羟酸/脂肪醇羟基肉桂酰转移酶（FHT）是催化木栓质合成中脂肪类物质和芳香类物质交联的关键酶。植物激素脱落酸（ABA）在正向调控木栓质合成中具有重要作用，然而其对FHT基因的调控机理尚不清楚。本研究从猕猴桃果实中克隆出AchnFHT基因，亚细胞定位显示，AchnFHT蛋白定位在细胞溶质中。将AchnFHT基因在本氏烟草中超表达可显著诱导叶片中木栓质阿魏酸、ω-羟酸和伯醇的积累；体外蛋白活性实验表明，AchnFHT蛋白可催化阿魏酰辅酶A中的阿魏酰基转移到ω-羟基十六烷酸和十四烷醇上。通过酵母单杂交筛选出四个均可直接结合在AchnFHT基因启动子上的转录因子AchnABF2、AchnMYB41、AchnMYB107和AchnMYB4；双荧光素酶活性报告显示，AchnABF2、AchnMYB41、AchnMYB107均可显著激活AchnFHT基因的表达，而AchnMYB4则抑制该基因的表达，此外，亚细胞定位表明，四个转录因子均定位在细胞核中。将四个转录因子基因在烟草中超表达，结果表明AchnABF2、AchnMYB41、AchnMYB107可显著上调烟草叶片中木栓质合成基因（包括烟草FHT基因）的表达以及木栓质的积累，而AchnMYB4则具有抑制作用。利用外源ABA和FLD（氟啶酮，ABA合成抑制剂）分别处理损伤猕猴桃果实，结果表明外源ABA可诱导AchnFHT、AchnABF2、AchnMYB41和AchnMYB107基因的表达以及木栓质单体的合成，同时抑制AchnMYB4的表达；而FLD处理的效果与外源ABA处理相反。综上结果表明，植物激素ABA通过调控AchnABF2、AchnMYB41、AchnMYB107和AchnMYB4，激活AchnFHT基因参与木栓质的合成，进而促进猕猴桃果实损伤愈合。