甜瓜果实呼吸跃变过程中差异表达基因的分离鉴定及功能分析

Fruit ripening is an important period in the higher plant life cycle. It is a genetically programed event that is characterized by a series of physiological and biochemical changes, which is caused by temporal and spatial expression of the genes involved. Respiratory climacteric is unique physiological phenomenon during climacteric fruit ripening, and that is the turning point of the fruit from mature to senescence. At present, the physiological and biochemical processes during the fruit ripening and the mechanisms of ethylene regulation of climacteric fruit ripening have been partly understood. However generally speaking, the molecular mechanisms of the fruit respiratory climacteric is not yet clear. In this study, the digital gene expression(DGE) profile of climacteric and pre & post-climacteric stages of melon fruit will be sequenced with Illumina high-throughput sequencer, so as to determine the differentially expressed genes of unknown function during the respiratory climacteric stage of melon variety Hetao, which is a typical respiratory climacteric fruit. Then the expression patterns of some candidate genes at different developmental stage of fruits will be analyzed by RT-qPCR. The function of some key candidate genes will be analyzed by melon fruit transient expression. The results will have important scientific significance for deeply realizing and laying the foundation for further elucidating the molecular mechanism of melon fruit respiratory climacteric. In addition, the results of this study will be expected to provide new functional genes in genetic engineering to improvement of melon fruit quality, and will have important application prospects.

果实成熟是高等植物生命周期中的一个重要环节，是一个复杂的生理生化变化过程，是生物体基因时空特异表达的结果。呼吸跃变是果实成熟过程中特有的重要生理现象，是果实从成熟到衰老的转折期。目前人们虽然对果实成熟的生理生化过程和乙烯调控跃变型果实成熟的分子机理已有一定的了解，但总体上讲，人们对果实呼吸跃变分子机理的认识还十分有限。本研究以典型的呼吸跃变型甜瓜品种河套蜜瓜为研究对象，应用新一代高通量测序技术分析呼吸跃变期和跃变前后各时期果实数字基因表达谱，鉴定呼吸跃变过程中差异表达的未知功能基因，应用RT-qPCR技术对表达差异显著的候选基因进行表达特性分析，采用甜瓜果实瞬时表达技术验证其功能。研究结果将加深对甜瓜果实呼吸跃变分子机理的认识，并为进一步全面深入阐明甜瓜果实呼吸跃变的分子机理奠定基础，具有重要的科学意义。此外，研究结果有望为甜瓜果实性状改良的基因工程提供新的功能基因，具有应用前景。

本研究以典型的呼吸跃变型甜瓜品种河套蜜瓜为研究材料，采集其果实生长期（G期）、成熟期（R期）、呼吸跃变期（C期）和呼吸越变后期（P期）四个典型发育时期果肉样品，进行了转录组、lncRNA和miRNA测序，并进行相关生物信息学、表达谱、基因超表达和编辑等功能相关分析。结果表明：（1）转录因子基因CmNAC34、CmbHLH93/130，热休克蛋白基因CmHsp70-9和CmHsp83，酶蛋白基因CmEXPA8、CmXTH28-2，生长素初级相应基因CmIAA16具有促进果实成熟的功能；CmEXPA12基因具有抑制果实成熟并促进果实膨大的功能。（2）鉴定了142个高表达lncRNAs（平均FPKM >100），对聚类在植物激素信号转导通路、乙烯合成通路、糖类合成信号通路以及部分转录因子的差异lncRNA和mRNA进行了进一步分析，发现基因CmACO、CmSAPK、CmETR，转录因子基因ERFs、NAC56、NAC73和WRKY70和部分lncRNAs（LNC_000987、LNC_000693、LNC_001323、LNC_003610 和LNC_003380）与甜瓜果实发育及成熟密切相关；LNC-003380与PDC1、SAPK4、AG2三个基因存在共表达关系；LNC-003610是甜瓜果实成熟的负调控因子。（3）共鉴定得到61个保守和24个新miRNAs，其中32保守miRNAs在果实发育过程中差异表达，从降解组测序数据中鉴定得到43个保守和1个新miRNA的靶基因共计62个，Cme-miR393具有正调控果实成熟的功能，其靶基因为CmTIR1 和CmAFB2。上述研究结果丰富了人们对甜瓜果实发育成熟及呼吸跃变分子机理的认识，具有重要的科学意义，所鉴定的部分功能基因在甜瓜果实性状改良的基因工程中具有应用前景。已发表3篇期刊论文，会议报告论文1篇，培养博士生2名、硕士生11名。