甜瓜非乙烯响应CmADH4表达调控相关转录因子的筛选与功能分析

Aliphatic aromatic substances are important components of fruit flavor. Most of aliphatic aroma compounds are derived from Lipoxygenase (LOX) pathway in plants and ethylene induction is a common feature of LOX pathway in climacteric fruits. Alcohol dehydrogenase (ADH) is a key gene in LOX pathway. Previous studies showed that CmADH4 participated in aroma synthesis of respiratory climacteric melon fruits, but did not respond to ethylene induction. So its regulatory mechanism needs further study. Transcription factors are important pathways to regulate secondary metabolism in plants. In this study, the promoter of CmADH4 will be obtained by the sequence information on melon genome website and 5'RACE technology. The bait sequence was designed by bioinformatics analysis, and the regulatory transcription factors of CmADH4 in melon fruit were screened by yeast single hybridization. The temporal and spatial expression patterns of candidate transcription factors (CTFs) were analyzed by real-time quantitative PCR. The interaction between candidate transcription factors and CmADH4 promoter was detected by immunoprecipitation technique. The function was verified by transient expression in melon fruit mediated by Agrobacterium tumefaciens. Based on the above results, we hope to elucidate the regulation mechanism of non-ethylene response CmADHs in the fruit of respiratory climacteric melon, and improve the development regulation network of melon.

脂肪族香气物质是果实风味的重要组成部分，众多呼吸跃变型植物果实中的脂肪族香气物质主要来源于脂氧合酶（LOX）途径的代谢，受乙烯调控是该途径的特征之一。醇脱氢酶（ADH）是LOX途径的关键基因，前期研究表明部分CmADH4参与呼吸跃变型甜瓜果实的香气合成，但不响应乙烯诱导，其调控机制尚未明确。转录因子是调控植物次级代谢的重要途径，已有报道参与植物果实的香气合成调控。本研究拟采用甜瓜基因组网站上的序列信息结合5’RACE技术，获得非乙烯响应CmADH4的启动子；利用酵母单杂交技术筛选甜瓜果实内CmADH4的调控转录因子；实时荧光定量PCR分析候选转录因子 (CTFs) 的时空表达模式；免疫共沉淀技术检测候选转录因子与CmADH4的互作；并在根癌农杆菌介导甜瓜果实内瞬时表达验证其功能。综合上述结果，期待阐明呼吸跃变型甜瓜果实中非乙烯响应CmADHs基因的调控机制，完善甜瓜果实发育的调控网络。

甜瓜(Cucumis melon L.)是全世界广泛栽培的园艺作物，是世界十大水果之一，其薄皮甜瓜亚种（ssp.agrestis Jeffrey）下的香瓜变种（var. makuwa Makino）风味独特，是我国特色的甜瓜类型。目前，我国是甜瓜栽培面积和产量最大的国家，但薄皮甜瓜果实品质调控有关的研究相对滞后。前人对薄皮甜瓜品质调控的研究多集中在乙烯调控的脂氧合酶途径等相关通路上，而本研究基于前期从薄皮甜瓜越瓜变种（var. conomon Thunberg）“八棱脆”中筛选出的非乙烯响应的香气物质合成关键基因CmADH4，从非乙烯调控的角度进行薄皮甜瓜果实生长发育及果实品质调控的研究，以期进一步完善薄皮薄皮甜瓜果实成熟调控的理论体系。本项目中，我们通过染色体步移获得了薄皮甜瓜中的非乙烯响应CmADH4启动子序列，并通过酵母单杂交结合生物信息学分析筛选到了其互作的ABA响应的转录因子ASR，由凝胶迁移试验和果实内瞬时表达验证了其对CmADH4启动子的互作和在果实内的调控效果。最后通过转录组分析初步评估了ASR对果实发育进程的影响。结果表明在薄皮甜瓜果实发育品质形成过程的前中期，ASR发挥了一定的调控作用，这也暗示了ABA参与了薄皮甜瓜果实成熟前期发育的进程。这与近几年部分果实的发育成熟过程由多激素、分阶段调控的研究结论相似。尽管ABA及ASR具体影响的薄皮甜瓜果实发育成熟代谢通路和具体进程仍需要进一步验证明确，但相对于原有的着眼于乙烯调控途径而言，本研究结果为薄皮甜瓜及其它呼吸跃变果实的发育过程和果实品质调控提供了一个新的思路。