黄皮甜瓜低温/抗冷诱导过程中多胺（PAs)代谢与转录因子CmCBFs之间的关系研究

Yellow melon fruits are very sensitive to low temperature and will develop chilling injury when temperature is below 5℃, resulting in reduced commercial quality. Therefore, studying on the incidence and regulation mechanism of chilling injury in yellow melon is theoretical and practical. Polyamines (PAs) and the transcription factor CBF (C-repeatbinding factor) play the important role in plant resistance to cold, and are closely related to the incidence of chilling injury in fruits and vegetables. Our previous research has shown that there are the relationship between putrescine (Put), spermine (Spm), CmCBF1 and CmCBF3 and chilling injury, but it is unknown whether there is the relationship between PAs and CmCBFs under low temperature .Thus, ‘Xinmi No.3’ fruit was selected as materials, the different temperatures, exogenous polyamines treatment and overexpression of CmCBFs were carried out on the chilling injury, polyamines, key enzymes of metabolism, and CmCBFs expression in yellow melons using the techniques/methods of traditional postharvest physiology, and combining modern PCR amplification techniques, real-time fluorescence quantitative qPCR, and Agrobacterium-mediated , In order to explore the relationship between polyamine metabolism and the transcription factor CmCBFs during low temperature/cold resistance . it provides a new idea for the directional regulation of postharvest chilling injury of yellow melon fruit, and lays a theoretical foundation for molecular breeding of melon.

黄皮甜瓜对低温较为敏感，低于5℃即会出现冷害症状，降低果实商品性。因此，研究黄皮甜瓜果实冷害发生及其调控机制，具有十分重要的理论和现实意义。多胺（PAs）和转录因子CBF（C-repeatbinding factor）在植物抗冷过程中发挥了重要作用，与果蔬冷害发生密切相关。本团队前期研究发现，黄皮甜瓜冷害发生与腐胺（Put）、精胺（Spm）、CmCBF1及CmCBF3有关，然而，多胺与CmCBFs之间是否有关联，还不清楚。为此，本研究以‘新密3号’为试材，采用传统采后生理学，结合现代PCR扩增技术、实时荧光定量qPCR和农杆菌介导技术/方法，研究不同温度、外源多胺处理及CmCBFs过表达对黄皮甜瓜冷害、多胺、代谢关键酶及CmCBFs表达的影响，以期探索低温/抗冷诱导过程中多胺代谢与转录因子CmCBFs之间的关系，为黄皮甜瓜果实采后冷害定向调控提供新思路，为甜瓜抗冷分子育种奠定理论基础。

本项目以‘新密3号’为试材，采用传统采后生理学，结合现代PCR扩增技术、实时荧光定量qPCR和农杆菌介导技术/方法，研究了不同温度、外源多胺处理及CmCBFs过表达对黄皮甜瓜冷害、多胺、代谢关键酶及CmCBFs表达的影响，筛选出调控黄皮甜瓜‘新密3号’果实采后冷害的多胺类型为腐胺（Put），其适宜的浓度为2.0 mmol/L ；克隆获得了黄皮甜瓜冷诱导转录因子CmCBF1-6基因全长，即CmCBF1因全长序列分别为639 bp，编码212个氨基酸，编码蛋白质分子量为23.89 kDa，等电点为5.23；CmCBF2全长651bp，编码216个氨基酸，蛋白质分子量为54.64kDa，等电点为5.16；CmCBF3基因全长序列分别为615bp，编码204个氨基酸，蛋白质分子量为22.62 kDa，等电点为8.42；CmCBF4全长675bp，编码224个氨基酸，蛋白质分子量为56.35kDa，等电点为5.19；CmCBF5全长1683 bp，编码560个氨基酸，蛋白质分子量为138.99 kDa，等电点为5.01；CmCBF6全长726 bp，编码241个氨基酸，蛋白质分子量为61.08 kDa，等电点为5.09，其中CmCBF1-3受低温诱导瞬时表达；解析了外源Put处理调控黄皮甜瓜果实采后冷害一方面通过调控精氨酸脱羧酶（ADC）、鸟氨酸脱羧酶（ODC）、N-氨基甲酰腐胺水解酶（CPA）、S-腺苷甲硫氨酸脱羧酶（SAMDC）、二胺氧化酶（DAO）和多胺氧化酶（PAO）活性，诱导亚精胺（Spd）水平，另一方面通过诱导果皮组织CmCBF-3基因的短时表达，从而提高其采后抗冷性；明确了转录因子CmCBF1在果实抗冷中的作用，证实了黄皮甜瓜抗冷诱导过程中內源腐胺与CmCBF1基因之间的正相关关系，以上结果为黄皮甜瓜果实采后冷害定向调控提供科学依据，为甜瓜抗冷分子育种提供新思路。..