转录因子CsSHN3提高采后柑橘果实耐冷性的分子机制

Citrus fruit is one of the most important fresh fruits in south China, and a whole cold chain logistics process is required for its year-round supply. Improving cold tolerance of citrus fruit is an urgent issue to be addressed in citrus postharvest production. Previous studies showed that two bud mutants ‘Gannan No.1’ and ‘Long Huihong’ respectively exhibited lower and higher cold tolerance under orchard or postharvest-storage conditions, compared with original cultivar ‘Newhall’ navel orange (Citrus sinensis Osbeck). To analyze the mechanism for the differences in cold tolerance among ‘Newhall’ navel orange, ‘Long Huihong’ and ‘Gannan No.1’, the analysis of transcriptome data was carried out. The results showed the wax related transcription factor CsSHN3, which significantly responded to cold signal. Based on the results, we speculated whether the cold stress regulated fruit surface wax synthesis contributed to the cold tolerance of citrus fruit. To verify it, this project aims to analyze the cold tolerance function of CsSHN3 in citrus fruit, by suppressing or overexpressing CsSHN3 in a short-lived mountain kumquat, and silencing CsSHN3 in ‘Long Huihong’ fruit by virus-induced gene silencing (VIGS) technology, and measure the cold tolerance parameters such as wax and hormone of transgenic fruit. Additionally, yeast two-hybrid,Co-Immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) methods will be adopted to further confirm the interacting and cold-response proteins of CsSHN3 , and clarify the influence and regulation mechanism of this gene in cold tolerance during postharvest storage. The results may be of important theoretical significance for cold chain technology in citrus production.

柑橘是我国南方最重要的水果，为实现鲜食果实周年供给的目标，全程冷链贮运已成为柑橘产业的现实需求，如何提高果实对低温的耐受性是迫切需要解决的科学问题。前期对纽荷尔脐橙芽变材料‘赣南1号’和‘龙回红’田间观察和贮藏试验发现，‘赣南1号’果实耐冷性降低，而‘龙回红’果实耐冷性升高；基于上述果实耐冷性差异，转录组测序分析，筛选到一个与蜡质调控相关且显著响应低温信号的转录因子CsSHN3。由此推测，柑橘果实在低温胁迫下可能通过诱导蜡质合成提高果实耐冷性。为证实此推测，本项目拟在山金柑中超量和抑制表达CsSHN3、通过病毒诱导的基因沉默（VIGS）技术沉默‘龙回红’果实CsSHN3验证其功能，测定转基因果实蜡质、激素等耐冷相关指标；利用酵母双杂交、免疫共沉淀和双分子荧光互补技术筛选CsSHN3互作且响应低温的蛋白，以期阐明CsSHN3调控果实耐冷性的分子机理，为优化柑橘冷链技术提供理论依据。

在柑橘生产中，探讨调控果实品质和抗逆性如低温、高盐和干旱的协同机制，对改善柑橘果实风味品质，提升果实耐贮性，实现果实周年供应尤为重要。大量研究表明AP2/EREBP转录因子是调控植物非生物胁迫响应的关键转录因子，但是关于其调控果实品质的研究报道甚少。本项目发现转录因子CsSHN3编码246个氨基酸，具有一个由3个β折叠和1个α螺旋组成的AP2保守结构域，且第14位氨基酸为缬氨酸，为典型的CBF/DREB成员。CsSHN3在果实中基因表达量显著高于叶和花，在花后150天显著升高；且被外源褪黑素和赤霉素处理诱导；CsSHN3蛋白在膜上和核上均有表达，具有转录激活特性。在拟南芥超量CsSHN3植株中，与野生型相比，超量植株在200 mM NaCl生长无白化现象、生长状态更强，但没有呈现更耐低温；在番茄中CsSHN3超量表达也不能提高植株和采后果实耐冷性。有趣的是，超量CsSHN3番茄果实的可溶性糖如蔗糖和果糖、氨基酸如谷氨酸、脂类如棕榈酸单甘油酯含量显著升高；结合转录组发现，超量株系糖积累相关基因被显著升高，如蔗糖转化基因和糖转运基因。将CsSHN3基因瞬时超量转化“纽荷尔”脐橙果实发现超量果实蔗糖和脯氨酸含量显著高于空载果实。将CsSHN3基因沉默后，植株蔗糖含量（2.86 mg/g）显著低于野生型，果糖含量降低到野生型33.31%，氨基酸如脯氨酸和亮氨酸也显著降低。基于转录组、DAP-seq和双荧光素酶等技术初步筛选SHN3调控蛋白CsSUC3-like1。利用非洲爪蟾卵母细胞表达体系证实CsSUC3-like1在pH5.5条件下有较强的蔗糖和果糖的共质子转运能力；在番茄中超量CsSUC3-like1表达后果实的果糖和蔗糖含量均显著升高。本项目初步验证了CsSHN3与CsSUC2-like3模块参与柑橘果实糖积累进而调控品质形成，为生产高品质和抗逆性柑橘提供理论依据。