糖响应转录因子ClATB2调控西瓜果肉有色体发育的机制

In watermelon fruit development, the sugar accumulation and flesh color development are collaborative pathways. While effectors of this crosstalk between the two have not been identified. In red-flesh elite Chinese cultivar 97103 (C. lanatus var. lanatus) the sugar accumulation with the chromoplast formation during fruit development. But in white-flesh U.S. Plant Introduction (PI) 296341FR (C. lanatus var. citroides) fruit, these two products are missing. Our preliminary study found a fruit-special phosphate transporter gene ClPHT4;2, whose expression level was greatly consistent with watermelon chromoplast development. It’s transcription level increased obviously as fruit ripening in 97103 whereas the level was always low in PI296341FR. Moreover, we found a key bZIP-type transcriptional factor ClATB2 which own a sucrose-controlling uORF can regulate this different expression. All the results will explore the functions of ClATB2 in crosstalk between chromoplast development and sugar accumulation. We try to use RACE, EMAS, and tobato leaf transformation system to analysis trans-activation activity of the ClATB2 protein. RNAi and overexpression of ClATB2 will be constructed to verify their biological function in chromoplast development. The sucrose-controlled translation of this gene will be testified using transform the UBQ10:ClATB2 5’UTR-GUS/GFP to Arabidopsis. Our research will provide new insights into understanding the important effectors in the crosstalk between watermelon chromoplast development and sugar accumulation pathways and gives a new theoretical basis for watermelon breeding.

栽培西瓜果实糖分积累及瓤色形成是一个精密协同发育的过程，但两条途径的交联（crosstalk）作用机制尚不清楚。红瓤西瓜果实糖分积累由低到高，其有色体发育导致瓤色由白到红，而白瓤野生无糖西瓜却无上述变化。本项目前期研究发现磷转运蛋白ClPHT4;2为有色体发育的关键基因，在红瓤与白瓤品种中表达差异显著；并发现bZIP类转录因子ClATB2可以调控这种差异转录，最引人关注的是ClATB2具有糖调控上游开放阅读框序列。那么ClATB2是如何响应糖浓度变化，并协调有色体发育与糖代谢呢？本项目拟采用RACE、EMSA、瞬时转化及西瓜遗传转化技术分析ClATB2对西瓜有色体发育的影响；利用uORF连接报告基因技术验证糖分对ClATB2的转录后调控作用；明确糖信号通过ClATB2调控果肉有色体发育的分子机制。本研究将为全面揭示果实发育调控网络提供节点基因，并为西瓜品质育种提供理论指导。

本项目研究关注西瓜果实品质中瓤色形成的关键基因及果实糖分积与瓤色形成两条途径精密协同发育的节点基因。西瓜饱满鲜艳的果实瓤色是由于西瓜果瓤细胞中的有色体不断发育并大量积累类胡萝卜素类物质形成的。西瓜有色体的发育伴随着剧烈的能量代谢过程，类胡萝卜素物质的合成及代谢等所需的能量需要经过物质转运通道与细胞质中发生交换。红瓤西瓜果实糖分积累由低到高，其有色体发育导致瓤色由白到红，而白瓤野生无糖西瓜却无上述变化。这两种材料为我们研究果实瓤色及糖分的协同发育提供了良好的实验对象。本课题首次明确了西瓜有色体质膜上的一个新型的磷转运蛋白在有色体发育中的重要作用。阐明了其转运功能及对西瓜瓤色发育的影响，该基因的大量表达是西瓜鲜艳瓤色形成的物质基础之一。为了明确该磷转运蛋白的转录调控作用，利用了酵母单杂交筛选候选转录因子，发现了ClbZIP2转录因子可以调控该有色体磷转运蛋白的表达。分析了这个转录因子的序列及表型情况，发现该转录因子具有被糖调控的SC-uORF结构，通过拟南芥转基因试验证实该结构具有对外源糖分的响应功能。各项实验表明糖分对ClbZIP2具有转录后调控作用。本项研究明确了糖信号通过ClbZIP2调控果肉有色体发育的分子机制。且该转录因子自身的表达又收到了糖信号的调控。本研究揭示果实发育调控网络提供节点基因，并为西瓜品质育种提供了候选基因。