马铃薯淀粉酶StBAM9在低温糖化过程中的功能解析

Accumulation of reducing sugars in potato tubers exposed to low temperature is an important factor that strongly impacts on quality of the processing products. Starch degradation plays a vital role during this process known as cold-induced sweetening (CIS). Based on identifiction of amylase, StBAM9, that functions in favor of starch hydrolyzation of cold stored potato tubers, the present research will look insight the regulatory mechanism uncoving transcriptional and posttranslational function modulation of StBAM9. The project will be implemented firstly by cloning, cis-elements analysing of the promoter of StBAM9 and dissecting its transcription factors. This part will provide evedences if and how StBAM9 is transcriptionally controlled. Simultaneously, the interacted proteins of StBAM9 will be clarified, for this the strategies of Y2H for candidate target proteins, Pull-down for direct counterparts, immunoelectron microscopy and transient gene expression assays for subcellular localization of StBAM9 and the target proteins will be taken. The interaction pattern between StBAM9 and the target proteins will be dissected by multiple approsches such as structural simulation, x-ray crystallography, NMR and site directed mutagenesis. The analyses of the affinity to substrate of StBAM9 and the target proteins will be applied to compensate the regulatory mechnism of StBAM9. This study will enrich our understanding of the molecular mechanism of potato CIS, and also provide important genetic resources and theoretical basis for improving the processing quality of this economically important crop.

马铃薯块茎低温贮藏时还原糖的累积是影响油炸加工品质的重要因素，其中淀粉降解起着重要作用。本项目在已完成淀粉酶基因功能鉴定的基础上，拟完成马铃薯淀粉酶StBAM9基因启动子顺式元件分析、转录因子分离及验证等，明确StBAM9转录水平调控机制。利用Pull-down和酵母双杂交等技术，分离StBAM9互作蛋白；通过免疫电镜、基因瞬时表达等方法，明确StBAM9和目标蛋白互作的亚细胞定位；并利用结构模拟、X-晶体衍射和NMR等技术，明确互作位点，通过定点突变、体外添加活性测定等试验，确定其与互作蛋白的互作模式，明确其翻译后调节机制。在此基础上，通过对StBAM9结构域分析及底物亲和力检测以及其与互作蛋白及底物共孵育等，明确StBAM9特异靶向底物的结构域及其与互作蛋白、底物三者的作用方式。研究结果将进一步明确马铃薯抗低温糖化淀粉降解途径的调控机理，为马铃薯加工品质改良提供理论指导和新的基因资源。

马铃薯是我国重要的粮食作物，低温贮藏导致块茎还原糖的累积即低温糖化现象严重制约了马铃薯加工品质。淀粉降解在低温糖化过程中起着重要作用，本实验室前期研究表明StBAM9在低温贮藏过程中淀粉降解起着重要作用，然而StBAM9的转录调控机制及其翻译后水平调节机制还不明确。为了探究StBAM9的作用机制，本研究通过构建低温储藏马铃薯块茎的酵母文库，对StBAM9的互作蛋白进行了分离和筛选。并对相关蛋白进行了互作验证，结果显示StBAM9与StBAM1和StTPR01660互作，进一步通过截短对互作区段进行了鉴定。此外，还对互作蛋白StTPR01660进行了功能鉴定，初步阐明了其在马铃薯叶片淀粉降解过程中的重要作用。同时还对StBAM9的启动子进行了克隆和分析，结合其启动子元件预测和表达模式分析，我们筛选了可能结合并调控StBAM9的转录因子StAREB/ABF/ABI5家族，通过转录激活、表达模式分析、亚细胞定位和双荧光素酶验证，初步明确StAREB2通过识别并结合StBAM9启动子上的ABRE元件从而实现对StBAM9转录水平的调控机制。本项目基本阐明了StBAM9转录水平和翻译后水平的调控机制，为进一步阐明低温诱导块茎淀粉降解机理奠定了基础，也为马铃薯品质改良提供了新的基因资源。