香蕉果实直链淀粉合成关键酶基因MaGBSSI-3表达的分子调控机制

Amylose is a major component of banana fruit, which directly determines the resistant starch content, fruit waxy, texture, and other quality. Granule-bound starch synthase (GBSS) is a key enzyme in amylose synthesis. Our preliminary findings showed that GBSS and MaGBSSI-3 expression in banana fruit is closely related to the amylose synthesis, which is a key factor regulating amylose biosynthesis. However, the regulatory mechanism of MaGBSSI-3 expression in banana fruit during development is not clear, amylose biosynthesis do not effectively regulate in banana fruit production. This study intends to study the function of MaGBSSI-3 in amylose synthesis using yeast expression system. Key cis-acting regulatory elements of MaGBSSI-3 were explored by promoter clone and 5′end deletion mutants. The yeast one-hybrid experiment was performed to screen transcription factors directly regulating the expression of MaGBSSI-3 gene. The role of regulatory of target transcription factors for MaGBSSI-3 gene was determined by co-transformation in vitro. These results will reveal the regulatory mechanisms of MaGBSSI-3 expression during development of banana fruits, and provide a theoretical basis for regulating the amylose biosynthesis of banana fruit during development by biotechnology.

直链淀粉是香蕉果实的主要成分之一，它的含量直接决定着抗性淀粉含量和果实糯性、质地等品质。颗粒结合淀粉合成酶(GBSS)是决定直链淀粉合成的关键酶，我们前期研究结果表明香蕉GBSS及果实中表达的MaGBSSI-3基因与直链淀粉合成密切相关，是影响果实直链淀粉合成的主要因素，但MaGBSSI-3在香蕉果实发育过程中表达的调控机制不清楚，生产中不能够在果实发育过程中有效调节直链淀粉的生物合成。本项目拟采用酵母表达系统鉴定MaGBSSI-3在直链淀粉合成中的作用，通过MaGBSSI-3启动子克隆和5′端片段缺失突变发掘调控MaGBSSI-3表达的关键响应元件，应用酵母单杂交方法分离直接调控该基因表达的转录因子，体外共转化鉴定目标转录因子对MaGBSSI-3基因的调控作用。本研究将揭示MaGBSSI-3基因在香蕉果实直链淀粉合成中表达的调控机制，为通过生物技术调节香蕉果实直链淀粉的合成提供理论依据。

香蕉GBSS及果实中表达的MaGBSSI-3基因是影响果实直链淀粉合成的关键因子，但MaGBSSI-3在香蕉果实发育过程中表达的调控机制不清楚，生产中不能够在果实发育过程中有效调节直链淀粉的生物合成。本项目采用酵母表达系统体外证明了MaGBSSI-3 蛋白在香蕉果实直链淀粉合成中具有活性，体内过表达MaGBSSI-3基因发现其影响了淀粉颗粒数目及直链淀粉含量。通过MaGBSSI-3启动子克隆和5′端缺失突变确定了不同调控元件的诱导活性，田间试验获得了调控MaGBSSI-3 基因表达的ABA、GA、IAA等关键响应元件。应用酵母单杂交方法分离了30个与MaGBSSI-3基因启动子互作的候选转录因子或基因，体外共转化鉴定了2个目标转录因子MaMYB44和Ma14-3-3在香蕉果实直链淀粉合成早期互作调控MaGBSSI-3基因上调表达的作用，且MaMYB44的调控强度高于Ma14-3-3。本研究揭示了MaGBSSI-3基因在香蕉果实直链淀粉合成中表达的调控机制，为通过生物技术调节香蕉果实直链淀粉合成提供了理论依据和技术支持。