玉米淀粉合成酶V基因(ZmSSV)的功能解析

Starch synthases (SSs), a group of key enzymes in maize starch biosynthesis, elongate dextran chains, which influence the content and structure of endosperm starch, and eventually determine crop yield and quality. Maize SSs are grouped into five classes, including SSI-Ⅳand granule-bound starch synthase (GBSS). GBSS synthesizes amylose, SSI-III synthesize amylopectin with different chain lengths, and SSⅣ is responsible for initiation of starch granule formation. In previous study, we have identified a SS isoform encoding the SSV class in maize by a comparative genomic analysis, designated ZmSSV. SSV has been found in all green plants with conserved sequences and gene structures. Our phylogenetic analysis has suggested that SSIV and SSV resulted from a gene duplication event. An expression profile analysis of ZmSSV in maize has indicated that ZmSSV is mainly transcribed in the kernel and ear leaf during the grain filling stage. The protein encoded by ZmSSV with SS activity can elongate dextran chains. In this project, the optimum temperature, pH and substrate of ZmSSV will be determined, and the function of ZmSSV in maize starch content, structure and morphology of starch granule will be analyzed by transgene-induced mutants.

玉米淀粉合成酶是淀粉生物合成中的关键酶之一，催化葡聚糖链的延伸，影响胚乳淀粉含量和结构，决定作物产量和品质。玉米中已知的淀粉合成酶有五种亚型（GBSS、SSI、SSII、SSⅢ和SSⅣ），分别负责合成直链、不同链长的支链淀粉，以及淀粉粒的起始形成等。申请人利用比较基因组技术新鉴定了一个玉米淀粉合成酶基因ZmSSV，编码ZmSSV亚型。该基因存在于所有绿色植物中，与已知的SSⅣ共同起源于一次祖先基因复制，在灌浆期的叶片和籽粒中高表达，其编码蛋白具有淀粉合成酶活性，能延伸葡聚糖链。在此基础上，本项目拟经体外实验分析 ZmSSⅤ的最适温度、pH和底物等，明确基本酶学性质；采用转基因技术创制突变体，分析ZmSSⅤ对淀粉含量、结构和淀粉粒形态的影响，揭示ZmSSV的功能。

玉米淀粉合酶是淀粉生物合成中的关键酶之一，影响胚乳淀粉含量和结构，决定产量和品质。经比较基因组学分析,鉴定了一个玉米淀粉合酶基因ZmSSV，编码ZmSSV亚型，不同于五种已知的淀粉合酶亚型（GBSS、SSⅠ-Ⅳ），在此基础上，本项目主要开展ZmSSV的功能研究。经原核表达和体外酶学特性分析，表明其最适温度为37℃，最适pH为7.5，最适底物为牡蛎糖原。双倒数作图法计算其米氏常数为0.116±0.012 mmol/L，最大反应速率为0.117±0.004 nmol/min。从玉米突变体库获得的突变体其Mu元件插入起始密码子上游约60bp处，qPCR分析发现ZmSSV的表达并未改变，不能用于后续的功能分析。因此采用转基因技术创制突变体和过表达材料，剖析ZmSSV对淀粉合成的影响。通过设计、构建和转化gRNA-Cas9 双元载体和过表达载体，获得了3个在籽粒中高表达的阳性株系。分析其淀粉合成特性，发现ZmSSV在籽粒灌浆的中后期表达量增高，最高表达量出现在DAP30天，相较对照表达量提高了10-90倍；ZmSSV过表达使成熟籽粒总淀粉含量提高了4%-7%，籽粒中的短链和中长链淀粉含量略微增多；扫描电镜分析表明籽粒淀粉粒形态、大小无明显变化。综合分析ZmSSV体内外催化产物的链长，发现可延伸短链、中长链和超长链淀粉，链长催化谱较宽。推测过表达的ZmSSV可能协同其他淀粉合成相关酶类促进籽粒淀粉合成，提高淀粉含量。研究结果为深入揭示玉米淀粉合成分子机理，改良作物产量和品质奠定了理论基础。