马铃薯糖苷生物碱合成的分子调控研究

The potato (Solanum tuberosum L.) glycoalkaloids (GAs) are important components of plant resistance against pathogens and pests as well as osmotic adjustment but can be toxic to humans at high levels. The study of space genetic regulation of potato GAs biosynthesis and distribution will has important theoretical and practical significance for exploring molecular regulation strategies and methods of plant secondary metabolite, improving resistance to biotic or abiotic-stress and quality of plant. In order to accurately control the GAs biosynthesis and accumulation in different organs of potato, the project will clone the key enzyme genes associated with GAs biosynthesis and leaf-specific and tuber-specific promoters by molecular techniques, construct vectors that have efficient leaf-specific expression of the target gene and tuber-specific inhibited expression respectively, and gain genetically modified material by transforming model plant or potato. We will analyze the target-gene expression levels by real-time quantitative polymerase chain reaction (RQ-PCR) method, meanwhile determine the GAs content by HPLC method in different organs of the transformation materials and according to the results we will screen out the highly organ-specific promoters and their effective gene expression vector structure.Eventually,the project will construct an integration vector that can make the key enzyme gene overexpressed in leaves but inhibited in tubers, and gain potato germpalsm resources with GAs content enriched in the foliage but diminished in the edible tubers by agrobacterium-mediated genetic transformation.This will develop a new way of creating germpalsm with stress tolerance for potato breeding.

马铃薯糖苷生物碱（GAs）是抗虫、抗病原体并具有渗透调节功能的重要成分，但高浓度GAs对人体有毒。开展马铃薯GAs合成和分布的空间遗传调控研究，对于探索次生代谢物分子调控策略和方法、提高植物抗逆性和改良品质将具有重要的理论和实践意义。为了精确地控制马铃薯不同器官中GA的合成和积累，本项目拟采用分子克隆技术分离GAs合成代谢途径的关键酶基因及叶片和块茎特异性启动子，分别构建叶片特异表达和块茎特异沉默载体，转化模式植物或马铃薯以获得转基因材料；采用实时定量PCR技术和HPLC方法鉴定转化材料中不同器官和组织中关键酶基因的表达水平和GAs含量，以此为依据筛选出具有器官高度特异性启动子及其有效的基因表达载体结构，最终构建能够使GAs合成的关键酶基因在叶片中过表达和块茎中受到抑制的整合载体，通过农杆菌介导法获得叶片GAs含量丰富而食用块茎含量低的材料，为马铃薯育种创新抗逆种质资源开辟一条新的途径。

本项目对马铃薯GAs合成的分子机理、关键酶基因和组织特异启动子克隆、关键酶基因表达载体和RNAi载体及调控GAs的效果进行了研究。取得的主要成果有：. 1. 通过马铃薯GAs合成代谢相关酶基因的表达水平研究，明确了GAs合成代谢途径关键酶为葡萄糖基转移酶（SGT1）、半乳糖基转移酶（SGT2）和鼠李糖基转移酶（SGT3）。采用RT-PCR方法，克隆到3个糖基转移酶基因（sgt1、sgt2和sgt3）全长cDNA。采用PCR技术，克隆了绿色组织特异rbcS启动子和块茎特异Patatin启动子；采用染色体步移方法，分离到SGT1、SGT2和SGT3基因的启动子，经分析和功能鉴定这3个启动子具有光诱导、脱水胁迫和干旱诱导的特性，序列已注册到GenBank。. 2. 构建了rbcS P驱动的SGT3基因植物表达载体pCERS3，用于提高马铃薯地上部绿色组织GAs含量。同时，成功构建以3个SGT末端酶基因为靶标的块茎特异RNAi载体pCEI-PFR，用于降低块茎中GAs的积累的种质创新。. 3. 以提高地上部绿色组织中GAs含量为目标，采用pCERS3载体对丰产优质、但抗病性较差和易退化的2个优良品种进行转化，12份转基因株系；同时，采用pCEI-PFR载体对丰产抗病性较强、但块茎GAs含量高的品种2个品种进行转化，获得了地上部GAs未显著改变、而块茎GAs含量下降的8个株系。. 4. 为了识别光响应的miRNA / mRNA，由此miRNA-mRNA对GAs调节，对红光刺激下的马铃薯进行了整合组学（sRNAome和转录组）分析。在叶和块茎中分别鉴定出了差异表达的31个和48个miRNA。这些发现将启发我们对次生代谢途径的遗传调控，为应用基因工程调控马铃薯GAs开辟新的途径。. 本研究建立GAs合成和空间分布分子调控技术，对于创造高抗逆性和食用安全的马铃薯种质资源具有重要的作用和意义。同时，这一研究成果对于植物次生代谢物的调控也具有一定的参考价值。