基于转录组学的山药 (Dioscorea opposita) 块茎膨大期淀粉合成的机理研究

Yam (Dioscorea spp.) is the most important and characteristic crops for vegetable, food and Chinese medicine. Starch synthesis and accumulation in yam tubers with the tuber development. So starch is the principle constituent of yam tubers and is one of considerable importance factor during tuber swelling period of yam. Starch is also the important factor of yam tuber quality and yield. .However, a description of the complete inventory of genes involved in starch metabolism and their genome organization in yam plants is still missing. Moreover, mechanisms regulating the expression of starch genes in tubers remain elusive with regard to complicated in starch metabolism. There are not sufficient transcriptomic and genomic data in public databases for understanding of the molecular mechanism underlying the tuberous formation and development. Thus, high throughput transcriptome sequencing is need to generate enormous transcript sequences from yam tuber for gene discovery and molecular marker development. In addition to its agricultural importance, the yam tuber, involved in carbohydrate storage and vegetative propagation, is also a unique organ, which has the value of biological research for organogenesis and evolution. .This program aimed at identifying and mapping the complete set of yam starch genes and studying their expression pattern in tubers by different sets of transcriptome data. On the base of our previous research about yam, the material Dahechangyu yam (Dioscorea opposita Thunb.) , a high starch yam and Bikeqi yam (Dioscorea opposita Thunb.), a low starch yam as control were selected respectively. The project intends to sequences cDNA fragment sequences of expresses by RNA-sequence technique and to construct gene expression pool then to isolate different expression genes about starch synthesis in yam tuber. Function and expression of these differential genes were primarily elucidate by bioinformatics technologies such as GO classification and KEGG Pathway analysis. Complete length sequence of genome DNA and cDNA of these genes related starch synthesis in yam tuber will be clone by RACE techniques with designed primers based on differential fragment sequenced known. These starch synthesis related genes will further testified in express pattern by real-time fluorescence quantitative PCR, subcellular localization and the structures of these gens will be analysis too. .In this project will offering valuable sequence resource to yam community, an efficient, inexpensive and reliable approach for transcriptome sequencing that could be adopt by researchers studying non-model organisms will be provided.

山药(Dioscorea spp.)是我国粮、菜、药兼用的重要特色作物。淀粉是山药块茎的主要成分，随山药块茎的膨大而合成积累。块茎中淀粉含量及性质决定山药品质，也是研究块茎膨大机制的重要指标之一。本研究以高淀粉含量品种大和长芋山药(Dioscorea opposita Thunb.)为材料，低淀粉含量品种内蒙古毕克齐山药(Dioscorea opposita Thunb.)为对照，用转录组测序技术对山药块茎形成过程中淀粉积累表达基因测序，筛选差异表达基因，通过生物信息学解析相关基因的功能和表达；根据已知基因片段设计引物，克隆淀粉合成相关基因cDNA全长序列和对应的gDNA序列，进行生物信息学分析和基因结构分析；构建植物瞬时表达载体；对候选基因进行亚细胞定位；及山药不同组织和外源蔗糖处理叶片的表达分析。旨在明确山药块茎淀粉合成机制，挖掘功能基因，为山药优良资源的利用发挥重要的作用。

山药（Dioscorea opposita Thunb.）是我国粮、菜、药兼用的重要特色作物。淀粉是山药块茎的主要贮藏物质，占干物质含量的65%以上，直接影响山药的产量和品质。内蒙古中西部地区因其生产的山药高产优质，已成为我国山药种植的新兴产区之一。因此，利用现代分子技术研究山药块茎膨大期淀粉合成积累机制对优良基因的发掘和品种的培育具有重要意义。.根据实验室前期研究得到的内蒙古地区山药淀粉积累规律，对山药块茎不同膨大时期的形态及生理指标进行测定；采用RNA-seq技术，对山药块茎形成过程中不同时期的样品进行转录组深度测序，获得大量与淀粉合成、积累相关的表达基因信息，并采用生物信息学分析方法对转录组数据的比较分析；通过差异表达基因（Differentially expressed genes，DEGs）的筛选，对这些DEGs的GO（Gene ontology）功能富集分析、KEGG Pathway富集分析和差异基因共表达网络分析，将在淀粉合成、积累过程中起关键作用的DEGs 作为候选基因进行克隆及生物信息学分析，克隆了候选基因对应的gDNA序列，并进行基因结构分析；构建植物瞬时表达载体转化烟草细胞对候选基因进行亚细胞定位；对候选基因进行山药不同组织和不同发育时期的表达分析。探究了山药块茎不同膨大时期的形态及生理指标的变化规律；通过转录组分析，筛选得到了与淀粉合成、积累相关的表达基因信息；分离克隆与山药淀粉相关的基因7个，并对基因在山药中的表达模式进行分析。.本研究从山药块茎膨大的形态、生理及分子水平上对山药淀粉合成、积累的机制进行深入解析，明确了山药块茎淀粉合成积累的分子调控机制，为山药育种建立一个丰富的基因资源库，为山药品质遗传改良和优质品种的选育提供技术和理论依据。