芋头淀粉合成酶的基因分析及分子标记开发与验证

Taro (Colocasia esculenta (L.) Schott) is one of the major monocotyledons, and now as an important vegetable and crop in the world. With its tiny starch granule, taro displays special medicinal values and plays important roles in food industry. A lot of researches have been done in the starch biosynthesis process of Arabidopsis, and four gene families, including ADP-glucose pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and debranching enzymes (DBE), have been found to be involved in the starch biosynthesis of plants. However, the process of starch biosynthesis in taro is unclear, and information of taro starch synthase genes is still unknown. As one of the centers of origin and diversity of taro, China has a large number of genetic resources of taro. However, the lack of knowledge in taro starch synthase slows down developments of detection methods on taro starch, and limits the exploitation and utilization of taro starch in China. In this study, the bioinformatic method is applied in the research of taro SS gene family. Based on the transcriptome data of taro cultivar ‘Jingjiang xiangsha’, informations of taro SS genes are screened and identified, and then full-length gene sequences of these genes are cloned and sequenced. The phylogenetic analysis and functional domains analysis will show potential clusters of these SS genes in taro, and expression levels of these genes in different growth stages of taro are detected. Informations of simple sequence sepeats (SSR) and single nucleotide polymorphisms (SNP) sites in taro SS genes, including numbers, types and distribution, are analyzed by the MISA program. Based on polymorphism fragments from 50 taro cultivars with different genetic backgrounds, SSR and SNP molecular markers related with contents of taro starch are screened. Our study will be benefit for the determination of taro starch biosynthesis process, and further promote the full utilization of taro starch resources in China.

芋头富含淀粉，颗粒较小且易于消化，具有较好的食疗保健功能，在食品工业中的应用也很广泛。作为芋头的起源地之一，中国拥有丰富的种质资源，但是目前对于芋头淀粉的开发程度较低，而且资源品种间的淀粉性状存在较大差异，缺乏有效方法对芋头淀粉含量进行快速、可靠的高通量检测鉴定。本研究基于已完成的靖江香沙芋转录组测序，通过对芋头转录组数据进行功能注释，从中筛选并克隆获得芋头淀粉合成酶SS的基因全长序列，明确芋头SS基因在不同组织和生长时期的基因表达趋势。根据芋头SS基因序列中的SSR位点和SNP的分布，设计开发芋头淀粉含量相关SSR和SNP标记，从50份具有代表性的芋头种质资源中，筛选获得芋头淀粉含量特异分子标记，为芋头淀粉合成途径的研究提供理论基础，进而为我国芋头淀粉资源的充分开发利用提供保障。

芋头是一种高淀粉作物，口感好，营养高，易于消化，是热带和非洲地区的主粮食物。我国是芋头起源中心之一，拥有大量芋头野生资源和地方品种，但是对于芋头高品质淀粉资源的开发程度较低，缺乏可靠有效的种质资源检测方法。本研究通过对靖江香沙芋转录组数据的功能注释和分析，获得了芋头淀粉合成相关基因序列信息。从靖江香沙芋中克隆获得了1个ADP–葡萄糖焦磷酸化酶CeAps1和3个淀粉合成酶CeSS1、CeSS2、CeSS3的基因序列全长，并进行了氨基酸序列结构和同源性分析。基因表达结果表明，CeApS1、CeSS1、CeSS2、CeSS3基因在‘靖江香沙芋’的母芋和子孙芋中表达量较高，在叶片、叶柄和根中表现为低表达，其中CeSS2的基因表达量最高。在球茎生长过程中，3个淀粉合成酶基因表现出表达时间差异，但总体表达水平与淀粉含量之间存在显著正相关。根据转录组位点序列信息，共检测到91,689个SNP位点和11,363个SSR位点，设计开发了4,350对芋特异的SSR引物。随机选择了150对引物进行引物可靠性检测，结果显示112对引物能够扩增获得有效结果，扩增有效率为74.7%。其中100对表现出多态性，共扩增出316条清晰条带，每对引物扩增条带数在2～8条之间，表明设计的SSR引物具有较高的可靠性和重复性，可以用于种植资源的筛查与鉴定。利用筛选获得的43对多态性好、重复性可靠的SSR引物，结合芋头淀粉含量和生长表型数据，对51份芋头种质资源进行了UPGMA聚类分析。本研究开发获得的SSR分子标记，可以对不同淀粉含量芋头种质资源进行分类和初步鉴定，为芋头种质资源的开发与应用提供了理论依据和技术支持。