苦荞全基因组11S种子储藏蛋白基因的鉴定及表达分析

以苦荞（Fagopyrum tataricum（L.）Gaertn）全基因组数据为平台，采用生物信息学方法，挖掘出9个11S种子储藏蛋白基因，并对其定位、蛋白结构、系统发育及表达模式进行了分析。结果表明，苦荞9个11S种子储藏蛋白基因编码的蛋白长度为189~914 aa，等电点位于5.18~9.82之间，分子量为21.27~103.33 kD；定位分析结果显示，这些成员位于苦荞基因组的6条连锁群上（Megascaffold2/5以及scaffold77/344/395/861）；序列比对分析发现，除了1个11S种子储藏蛋白sample1_00009513-RA具有1个cupin保守结构域外，其余8个都含有2个cupin结构域，并且在cupin保守结构域中，苦荞和拟南芥（Arabidopsis thaliana（L.）Heynh）共有14个保守的氨基酸残基；蛋白结构预测表明，苦荞11S种子储藏蛋白的结构具有2种类型；苦荞与其它6个物种拟南芥、花生（Arachis hypogaea Linn.）、大豆（Glycine max（Linn.）Merr.）、杏仁（Armeniaca vulgaris Lam.）、胡桃（Juglans regia L.）和芝麻（Sesamum indicum Linn.）]11S种子储藏蛋白以及苦荞过敏蛋白（TBb和TBt）系统发育分析结果表明，这些蛋白可以分为3类，共具有4对旁系同源蛋白和3对直系同源蛋白；与已报道的苦荞过敏性储藏蛋白以及其它5个物种（花生、大豆、杏仁、胡桃和芝麻）的11S过敏蛋白比较发现，5个11S种子储藏蛋白（sample1_00013128-RA、sample1_00013130-RA、sample1_00021677-RA、sample1_00021668-RA和sample1_00021674-RA）与苦荞2个过敏蛋白的同源性较高，同时它们与胡桃11S过敏蛋白的同源性最高，但尚需进一步实验来确定这5个成员是否为食物过敏原；RNA-Seq转录组数据显示，4个基因（sample1_00018411-RA、sample1_00026786-RA、sample1_00021674-RA、sample1_00022718-RA）在2种荞麦属植物的灌浆期种子中表达水平较高，且在‘大苦1号’中的表达水平要高于‘大甜1号’。

Genome-wide identification and expression analysis of 11S seed storage protein genes in tartary buckwheat (Fagopyrum tataricum)

Based on whole genome data of tartary buckwheat (Fagopyrum tataricum (L.) Gaertn), we used bioinformatics to exploit nine 11S seed storage protein genes, and analyzed their identification, location, protein structure, phylogenetic relationship, and expression. Results showed that the encoded protein lengths of these nine genes ranged from 189 to 914 aa, the isoelectric points ranged from 5.18 to 9.82, and the molecular weights ranged from 21.27 to 103.33 kD. Sequence alignment showed that one 11S seed storage protein (sample1_00009513-RA) contained only one cupin conservative domain, with the other eight members all containing two cupin conservative domains. In addition, there were 14 conservative amino acid residues in the cupin domain between tartary buckwheat and Arabidopsis. Location analysis demonstrated that these genes were mapped on six linkage groups of the tartary buckwheat genome (Megascaffold2/5 and scaffold77/344/395/861). Prediction analysis suggested that the tartary buckwheat 11S seed storage proteins exhibited two types of protein structure. The phylogenetic relationship of the 11S seed storage proteins from tartary buckwheat and six other species (Arabidopsis, peanut, soybean, almond, walnut, and sesame) indicated that these proteins could be classified into three groups, containing four pairs of paralogous and three pairs of orthologous. Compared with the reported allergic storage proteins in tartary buckwheat and 11S allergens from five other species (peanut, soybean, almond, walnut, and sesame), five 11S seed storage proteins demonstrated high similarity, with the highest similarity found with walnut; however, further experiments are needed to clarify whether these five members are food allergens or not. RNA-Seq analysis revealed that only four members (sample1_00018411-RA,sample1_00026786-RA,sample1_00021674-R,sample1_00022718-RA) had high expression levels in milk stage seeds of two kinds of buckwheat, and were more highly expressed in ‘Daku 1’ than in ‘Datian 1’.