苦荞种子灌浆期全长cDNA文库的构建及部分表达序列标签分析

摘要

苦荞种子富含氨基酸均衡的蛋白质和高生物活性的黄酮类物质,是典型的药粮兼用作物.灌浆期是苦荞营养物质积累和合成的关键时期,分析灌浆期的基因表达对于苦荞种子有效物质的积累和调控研究有重要的价值.本实验以苦荞(Fagopyrum tartaricum)榆6-21为材料,采用SMART(switching mechanism at 5' end of RNA transcript)技术构建种子灌浆期全长cDNA噬菌体文库.结果显示,所构建原始文库滴度为4.86× 105 pfu/mL,扩增文库滴度为6.2×109 pfu/mL,蓝白筛选证实文库的重组率为99.58％.插入片段的大小在500～3 500 bp.随机挑取环化的质粒克隆进行测序,获得文库中部分EST序列(GenBank登录号:GO477569,GO496285～GO496321).经生物信息学比对分析,54％ ESTs与功能已知基因有较高同源性,28％ ESTs与功能尚未确定的假设蛋白或未知蛋白高度同源,5％ ESTs无匹配的同源片段.其中储藏蛋白的EST占到已知功能EST的17.93％,其中包括10、16和22 kD储藏蛋白、11S、13S球蛋白、豌豆球蛋白以及油体蛋白等的基因,另外获得了2个苦荞种子发育期次生代谢相关的ESTs,分别为黄烷酮3-羧化酶和苯丙氨酸解氨酶的基因序列.这些结果说明构建的文库有较高的质量,可进一步从文库中分离苦荞种子蛋白以及次生物质代谢的相关基因.%Tartary buckwheat (Fagopyrum, tartaricum) seeds contain abundant protein with balanced amino acids and functional flavonoids. It is one typical crop for food and medical functions. Seed-filling development period is the crucial stage for the accumulation of those functional substances, so revealing the gene expression profile of this period is important for further regulation of functional component and improving the seed quality. For this reason, A full-length cDNA library was constructed by swithching mechanism at 5'end of RNA transcript(SMART) technology from filling stage Tartary buckwheat seeds and partial expressed sequence tags (EST) were analyzed. The results indicated that the titer of primary library and amplified library were 4.86×105pfii/mL and 6.2×109 pfu/mL, respectively. The blue-white screening showed that it had a recombination rate of 99.58%, and the length of cDNA inserts was range from 500 to 3 500 bp. Some ESTs of the cDNA library (GenBank accession No. GO477569 and GO496285-GO496321) were obtained by sequencing of the random circularized plasmid clones. Bioinformatics analysis indicated that 54% ESTs showed high homology with the functional known genes, 28% ESTs had high homology with unknown protein or hypothetical protein, and 5% ESTs had no-hits. Among all known genes, ESTs of storage protein accounted for 17.93%, including 10, 16 and 22 kD storage protein, 1 IS and 13S globulin, vicilin and oleosin,etc. In addition, two genesrelated to the secondary metabolism named flavonoid 3'-hydroxylase and phenylalanine ammonia-lyase were obtained. All those results can lay a foundation for further isolation the genes related with the metabolism of seed storage protein and secondary metabolites.