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本实验以三色堇(Viola wittrockiana)的花瓣为研究材料,提取其总RNA,通过RACE(rapid-amplification of cDNA ends)技术扩增4,5-多巴雌二醇双加氧酶(4,5-DOPA dioxygenase extradiol)基因的cDNA全长,并对其进行生物信息学分析。本实验成功克隆VwDODA的cDNA全长1 055 bp,其中3’端非编码区157 bp,包含29 bp Poly(A),5’端非编码区103 bp,其开放阅读框为795 bp,编码263个氨基酸。通过NCBI的BLAST比对表明VwDODA编码的氨基酸序列与其他植物相关基因的相似性达到80.29%,与大豆(Glycine max)亲缘关系最近。通过生物信息学分析软件对VwDODA的蛋白质结构与功能进行预测,结果表明Vw D-%ODA是一种亲水稳定蛋白,不存在跨膜区域和信号肽,分子式为C1337H2026N356O384S7,等电点为6.70,分子量为29 455.37。本研究为将来三色堇中甜菜色素与花色素的互斥现象研究打下了基础。
In this study, the petals of Viola wittrockiana were used as materials to extract total RNA, and 4, 5-D-estradiol dioxygenases were amplified by rapid amplification amplification cDNA ends (RACE) 5-DOPA dioxygenase extradiol) gene and its bioinformatics analysis. The full-length cDNA of VwDODA was cloned at length of 1 055 bp. The 3 ’untranslated region was 157 bp in length and contained 29 bp poly (A). The 5’ untranslated region was 103 bp. The open reading frame was 795 bp encoding 263 Amino acids. BLAST analysis by NCBI showed that the similarity of VwDODA-encoded amino acid sequence with other plant-related genes was 80.29%, which was the closest to Glycine max. Bioinformatics analysis software was used to predict the protein structure and function of VwDODA. The results showed that Vw D-ODA was a hydrophilic stable protein without transmembrane domain and signal peptide. The molecular formula was C1337H2026N356O384S7, the isoelectric point was 6.70, The molecular weight is 29 455.37. This study laid the foundation for the future study on the mutual exclusion of beet pigment and anthocyanidin in pansy.