紫玉兰‘红元宝'Ml3GT1基因的克隆及表达分析

Cloning and expression analysis of Ml3GT1 in Magnolia liliflora ‘Hongyuanbao'

关键词:  紫玉兰, 花色, 糖基转移酶, 基因克隆, 表达分析

Key words:  Magnolia liliflora, flower color, glycosyltransferase, gene cloning, expression analysis

摘要:

UDP-类黄酮3-O-葡萄糖基转移酶(3GT)是花青素生物合成途径的重要催化酶之一。为研究其在紫玉兰花青素苷合成途径中的作用,该文以紫玉兰品种‘红元宝'(Magnolia liliflora ‘Hongyuanbao')为材料,根据转录组测序获得的3GT序列设计引物,利用RT-PCR技术克隆花青素苷生物合成途径中的结构基因Ml3GT1,并对其进行生物信息学和表达模式分析。结果表明:(1)Ml3GT1基因的cDNA序列长度为1 863 bp,其中最长开放阅读框(ORF)为1 374 bp,编码一条457 aa的肽链,相对分子质量为49.37 kDa,理论等电点(pI)为6.04。(2)氨基酸序列比对显示其具备典型的植物次生产物糖基转移酶信号序列(PSPG box)。(3)系统发育分析结果表明,Ml3GT1蛋白与小苍兰、矮牵牛、番薯等物种的3GT蛋白聚在一支。(4)qRT-PCR结果显示Ml3GT1基因的表达具有时空特异性,在花中的表达量最高,在嫩叶和老叶中有少量表达,而在根和茎中几乎不表达; 随着花的发育,Ml3GT1基因的表达量呈现先降低后升高的趋势,并在盛花期达到最高。上述结果表明,Ml3GT1可能参与类黄酮3-O的糖基化修饰,本研究结果将为木兰属植物花色育种研究奠定基础。

Abstract:

UDP-flavonoid 3-O-glucosyltransferase(3GT)is one of the important catalytic enzymes in the anthocyanin biosynthesis pathway. To study the function of 3GT in anthocyanin biosynthesis of Magnolia liliflora, M. liliflora ‘Hongyuanbao' was employed as materials. Primers were designed based on the 3GT sequence obtained from the transcriptome database of M. liliflora ‘Hongyuanbao', and the structural gene Ml3GT1 in anthocyanin biosynthesis pathway was cloned by RT-PCR(reverse transcription-PCR), and its bioinformatics and expression pattern were analyzed. The results were as follows:(1)The cDNA sequence length of Ml3GT1 was 1 863 bp, and the open reading frame was 1 374 bp, encoding 457 amino acid residues. The relative molecular weight of Ml3GT1 was 49.37 kDa, and its isoelectric point was 6.04.(2)The deduced amino acid sequence of Ml3GT1 contains a conserved plant secondary product glycosyltransferase signature sequence(PSPG box).(3)Results of the phylogenetic analysis showed that Ml3GT1 protein was closely relative to 3GT proteins from Freesia hybrida, Petunia × hybrida, and Ipomoea batatas.(4)Results of fluorescence quantitative PCR(qRT-PCR)revealed that Ml3GT1 has spatio-temporal specificity, with the highest expression level in flowers, the lower expression level in young leaves and old leaves, and little expression in roots and stems; With the development of flowers, the expression level of Ml3GT1 gene decreased first, then increased, and showed the highest expression level at the fully-flowering period. These results suggest that Ml3GT1 may be involved in flavonoid 3-O-glycosylation. This study will lay a foundation for the flower and color breeding of Magnolia plants.