黑莓RuCYP73A基因的克隆及表达分析

Cloning and Expression Analysis of RuCYP73A Gene in Blackberry

黑莓果实富含黄酮类化合物,具有清除自由基、抗氧化和抗肿瘤等功效。本研究基于黑莓未成熟和成熟果实的转录组数据,克隆了黄酮合成通路中显著差异的CYP基因(与蔷薇科植物CYP73A同源,命名为RuCYP73A),利用RT-qPCR分析RuCYP73A基因在黑莓不同组织和果实不同发育时期的相对表达量,结果表明RuCYP73A在果实中的表达量最高,在果实发育过程中呈先升高后逐渐降低的规律,其中15 d果龄时表达量最高。基因结构分析显示,RuCYP73A含1 497 bp开放阅读框,编码499个氨基酸,终止密码子为TAA。生物信息学分析表明,RuCYP73A编码蛋白的分子量为56.80 kD,理论等电点为8.53,具有跨膜结构域,为疏水碱性蛋白,属于细胞色素P450超家族。系统进化分析表明,RuCYP73A与蔷薇科月季和野草莓的亲缘关系较近,与梧桐科可可树和桃金娘科尾叶桉亲缘关系较远,符合植物分类学特征。研究结果为黑莓黄酮类物质生物合成关键基因CYP73A的深入研究奠定了基础。

Blackberry has rich flavonoids with medicinal value,such as scavenging free radicals,anti-oxidant,anti-tumor,anti-bacterial and so on.To study the mechanism of the key genes of flavonoid biosynthesis during the ripening of blackberry fruit,the significantly different expressed CYP gene of the flavonoid biosynthesis pathway was cloned based on the transcriptome data of the immature and mature blackberry fruit,which is homologous to CYP73A and named as RuCYP73A.The results of real-time fluorescent quantitative PCR showed that RuCYP73A was expressed at a higher level in fruits than other organs,and the expression level in fruit development process increased first,reached the highest level in red fruits(15-day age),then gradually reduced.The analysis of the cloned RuCYP73A gene indicates that total length of the opening reading frame is 1497 bp,encoding 499 amino acids,the stop codon is TAA.Bioinformatics analysis shows that the molecular mass of RuCYP73A protein is 56.80 kD,and the theoretical isoelectric point is 8.53,so it is a hydrophobic basic protein.RuCYP73A has a transmembrane domain and belongs to the cytochrome P450 superfamily.Phylogenetic analysis shows that RuCYP73A is close to Chinese rose(Rosaceae)and wild strawberry(Rosaceae)CYP73A100-like,but is distant to Theobroma cacao(Sterculiaceae)and Eucalyptus urophylla(Myrtaceae)CYP73A100,which conforms to the characteristics of plant classification.This research provides a basis for further research on the key gene CYP73A related to flavonoid biosynthesis in blackberry.