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[摘要]
目的 克隆菘蓝中肉桂酸-4-羟基化酶(C4H)基因,并进行生物信息学和表达分析.方法 采用cDNA末端快速扩增(RACE)技术克隆C4H基因全长cDNA序列,进而获得其基因组DNA序列.通过RT-PCR法检测C4H蛋白在不同器官中及受到相应刺激因素下的表达情况.结果 菘蓝C4H基因全长cDNA为1 674 bp(GenBank登录号:GU014562),包含一个1 530 bp的开放阅读框(ORF),编码509个氨基酸残基.对应的基因组分析表明,C4H基因含2个内含子,3个外显子.在根、茎、叶各器官中均有表达,其中根中最多.胁迫因素紫外照射(UV-B)、茉莉酸甲酯(MeJA)、脱落酸(ABA)和赤霉素(GA3)在一定程度上能够诱导C4H的表达上调.结论 首次从菘蓝植物中克隆得到C4H基因的cDNA全长序列,并证实其在根中表达量最高,且在胁迫因素下表达量上调,为进一步研究菘蓝中抗病毒活性成分木质素类成分的次生代谢工程奠定基础.
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[Abstract]
Objective To clone cinnamate 4-hydroxylase (C4H) gene from Isatis indigotica and to analyze its bioinformatics and expression. Methods The full-length cDNA of C4H was 1 674 bp (GenBank accession No. GU014562) long with an open reading frame (ORF) of 1 530 bp encoding a polypeptide of 509 amino acid residues. The comparison of C4H genomic DNA sequences and C4H cDNA sequence revealed that the C4H genomic DNA contained two introns. Southern-blotting analysis indicated that C4H was a multiple copy gene. C4H expression could be detected in all tissues at different expression levels, with the strongest expression in the roots. Further expression analysis revealed that the signaling components of defense/stress pathways, such as ultraviolet-B radiation (UV-B), methyl jasmonate (MeJA), abscisic acid (ABA), and Gibberellins (GA3) could up-regulate the C4H transcript levels compared with the control. Conclusion We have first extracted the full length cDNA of C4H gene from I. indigotica, and its structural and bioinformatic analyses are carried out, which will help us to further illuminate this pathway. The research also provides a possibility to study the antiviral active constiuents in I. indigotica by plant secondary metabolic engineering.
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[基金项目]
上海市科学技术委员会登山行动计划项目(07DZ19721)