朱家红 夏栋楠 王颖 郭冬 李辉亮 梅文莉 彭世清

摘 要：海南龙血树是国产血竭的主要基源植物，其血竭主要化学成分为类黄酮化合物。为进一步了解DcWD40-1在类黄酮生物合成中的潜在功能和作用机制，该研究根据海南龙血树转录组数据，利用RT-PCR技术在海南龙血树中克隆了一个WD40基因DcWD40-1，该基因全长1 550 bp，包含一个1 353 bp的开放阅读框，编码450个氨基酸，蛋白质分子量50.77 kD，理论等电点5.71。生物信息学分析显示，DcWD40-1属于WD40蛋白家族成员，具有5个保守的WD40结构域，和其他植物WD40蛋白同源性高，保守性强。利用Genome Walking方法分离了1 503 bp的DcWD40-1启动子序列，该区域具有典型真核生物启动子结构特征，并含有多个应答激素和胁迫的响应元件。表达分析显示，血竭诱导剂能够诱导DcWD40-1的表达，DcWD40-1的变化与血竭形成及类黄酮积累正相关。此外，DcWD40-1也能对茉莉酸甲酯、细胞分裂素、油菜素内酯和UV-B处理做出积极响应。

关键词：海南龙血树，血竭，类黄酮，WD40转录因子，基因表达

中图分类号：Q949.45文献标识码：A

Abstract：Hainan Dragon trees （Dracaena cambodiana） are the main plant resources of dragons blood in China，and the main chemical constituents of the dragons blood are flavonoids. The recent studies about dragons blood mainly focus on the chemical constituents and pharmacological activities，while the molecular mechanisms of dragons blood formation remain unknown. WD40 transcription factor plays an important role in flavonoid accumulation. In this study，a WD40 gene named DcWD40-1 was cloned in Dracaena cambodiana based on transcriptome data and RT-PCR techno-logy. The full-length of cDNA of DcWD40-1 was 1 550 bp，containing 1 353 bp opening reading frame （ORF），and encoding 450 amino acids with the calculated molecular weight of 50.77 kD and calculated pI 5.71. Bioinformatics analysis showed that DcWD40-1 belonged to a member of WD40 superfamily，had five conserved WD40 domains，and shared high identities to WD40 proteins with other plants. A 1 503 bp-length promoter region of DcWD40-1 was isolated by Genome Walking method，which had structural characteristics of typical eukaryotic promoters. The promoter region of DcWD40-1 contained lots of hormone responsible elements，such as abscisic acid-responsive element，auxin-responsive element，salicylic acid-responsive element and jasmonic acid-responsive element; also had many cis acting elements related stress such as light，cold，hot and anaerobic inducer. Expression analysis showed that DcWD40-1 was induced by dragons blood inducers，positively related to flavonoids accumulation and formation of dragons blood. In addition，DcWD40-1 can also respond positively to jasmonic acid，cytokinin，brassinosteroid and UV-B treatment. These results will lay the foundation for further study of the potential functions and mechanisms of DcWD40-1 in flavonoid biosynthesis in Dracaena cambodiana.

Key words：Dracaena cambodiana，dragons blood，flavonoids，WD40 transcription factor，gene expression

血竭是一種传统名贵中药，具有活血化瘀、消炎镇痛和止血生肌等功效 （Gupta et al.，2008; Wang et al.，2011）。海南龙血树 （Dracaena cambodiana）是国产血竭的基源植物 （郑道君等，2009; Wang et al.，2013）。前期研究表明海南龙血树血竭中的主要化学成分以类黄酮化合物为主（Chen et al.，2012; Zheng et al.，2012; Wang et al.，2017）。海南龙血树及其血竭中的类黄酮化合物具有多种生物学活性，如抗癌、抗肿瘤、抗炎、抗真菌、凝血酶抑制和抗增殖活性等（Luo et al.，2011; Mei et al.，2013; Wang et al.，2017）。目前，人们对血竭的化学成分、药理活性及临床应用的研究较多，但对类黄酮积累和血竭形成机制并不了解 （Zhu et al.，2016）。

植物类黄酮生物合成主要受结构基因（编码类黄酮生物合成的各种酶类）和调节基因（转录因子）的控制 （Tohge et al.，2017）。……