颠茄AbCYP80F1基因的克隆与功能分析

Cloning and functional analysis of AbCYP80F1 gene in Atropa belladonna

目的克隆颠茄Ab CYP80F1基因和启动子序列,分析启动子上与调控信号和转录因子相关的调控元件,研究Ab CYP80F1基因超表达对颠茄发根中东莨菪碱积累的影响。方法以莨菪CYP80F1为探针,在颠茄转录组中进行Blastn检索获得同源的unigene。采用RACE技术克隆Ab CYP80F1全长,热不对称PCR技术克隆启动子序列,发根农杆菌浸染法获得超表达AbCYP80F1基因的颠茄发根,对培养的发根中东莨菪碱含量进行HPLC分析。结果克隆得到1 675 bp的全长Ab CYP80F1基因,其编码蛋白与铃铛子的CYP80F1亲缘关系最近。基因上游2 000 bp启动子上包含的顺式作用元件涉及光、无氧、生长素、茉莉酸、赤霉素和低温信号响应,同时包含有MYB、AP2/ERF、WRKY和bHLH转录因子的识别位点。超表达Ab CYP80F1基因使颠茄发根中东莨菪碱含量平均提高了1.8倍。结论 AbCYP80F1是颠茄须根特异性表达的合成途径基因,超表达能提高终产物东莨菪碱含量,Ab CYP80F1可能受到WRKY和bHLH转录因子的调控。

Objective To clone AbCYP80F1 gene and its promoter, analyze the Cis-acting elements related to control signals and transcription factors in AbCYP80F1 gene promoter and study the effect of AbCYP80F1 overexpression on scopolamine accumulation in Atropa belladonna. Methods CYP80F1 cDNA from Hyoscyamus niger was used as query sequence to do blastn search in A. belladonna transcriptome database to retrieve homologous unigenes. Full-length of AbCYP80F1 cDNA and gene promoter were acquired by RACE and hiTAIL-PCR, respectively. Agrobacterium rhizogenes-dipping method was adopted to induce AbCYP80F1-overexpressed A. belladonna hairy roots, and scopolamine content in hairy roots was detected by HPLC. Results A full-length of 1 675 bp of AbCYP80F1 gene was obtained and the deduced protein had a closed homology with CYP80F1 from Anisodus luridus. A length of 2 000 bp promoter contained Cis-acting elements related to light, anaerobic induction, auxin, MeJA, gibberellins and low temperature, as well as recognition sites of transcription factor of MYB, AP2/ERF, WRKY and bHLH. Overexpression of AbCYP80F1 gene increased the scopolamine content in hairy roots of A. belladonna by an average of 1.8 times. Conclusion Ab CYP80F1 is a secondary root-specific gene in scopolamine biosynthetic pathway and its overexpression can enhance scopolamine accumulation. Expression of AbCYP80F1 may be regulated by WRKY and bHLH transcription factors.