百合转录因子MYB12的克隆与表达分析

Cloning and expression analysis of MYB12 in Lilium oriental hybrid 'Sorbonne'

以东方百合‘索邦’(Lilium oriental hybrid ‘Sorbonne’)为材料,克隆获得花青素苷生物合成通路中的关键转录因子Lhsor MYB12基因。序列分析结果显示,Lhsor MYB12最大开放阅读框长720 bp,编码239个氨基酸,具有2个典型的DNA结合结构域;该基因包括3个外显子和2个内含子。该基因的氨基酸序列与郁金香(Tulipa fosteriana W. Irving)中的MYB氨基酸序列相似性最高。系统进化分析结果表明,Lhsor MYB12在MYB基因家族中与已报道的控制花青素苷合成的基因形成一簇。进一步采用染色体步移技术,获得了Lhsor MYB12基因起始密码子上游2143 bp的启动子序列,顺式作用元件预测结果显示,该序列中除核心启动子元件(TATA box)外,还包含有MYB蛋白的绑定位点、光反应元件以及参与昼夜节律等反应的相关元件。基因表达分析结果表明,Lhsor MYB12仅在‘索邦’花丝、花柱和花被片中表达;且在花蕾发育过程中表达量逐渐增高,花蕾盛开时表达量最大,但内、外花被的表达起始阶段不同。黑暗处理可导致Lhsor MYB12表达水平降低;光照条件下该基因的表达水平随处理时间的延长表现出先上升后下降再持续上升的趋势。研究结果提示Lhsor MYB12的表达变化规律可能与其启动子中相应的顺式作用元件相关。

To reveal the regulatory mechanism of the anthocyanin synthesis pathway in Lilium,we isolated cDNA,gDNA,and the MYB12 gene promoter from the petals of the L.oriental hybrid‘Sorbonne’.Results showed that the cDNA sequence contained a 720 bp open reading frame(ORF)encoding a 239 amino acid polypeptide with two typical DNA-binding domains.The 863 bp nucleotide sequence was amplified from the extracted DNA and the gene consisted of three exons and two introns.Furthermore,the amino acid sequence had highest similarity with MYB transcription factors in Tulipa fosteriana W.Irving.Phylogenetic analysis showed that the phylogenetic evolution of the transcription factor was in accordance with taxonomic classification,and it could be grouped with genes that regulate anthocyanin biosynthesis in the MYB gene family.The 2143 bp promoter sequence of the LhsorMYB12 gene was obtained using chromosome walking technology.The promoter element prediction results showed the existence of a core promoter element(TATA box),MYB protein binding sites,photoreactive elements,and other regulation motifs(e.g.,circadian rhythm response motif).RT-PCR analysis indicated that the expression of LhsorMYB12 could be detected in the tepals,filaments,stigmas,and styles.qRT-PCR analysis showed that the relatively high expression occurred in blooming period(12 cm)tepals,and then began to decrease.However,there was a difference between the initial stage of expression in the inner and outer perianths.Moreover,dark treatment resulted in a reduction in the expression of LhsorMYB12,with the lowest expression found after an hour of treatment.Under light conditions,the expression level of the gene increased at first,then decreased,and then continued to rise with increasing treatment time.The LhsorMYB12 expression pattern results might relate to the corresponding cis-acting elements in its promoter.