摘要
[目的]克隆夜香树(Cestrum nocturnum L.)萜类香气物质代谢途径关键限速酶DXS2(1-deoxy-D-xylulose-5-phosphate synthase 2)基因全长cDNA,并对该基因进行生物信息学分析,检测该基因在开花不同时间及叶片中表达量的动态变化.[方法]采用RT-PCR与RACE相结合的技术,获得夜香树DXS2的全长核苷酸序列,并进行生物信息学分析,qRT-PCR分析其在盛花期不同时间花和叶中的表达量.[结果]获得CnDXS2基因全长序列2 370 bp,其中ORF 2 145 bp,编码714个氨基酸,含DXP-synthase-N、TPP-PYR-DXS-TK-like和Transketolase-C等保守结构域,与绒毛状烟草(Nicotiana tomentosiformis L.)DXS2蛋白同源性达93%,推测属于Ⅱ类DXS蛋白;qRT-PCR分析表明,CnDXS2在叶中表达量总体高于花,花中22∶00表达量最高,14∶00表达量最低,具有节律性.[结论]萜类MEP代谢途径基因CnDXS2的克隆与表达分析为从分子方面揭示CnDXS2基因在萜类香气代谢途径中的功能提供了依据,有助于夜香树花香萜类代谢途径基因功能和花香基因工程的研究.
Abstract
[Objective] To clone the full-length cDNA of a critical rate limiting enzyme gene DXS2 in terpenoids pathway from Cestrum nocturnum L., analyse bioinformation and qRT-PCR of flowers and leaves of different time at blooming stage of the gene.[Method] The full-length of DXS2 gene was isolated using RT-PCR and RACE(rapid-amplification of cDNA ends) technology, then analyzed by bioinformatics.The expression of the gene in flower and leaves of different time from blooming stage was analyzed by qRT-PCR.[Result] The full-length of CnDXS2 was 2 370 bp and contained a 2 145 bp open reading frame(ORF) encoding a protein of 714 amino acid, including conserved domains such as DXP-synthase-N, TPP-PYR-DXS-TK-like and Transketolase-C,etc.The homology between CnDXS2 and DXS2 protein from Nicotiana tomentosiformis L.was 93%, maybe a Class Ⅱ DXS protein;qRT-PCR analysis showed that the expression level of CnDXS2 in leaves was generally higher than flowers, reached the peak at 22∶00 and lowest at 14∶00 in flowers, with rhythm.[Conclusion] Cloning and expression analysis of CnDXS2 form terpenoid metabolic pathway of MEP can provide the basis to reveal molecular function of CnDXS2 in terpene aroma metabolic pathway, promote the study on the gene function and the floral genetic engineering of terpenoid pathway in C.nocturnum L..
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