不同发育时期大麻素合成相关酶基因表达特征与大麻素含量的相关分析

Correlation Analysis Between Gene Expression Characteristics of Related Enzymes in Cannabinoids Biosynthesis and Cannabinoids Content at Different Developmental Stages of Cannabis sativa L.

本研究采用高效液相色谱法和实时定量PCR方法,分别测定了大麻植株不同发育时期主要大麻素含量变化以及大麻素合成途径16个相关酶基因的表达丰度,分析了大麻素含量与各基因表达量之间的相关性。结果表明,大麻素含量在幼苗期和快速生长期(简称快生期)接近零,生殖阶段(盛蕾期,盛花期和始果期)快速增加,以始果期苞片中含量最高。大麻素合成过程中4个合成途径内部存在基因协同表达特征,4个合成途径各自酶基因相对表达量变化规律相似。此外,发育过程合成相关酶基因表达量与大麻素含量相关性分析表明,CMK、MDS、HDS、HDR和GPP(lsu)等5个基因表达量和大麻素含量呈现显著正相关(相关系数>0.9)。结果说明大麻素合成相关酶基因可能通过协同作用来调控大麻素合成与积累,MEP和GPP途径后端的酶基因在大麻素合成过程中起着重要作用,而Cannabinoid途径中OLS、PT和THCAS三个大麻中特有的酶基因主要在始果期苞片腺毛中对大麻素合成积累起着关键作用。这些结果为今后利用基因工程手段改良大麻素含量提供了研究基础。

In this study,the HPLC and real-time PCR were used to determine the cannabinoids content and the expression levels of 16 related enzymes in cannabinoids biosynthesis at different developmental stages of Cannabis sativa L.,aiming to analyze the correlation between cannabinoids content and gene expression.The results showed that the cannabinoids content was close to zero at seedling stage and rapid-growing stage,but increased rapidly at the reproductive stage（budding,flowering and initial-fruiting）,with the highest content in the bract at initial-fruiting stage.In the process of cannabinoids synthesis,the 4 biosynthetic pathways were characterized by gene co-expression,and the relative expression of each enzyme gene in the 4 biosynthetic pathways was similar.Moreover,the correlation analysis between expression level of related enzymes and cannabinoids content showed that the expression levels of 5 enzyme genes（CMK,MDS,HDS,HDR and GPP（lsu）） had significant positive correlation with cannabinoids content（R〉0.9）.It was revealed that the related enzyme genes in cannabinoids biosynthesis might regulate the synthesis and accumulation of cannabinoids through coordinated expression.It alsodemonstrated that the back-end enzyme genes in MEP and GPP pathway played important roles in cannabinoids biosynthesis,while the three specific enzyme genes in C.sativa（OLS,PT and THCAS） played key roles in the biosynthesis and accumulation of cannabinoids in the glandular hairs of bract at initial-fruiting stage.These results would provide a basis for modifying cannabinoids content through genetic engineering in the future.