Cloning Of Key Enzyme Genes For Active Ingredients Of Ginseng Synthesis And The Relationship Study Of Expression Levels With Ecological Factors And Total Saponins

Ginseng is one of very rare Chinese medicinal materials in our country. The active ingredients of ginseng is ginsenoside, and its content is varied along with the change of cultivated area environment. The theory basis for elucidating the physiological and ecological mechanism of ginsenoside synthesis could be provided by the relationship study of key enzyme gene expression with ecological factors and content of ginsenosides. In this paper, HMGR, SS and SE genes were cloned in five-year-old ginseng. The expression levels of these genes and content of total saponins were detected in ginseng roots, and the relationship of expression levels with ecological factors and total saponins was analyzed. The main results were as follows:1. Five-year-old ginseng roots were used as test materials. HMGR, SS and SE genes of ginseng were cloned by RT-PCR technology. Nucleotide and amino sequences of those genes were analyzed by bioinformatics technology. The results showed that the full length c DNA of HMGR was 1750 bp that included a 1722 bp open reading frame(ORF)encoding 573 aa; HMGR-encoding protein contained two transmembrane regions and five conserved domains. The full length c DNA of SS was 1256 bp that included a 1248 bp open reading frame encoding 415 aa; SS-encoding protein contained two transmembrane regions and two conserved domains. The full length c DNA of SE was 1636 bp that included a 1611 bp open reading frame encoding 536 aa; SE-encoding protein contained four transmembrane regions and five conserved domains.2. Five-year-old ginseng roots were used as test materials. The amplified fragment of ginseng GAPDH gene was linked with p MD20-T vector to construct recombinant plasmids. The positive plasmids were diluted and used to established the standard curve by SYBR GreenⅠ. The melting curve was analyzed, and the specificity, sensitivity and repeatability were detected. The results indicated that the lowest copy number for detection of GAPDH gene was 36.0 copies/μL. A good linear relationship in a wide range from 36 to 3.6 × 107 copies/μL was established(R2 = 0.9964). The melting curve showed a single peak with the temperature of(84.57 ± 0.01) ℃. The coefficient of variation(CV) of five different concentration of positive plasmids was 0.54% to 2.77% in intra-assay and 2.60% to 4.39% in inter-assay respectively. This method has the advantages of sensitivity, specificity, rapidity and good repeatability. It provides a methodological basis for the quantitative analysis on the functional genes of ginseng when GAPDH gene is used as a reference gene.3. Five-year-old ginseng roots were used as test materials. The expression levels of HMGR, SS and SE genes were detected, and the content of total saponins was determinated. The correlation of expression levels with ecological factors and total saponins was analyzed. The results showed that the expression levels of HMGR, SS and SE increased gradually from flowering period to red fruiting period and declined slightly at fruit mature period, but still at a high level; the content of total saponins increased gradually from flowering period to fruit mature period. So later growth stage was the critical period for the formation of ginseng quality. The expression levels of HMGR, SS and SE genes and content of total saponins increased with the rising of 300 m to 800 m altitude in Ji’an, Dunhua and Fusong regions, and decreased with the rising of 900 m to 1200 m altitude in Changbai region. The best altitude that is suitable for key enzyme gene expression and accumulation of ginsenosides may be in the range of 700 m to 900 m. Average temperature, sunshine hours and monthly rainfall had significant effects on gene expression and the synthesis of saponins, and average temperature played a most important role. Relative humidity and monthly evaporation had little effect on gene expression and the synthesis of saponins. The expression levels of HMGR, SS and SE had significant positive correlations with the content of total saponins in different growth period, and they also had significant positive correlations with the content of total saponins in different altitudes, so key enzyme gene expression had important effects on the synthesis of ginsenosides.