MeJA对人参DS基因表达的影响及DS在酵母中的表达
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摘要
人参是珍贵的药用植物,由于具有显著的药理活性而得到十分广泛的应用。人参的主要有效成分为人参皂苷。至今已从人参中分离出至少60种单体皂苷,除R0外,均为达玛烷型三萜皂苷。而DS负责催化2,3-氧化角鲨烯生成达玛烯二醇,然后经一系列的羟基化、糖基化反应生成各种类型的达玛烷型皂苷。并且对人参DS基因的RNA干扰研究表明,DS基因的沉默导致人参皂苷合成量显著下降,这证明了DS是人参皂苷合成的关键酶,但其具体的调控机制还不清楚。基于以上分析,本论文对DS基因进行了研究。
本文首先检测了MeJA对人参发根生长和总皂苷的积累以及5种单体皂苷含量的影响。发现MeJA对人参发根生长和总皂苷的积累及人参皂苷单体Rb1、Rd、Re、Rg2的积累均有明显的促进作用。但是对人参皂苷单体Rg1有明显的抑制作用,且随着浓度的增高,这种积累(或抑制)的作用逐渐减弱。然后利用半定量RT-PCR法检测人参发根DS基因的表达量,发现DS基因的表达量得到提高。以上结果证明了,DS基因表达量的提高能够促进人参皂苷的积累；MeJA的诱导不仅能提高人参皂苷的含量,还能通过影响某些糖基转移酶的表达量来改变人参皂苷单体含量的比例。
提取了高质量的人参发根RNA,逆转录合成cDNA,扩增DS基因,建立克隆载体pMD-19T-DS,热转化到大肠杆菌JM109中,通过宝生物测序,发现改变的6个碱基并不引起目的氨基酸编码序列发生改变,证明所克隆的基因为目的基因。利用pAUR123载体构建表达载体pAUR123-DS,并转染酿酒酵母菌(CICC,31476)。利用SDS-PAGE法检测工程菌所表达的蛋白,发现得到85.5kDa的目的蛋白,证明目的基因片段在酵母菌中表达。用HPLC检测酵母代谢产物中达玛烯二醇的含量,发现达玛烯二醇含量较低。最后,检测了工程菌的遗传稳定性,结果证明本实验室构建的工程菌株yeast-pAUR123-DS在无筛选压力下存在质粒不稳定现象。
本研究对人参皂苷生物合成调控机制的研究和达玛烯二醇的工业化生产具有重要的指导作用,为调控人参皂苷的含量及人参代谢工程的研究奠定了理论和实践基础,具有广阔的应用前景。
Panax ginseng is one of the most famous medicinal plants and used intensively because of its potent pharmaceutical activities. Ginsenosides are the major active constituents including dammarene-type and oleanane-type saponins. So far, more than 60 kinds of ginsenosides were found. All the ginsenosides except ginsenoside R0 belong to dammarene-type saponins. Dammarene-type saponins have many physiological and pharmaceutical effects, including anti-fatigue, antihyperglycemic, anti-aging and anti-cancer effects. Dammarane-type ginsenosides are biosynthesized from dammarenediol by a sequence of two steps, namely hydroxylation and glycosidation. Dammarane type ginsenosides are biosynthesized from dammarenediol by a sequence of two steps, namely hydroxylation and glycosidation. RNA interference of DS in transgenic P. ginseng resulted in silencing of DS expression, which leads to a reduction of ginsenoside production. These results indicate that DS is a key enzyme involved in ginsenoside biosynthesis. However, mechanism of regulation in ginsenoside synthesis remains to be answered.
In this study, methyl jasmonate induced effect on the growth, content of total saponins and individual ginsenoside content of Panax ginseng hairy root and DS gene expression were investigated by HPLC and semi-quantitative reverse transcription and polymerase Chain reaction. Meantime, a recombinant S.cerevisiaes strain which can produce dammarenediol-Ⅱwere constructed as yeast-pAUR123-DS. The main contents and conclusions of this study are as follows:
1. First, we choosed the time of MeJA elicitation for 4 weeks from growth curve of Panax ginseng hairy root. Different concentration of MeJA elicitation could remarkably improve the growth of Panax ginseng hairy root and increase level of total ginsenoside and Rb1, Rd, Re, Rg2 ginsenoside by HPLC and colorimetry of vanillina. In the case of Rgl ginsenoside after MJ elicitation, the content was affected negatively in ginseng hairy root cultures. But the effects were weakened with increase of concentration. When the concentration of MeJA elicitation was 0.01mmol/L, growth, content of total saponins and Rb1,Rd, Re, Rg2, Rgl ginsenoside was 41.59g/L,24.1mg/g,2.4 mg/g,1.96 mg/g,5.82 mg/g,0.53mg/g,3.87 mg/g respectively.
2. In order to analyze the change of DS gene expression, concentration of Mg2+, annealing temperature and cycle number of PCR system were optimized analyzed, and then a stable and convenient semi-quantitative RT-PCR system which amplified DS andβ-actin gene at the same time in different tubes was established. The PCR condition for DS andβ-actin gene were as follows:concentration of Mg2+ was 1.5 mmol/L, annealing temperature was 55℃, the primes of P-actin gene was added after 6 cycles of DS gene amplication. When the 30th cycles, they arrived the exponential period at the same time.
3. The transcription of DS genes in hairy root cultures elicited by 0.1mm MeJA treatment remarkably increased as compared with the control. Compared with 24h,48h of MeJA elicitation, DS gene expression is the maximum at 12h. The difference is insignificant, so the upregulation of DS gene expression do not depend on time.
4. Total RNA was isolated from P.ginseng hairy roots. Total RNA were reverse-transcribed to produce cDNA. PCRs were performed with F and MR primer. After tailing and purifaction, the PCR product was subcloned to the plasmid vector (pMD-19T), namely pMD-19T-DS. Compared with sequence of DS gene in Genebank, the positive vector has 6 basic group changed by sequence analysis of TaKaRa. However, the mutated basic group did not mutate encode sequence of amino acids. After enzyme digestion, the DS fragment was ligated into the multi-cloning site of yeast expression vector pAUR123, namely pAUR123-DS. The plasmid pAUR123-DS was transferred to S. cerevisiae strain 31476. After screening on medium and enzyme digestion, the transformant had object,namely yeast-pAUR123-DS.
5. S. cerevisiae extract from transformed cells were applied onto SDS-PAGE, and then target protein of molecular weight 85.5kDa was found, whereas target protein was not found in the control. Meanwhile, dammarenediol-Ⅱwas found in secondary metabolite of S. cerevisiae by HPLC, and the content of dammarenediol-Ⅱwas 1.85mg/100g.
6. Plasmid stability of transgenic engineering bacteria was detected. The consequence showed that plasmid of recombinant yeast was not stable under no selective pressure. After 168h of culture, plasmid stability of recombinant yeast was 67% under no selective pressure, and plasmid stability of recombinant yeast was above 90% under selective pressure.
Analysis and prospect based on above result:First, DS is the key enzyme in biosynthensis pathway of ginsenoside, and upregulation of gene expression could improve level of dammarenediol-Ⅱand promote saponin accumulation ginsenoside. Second, MeJA elicitation could change glycosyltransferase gene expression, so the ratio of ginsenoside Rx was changed. Last, because the yield of dammarenediol-Ⅱof transgenic engineering bacteria is very low, we should knockout unwanted gene and optimize fermentation condition to improve level of dammarenediol-Ⅱ. This study is significant for further investigation of ginsenoside biosynthesis pathway and for the large-scale production of dammarenediol-Ⅱ.
本文首先检测了MeJA对人参发根生长和总皂苷的积累以及5种单体皂苷含量的影响。发现MeJA对人参发根生长和总皂苷的积累及人参皂苷单体Rb1、Rd、Re、Rg2的积累均有明显的促进作用。但是对人参皂苷单体Rg1有明显的抑制作用,且随着浓度的增高,这种积累(或抑制)的作用逐渐减弱。然后利用半定量RT-PCR法检测人参发根DS基因的表达量,发现DS基因的表达量得到提高。以上结果证明了,DS基因表达量的提高能够促进人参皂苷的积累；MeJA的诱导不仅能提高人参皂苷的含量,还能通过影响某些糖基转移酶的表达量来改变人参皂苷单体含量的比例。
提取了高质量的人参发根RNA,逆转录合成cDNA,扩增DS基因,建立克隆载体pMD-19T-DS,热转化到大肠杆菌JM109中,通过宝生物测序,发现改变的6个碱基并不引起目的氨基酸编码序列发生改变,证明所克隆的基因为目的基因。利用pAUR123载体构建表达载体pAUR123-DS,并转染酿酒酵母菌(CICC,31476)。利用SDS-PAGE法检测工程菌所表达的蛋白,发现得到85.5kDa的目的蛋白,证明目的基因片段在酵母菌中表达。用HPLC检测酵母代谢产物中达玛烯二醇的含量,发现达玛烯二醇含量较低。最后,检测了工程菌的遗传稳定性,结果证明本实验室构建的工程菌株yeast-pAUR123-DS在无筛选压力下存在质粒不稳定现象。
本研究对人参皂苷生物合成调控机制的研究和达玛烯二醇的工业化生产具有重要的指导作用,为调控人参皂苷的含量及人参代谢工程的研究奠定了理论和实践基础,具有广阔的应用前景。
Panax ginseng is one of the most famous medicinal plants and used intensively because of its potent pharmaceutical activities. Ginsenosides are the major active constituents including dammarene-type and oleanane-type saponins. So far, more than 60 kinds of ginsenosides were found. All the ginsenosides except ginsenoside R0 belong to dammarene-type saponins. Dammarene-type saponins have many physiological and pharmaceutical effects, including anti-fatigue, antihyperglycemic, anti-aging and anti-cancer effects. Dammarane-type ginsenosides are biosynthesized from dammarenediol by a sequence of two steps, namely hydroxylation and glycosidation. Dammarane type ginsenosides are biosynthesized from dammarenediol by a sequence of two steps, namely hydroxylation and glycosidation. RNA interference of DS in transgenic P. ginseng resulted in silencing of DS expression, which leads to a reduction of ginsenoside production. These results indicate that DS is a key enzyme involved in ginsenoside biosynthesis. However, mechanism of regulation in ginsenoside synthesis remains to be answered.
In this study, methyl jasmonate induced effect on the growth, content of total saponins and individual ginsenoside content of Panax ginseng hairy root and DS gene expression were investigated by HPLC and semi-quantitative reverse transcription and polymerase Chain reaction. Meantime, a recombinant S.cerevisiaes strain which can produce dammarenediol-Ⅱwere constructed as yeast-pAUR123-DS. The main contents and conclusions of this study are as follows:
1. First, we choosed the time of MeJA elicitation for 4 weeks from growth curve of Panax ginseng hairy root. Different concentration of MeJA elicitation could remarkably improve the growth of Panax ginseng hairy root and increase level of total ginsenoside and Rb1, Rd, Re, Rg2 ginsenoside by HPLC and colorimetry of vanillina. In the case of Rgl ginsenoside after MJ elicitation, the content was affected negatively in ginseng hairy root cultures. But the effects were weakened with increase of concentration. When the concentration of MeJA elicitation was 0.01mmol/L, growth, content of total saponins and Rb1,Rd, Re, Rg2, Rgl ginsenoside was 41.59g/L,24.1mg/g,2.4 mg/g,1.96 mg/g,5.82 mg/g,0.53mg/g,3.87 mg/g respectively.
2. In order to analyze the change of DS gene expression, concentration of Mg2+, annealing temperature and cycle number of PCR system were optimized analyzed, and then a stable and convenient semi-quantitative RT-PCR system which amplified DS andβ-actin gene at the same time in different tubes was established. The PCR condition for DS andβ-actin gene were as follows:concentration of Mg2+ was 1.5 mmol/L, annealing temperature was 55℃, the primes of P-actin gene was added after 6 cycles of DS gene amplication. When the 30th cycles, they arrived the exponential period at the same time.
3. The transcription of DS genes in hairy root cultures elicited by 0.1mm MeJA treatment remarkably increased as compared with the control. Compared with 24h,48h of MeJA elicitation, DS gene expression is the maximum at 12h. The difference is insignificant, so the upregulation of DS gene expression do not depend on time.
4. Total RNA was isolated from P.ginseng hairy roots. Total RNA were reverse-transcribed to produce cDNA. PCRs were performed with F and MR primer. After tailing and purifaction, the PCR product was subcloned to the plasmid vector (pMD-19T), namely pMD-19T-DS. Compared with sequence of DS gene in Genebank, the positive vector has 6 basic group changed by sequence analysis of TaKaRa. However, the mutated basic group did not mutate encode sequence of amino acids. After enzyme digestion, the DS fragment was ligated into the multi-cloning site of yeast expression vector pAUR123, namely pAUR123-DS. The plasmid pAUR123-DS was transferred to S. cerevisiae strain 31476. After screening on medium and enzyme digestion, the transformant had object,namely yeast-pAUR123-DS.
5. S. cerevisiae extract from transformed cells were applied onto SDS-PAGE, and then target protein of molecular weight 85.5kDa was found, whereas target protein was not found in the control. Meanwhile, dammarenediol-Ⅱwas found in secondary metabolite of S. cerevisiae by HPLC, and the content of dammarenediol-Ⅱwas 1.85mg/100g.
6. Plasmid stability of transgenic engineering bacteria was detected. The consequence showed that plasmid of recombinant yeast was not stable under no selective pressure. After 168h of culture, plasmid stability of recombinant yeast was 67% under no selective pressure, and plasmid stability of recombinant yeast was above 90% under selective pressure.
Analysis and prospect based on above result:First, DS is the key enzyme in biosynthensis pathway of ginsenoside, and upregulation of gene expression could improve level of dammarenediol-Ⅱand promote saponin accumulation ginsenoside. Second, MeJA elicitation could change glycosyltransferase gene expression, so the ratio of ginsenoside Rx was changed. Last, because the yield of dammarenediol-Ⅱof transgenic engineering bacteria is very low, we should knockout unwanted gene and optimize fermentation condition to improve level of dammarenediol-Ⅱ. This study is significant for further investigation of ginsenoside biosynthesis pathway and for the large-scale production of dammarenediol-Ⅱ.
引文
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