人参皂苷生物合成关键酶基因MVD和βAS的克隆及βAS的反义表达
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摘要
人参是我国道地中药植物,其活性成分主要是次生代谢过程中产生的人参皂苷。深入研究人参皂苷生物合成途径关键酶基因的结构与功能,以及利用基因工程手段调控这些关键酶基因活性,达到调控提高人参皂苷含量的目的,将具有重要的理论和实际应用价值。本论文选择二个具有代表性的基因为研究对象,开展了基因克隆及利用反义RNA技术调控人参皂苷生物合成的尝试工作。这二个基因分别是人参皂苷生物合成上游关键酶基因,既催化异戊二烯途径第一个前体物-异戊烯二磷酸(IPP)合成焦磷酸甲羟戊酸脱羧酶基因(MVD),另一个基因是人参皂苷生物合成下游关键酶基因β-香树素合成酶基因(βAS),该基因编码的β-香树素合成酶是催化人参皂苷R0(人参次要活性成分)合成的关键酶。
本论文用SMART RACE技术扩增人参MVD基因的保守序列,3’片段和5’片段,结果经过序列比对分析和拼接得到一个ORF为1254bp的片段,用此基因的ORF两端设计引物,扩增得到其cDNA全长。人参MVD基因在大肠杆菌中成功表达,其编码的蛋白分子量大小是46kd,与基因序列分析结果一致。(MVD基因在GenBank中的登录号:GQ455989)。
利用反义表达策略,首次研究了βAS基因在人参皂苷生物合成中的功能。通过构建反义pBI121-AβAS植物表达载体,转入工程菌A4后转化人参根,对转化条件进行了筛选,获得反义人参发根系A5,A9,A19,A24和A30,Southern杂交结果显示目的基因拷贝数都是单拷贝。Northern杂交显示5株反义发根中的βAS转录水平显著下降,也说明反义βAS的导入确实可降低βAS的转录量,最多比对照组降低1/3。对酶活分析显示,反义发根系βAS活性均降低,同时,转基因发根系DAS活性被上调,DAS活性在所有的转化组中都获得增加,尤其在A19中增加了1.2倍。化学分析结果显示皂苷合成前体物2,3氧化鲨烯的积累增加。人参皂苷含量分析结果表明,齐敦果烷型人参皂苷R0含量均降低,最多降40%,转化发根的达玛烷型人参皂苷含量最高增加到原来的1.3倍,并且单体皂苷Rb1, Rb2, Rc, Rd,Re,Rf和Rg1均不同程度增加。这些研究成果对于促进MVD的异源表达、提高人参皂苷含量的种质资源的研究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 a famous medicinal plant and used extensively because of its significant pharmaceutical activities. Ginsenosides are the major component including dammarene-type and oleanane-type saponins. So far, more than 60 kinds of ginsenosides have been found. All the ginsenosides except ginsenoside R B belong to dammarene-type saponins, which have many physiological and pharmaceutical effects, including immune system modulation, antistress, antihyperglycemic, anti-inflammatory, antioxidant and anticancer effects. Nowadays, pharmaceutical effects of ginsenosides and novel ginsenosides are still under research. Besides, Panax ginseng is consumed more and more all over the world. It is estimated that the raw Panax ginseng saled is beyond 1 billion dollars.
Due to the limitation of Panax ginseng source, it requires much cost to extract ginsenosides from root of Panax ginseng. Transgenic technique is an alternative method to improve ginsenosides content in Panax ginseng by regulating the biosynthesis pathway. Enzyme reactions involved in biosynthesis are of many steps, in which the downstream of biosynthesis is many researchers’focus. (a)Clone and identification of genes associated with upstream of the pathway is available only in a few number. In order to study the mechanism of upstream of ginsenosides biosynthesis, we chose Panax ginseng MVD gene as target to clone based on homologous alignment and RACE method. Panax ginseng MVD was obtained and expressed in Hescherichia coliH, which will support the further study. (b)In the downstream of ginsenosides biosynthesis pathway, we choseβAS from several HcandidateHs to achieve antisense technique transformation. That is becauseβAS catalyzes oleanane-type saponin from 2,3-oxidosqualene, which is the important ancestor for biosynthesis of dammarene-type saponins, major bioactive component in Panax ginseng. According to the current researches, antisense technique can successfully regulate the activity of key enzymes involved in biosynthesis pathway. The result in this study is as followed:
(1) Panax ginseng MVD gene, a key enzyme in the upstream of ginsenosides biosynthesis, was not isolated so far. Here conservative sequence, 3’fragment and 5’fragment were achieved by SMART RACE. After sequence aligning and assembling, a 1254bp of ORF was obtained.Using two primers designed according to the ORF, its total cDNA was received.
The predicted pi of Panax ginseng MVD(PgMVD) was 7.58 analyzed by software“Computer pI/Mw Tool”.Using software blast and DNAMAN, we found that PgMVD was highly homologous with plant MVDs. It has an identity of 77%、74%、72% and 68% to that of Hevea brasiliensis、Arabidopsis thaliana、Zea mays and Ginkgo biloba, respectively.
The analysis result of Panax ginseng MVD structure showed it has the classical characteristic of MVD gene family, haboring strictly conserved active sites including one leucine, one aspartate and three cysteine sites residues. Three dimensional structure of PgMVD was constructed based on the crystal structure of yeast MVD. Furthernmore, phylogenetic tree analysis exhibited that all the MVD genes were derived from the same ancestor. The MVD genes of Mus musculus, Rattus norvegicus, Homo sapiens and Danio rerio belong to one distinct animal branch differed from another plant branch of Panax ginseng, Ginkgo biloba, Hevea brasiliensis, Zea mays and Arabidopsis thaliana.
PgMVD gene was expressed successfully in Hescherichia coli H. The expressed protein exhibited a molecular weight of 46kd, which is consistent with the putative result. Thus the expressed protein supports the further analysis of MVD activities in vitro. It is also meaningful to learn the metabolic engineering of Panax ginseng and make it possible to improve quality of Panax ginseng. At the mean time, it is worthy that heterogenous expression of the new gene in other species could be exploited.
(2)Total RNA of Panax ginseng was isolated from modified Hguanidinium thiocyanate method. AntisenseβAS was subcloned into three plant expression vectors which all possessed GUS gene.
Factors influencing the Panax ginseng transformation were studied. The concentration value 0.5 of engineering bacteria is better at OD600 ,which resulted in the highest GUS positive rate; while the GUS rate was not obviously different among the infection period of 5min,10min,20min and 30min; Preculture is important because the GUS rate is more in 2 days preculture than in 1 or 3 days preculture; Three days coculture is appropriate and no Hagrobacterium H infection present; Light condition and AS concentration were researched during transformation: the result of no light and 100μM AS treatment showed efficient.
After antisenseβAS transformation, five positive hairy roots A5, A9, A19, A24 and A30 were screened out. Southern blot resulted in one copy ofβAS in all antisense hairy root lines. Nouthern blot exhibited a comprehensive decrease in all the antisenseβAS lines, which confirmed that introduction of antisenseβAS could inhibit the transcript ofβAS gene.βAS activities were all decreased significantly and the most decrease is 1/3. On the other hand, DAS were upregulated in all lines, especially in A19 with a 1.2 times as control. The oleanane-type saponin RB0 Bin AntisenseβAS Panax ginseng hairy roots lines A5, A9, A19, A24 and A30 had all dropped: it had a biggest reduction of 40% in A19, followed a 33% reduction in A30, and the least reduction of 13% was found in A5. The above result indicated that antisenseβAS could inhibit the transcript of endogenenousβAS so that it resulted in decrease of oleanane-type saponin RB0B in antisense lines. But totally inhibition ofβAS was not found because of the existance of the slight reduction in A5. Phytochemistry analysis showed that 2, 3-oxidosqualene content was elevated and dammarene-type saponins were most up to 1.3 times as control.
Dammarene-type ginsenosedes in antisense line A19 was accumulated most, thus we chose A19 as the target to detect the content changes of dammarene-type ginsenosedes over culture time. The growth curve of total ginsenosides was between ginsenoside group Rb and ginsenoside group Rg. The total ginsenosides in A19 was found 14.17mg/g DW in the ultimate culture cycle, increased 38.8% than that in initial culture cycle. Ginsenoside group Rb increased quickly over the last culture period. The Rb1 and Rb2 were increased obviously, with an increase of 20.3% and 23.3%, respectively. While Rc and Rd of ginsenoside group Rb had a slight increase. Besides, ginsenoside group Rg was elevated over culture and its growth trend was similar to the ginsenoside group Rb. Comparatively, the increase of ginsenoside group Rb in antisens line A19 was lower than that of ginsenoside group Rg.
Analysis and prospect based on above result: above all, we isolated Panax ginseng MVD by SMART RACE method. With the discover of the key enzymes involved in ginsenosides biosynthesis, scientist can master the metabolic network comprehensively so as to make it possible to further utilize the key enzymes or improve plant quality through biological technique such as genetic transformation. On the other hand, we researched the regulation of ginsenosides biosynthesis by antisense transformation. Nowadays, antisense genetic technique is turning into an important method and efficient approach in identification of gene function during the transition from genomics to post-genomics. Our research showed an inspiring prospect of antisense technique exploited in plant improvement.
本论文用SMART RACE技术扩增人参MVD基因的保守序列,3’片段和5’片段,结果经过序列比对分析和拼接得到一个ORF为1254bp的片段,用此基因的ORF两端设计引物,扩增得到其cDNA全长。人参MVD基因在大肠杆菌中成功表达,其编码的蛋白分子量大小是46kd,与基因序列分析结果一致。(MVD基因在GenBank中的登录号:GQ455989)。
利用反义表达策略,首次研究了βAS基因在人参皂苷生物合成中的功能。通过构建反义pBI121-AβAS植物表达载体,转入工程菌A4后转化人参根,对转化条件进行了筛选,获得反义人参发根系A5,A9,A19,A24和A30,Southern杂交结果显示目的基因拷贝数都是单拷贝。Northern杂交显示5株反义发根中的βAS转录水平显著下降,也说明反义βAS的导入确实可降低βAS的转录量,最多比对照组降低1/3。对酶活分析显示,反义发根系βAS活性均降低,同时,转基因发根系DAS活性被上调,DAS活性在所有的转化组中都获得增加,尤其在A19中增加了1.2倍。化学分析结果显示皂苷合成前体物2,3氧化鲨烯的积累增加。人参皂苷含量分析结果表明,齐敦果烷型人参皂苷R0含量均降低,最多降40%,转化发根的达玛烷型人参皂苷含量最高增加到原来的1.3倍,并且单体皂苷Rb1, Rb2, Rc, Rd,Re,Rf和Rg1均不同程度增加。这些研究成果对于促进MVD的异源表达、提高人参皂苷含量的种质资源的研究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 a famous medicinal plant and used extensively because of its significant pharmaceutical activities. Ginsenosides are the major component including dammarene-type and oleanane-type saponins. So far, more than 60 kinds of ginsenosides have been found. All the ginsenosides except ginsenoside R B belong to dammarene-type saponins, which have many physiological and pharmaceutical effects, including immune system modulation, antistress, antihyperglycemic, anti-inflammatory, antioxidant and anticancer effects. Nowadays, pharmaceutical effects of ginsenosides and novel ginsenosides are still under research. Besides, Panax ginseng is consumed more and more all over the world. It is estimated that the raw Panax ginseng saled is beyond 1 billion dollars.
Due to the limitation of Panax ginseng source, it requires much cost to extract ginsenosides from root of Panax ginseng. Transgenic technique is an alternative method to improve ginsenosides content in Panax ginseng by regulating the biosynthesis pathway. Enzyme reactions involved in biosynthesis are of many steps, in which the downstream of biosynthesis is many researchers’focus. (a)Clone and identification of genes associated with upstream of the pathway is available only in a few number. In order to study the mechanism of upstream of ginsenosides biosynthesis, we chose Panax ginseng MVD gene as target to clone based on homologous alignment and RACE method. Panax ginseng MVD was obtained and expressed in Hescherichia coliH, which will support the further study. (b)In the downstream of ginsenosides biosynthesis pathway, we choseβAS from several HcandidateHs to achieve antisense technique transformation. That is becauseβAS catalyzes oleanane-type saponin from 2,3-oxidosqualene, which is the important ancestor for biosynthesis of dammarene-type saponins, major bioactive component in Panax ginseng. According to the current researches, antisense technique can successfully regulate the activity of key enzymes involved in biosynthesis pathway. The result in this study is as followed:
(1) Panax ginseng MVD gene, a key enzyme in the upstream of ginsenosides biosynthesis, was not isolated so far. Here conservative sequence, 3’fragment and 5’fragment were achieved by SMART RACE. After sequence aligning and assembling, a 1254bp of ORF was obtained.Using two primers designed according to the ORF, its total cDNA was received.
The predicted pi of Panax ginseng MVD(PgMVD) was 7.58 analyzed by software“Computer pI/Mw Tool”.Using software blast and DNAMAN, we found that PgMVD was highly homologous with plant MVDs. It has an identity of 77%、74%、72% and 68% to that of Hevea brasiliensis、Arabidopsis thaliana、Zea mays and Ginkgo biloba, respectively.
The analysis result of Panax ginseng MVD structure showed it has the classical characteristic of MVD gene family, haboring strictly conserved active sites including one leucine, one aspartate and three cysteine sites residues. Three dimensional structure of PgMVD was constructed based on the crystal structure of yeast MVD. Furthernmore, phylogenetic tree analysis exhibited that all the MVD genes were derived from the same ancestor. The MVD genes of Mus musculus, Rattus norvegicus, Homo sapiens and Danio rerio belong to one distinct animal branch differed from another plant branch of Panax ginseng, Ginkgo biloba, Hevea brasiliensis, Zea mays and Arabidopsis thaliana.
PgMVD gene was expressed successfully in Hescherichia coli H. The expressed protein exhibited a molecular weight of 46kd, which is consistent with the putative result. Thus the expressed protein supports the further analysis of MVD activities in vitro. It is also meaningful to learn the metabolic engineering of Panax ginseng and make it possible to improve quality of Panax ginseng. At the mean time, it is worthy that heterogenous expression of the new gene in other species could be exploited.
(2)Total RNA of Panax ginseng was isolated from modified Hguanidinium thiocyanate method. AntisenseβAS was subcloned into three plant expression vectors which all possessed GUS gene.
Factors influencing the Panax ginseng transformation were studied. The concentration value 0.5 of engineering bacteria is better at OD600 ,which resulted in the highest GUS positive rate; while the GUS rate was not obviously different among the infection period of 5min,10min,20min and 30min; Preculture is important because the GUS rate is more in 2 days preculture than in 1 or 3 days preculture; Three days coculture is appropriate and no Hagrobacterium H infection present; Light condition and AS concentration were researched during transformation: the result of no light and 100μM AS treatment showed efficient.
After antisenseβAS transformation, five positive hairy roots A5, A9, A19, A24 and A30 were screened out. Southern blot resulted in one copy ofβAS in all antisense hairy root lines. Nouthern blot exhibited a comprehensive decrease in all the antisenseβAS lines, which confirmed that introduction of antisenseβAS could inhibit the transcript ofβAS gene.βAS activities were all decreased significantly and the most decrease is 1/3. On the other hand, DAS were upregulated in all lines, especially in A19 with a 1.2 times as control. The oleanane-type saponin RB0 Bin AntisenseβAS Panax ginseng hairy roots lines A5, A9, A19, A24 and A30 had all dropped: it had a biggest reduction of 40% in A19, followed a 33% reduction in A30, and the least reduction of 13% was found in A5. The above result indicated that antisenseβAS could inhibit the transcript of endogenenousβAS so that it resulted in decrease of oleanane-type saponin RB0B in antisense lines. But totally inhibition ofβAS was not found because of the existance of the slight reduction in A5. Phytochemistry analysis showed that 2, 3-oxidosqualene content was elevated and dammarene-type saponins were most up to 1.3 times as control.
Dammarene-type ginsenosedes in antisense line A19 was accumulated most, thus we chose A19 as the target to detect the content changes of dammarene-type ginsenosedes over culture time. The growth curve of total ginsenosides was between ginsenoside group Rb and ginsenoside group Rg. The total ginsenosides in A19 was found 14.17mg/g DW in the ultimate culture cycle, increased 38.8% than that in initial culture cycle. Ginsenoside group Rb increased quickly over the last culture period. The Rb1 and Rb2 were increased obviously, with an increase of 20.3% and 23.3%, respectively. While Rc and Rd of ginsenoside group Rb had a slight increase. Besides, ginsenoside group Rg was elevated over culture and its growth trend was similar to the ginsenoside group Rb. Comparatively, the increase of ginsenoside group Rb in antisens line A19 was lower than that of ginsenoside group Rg.
Analysis and prospect based on above result: above all, we isolated Panax ginseng MVD by SMART RACE method. With the discover of the key enzymes involved in ginsenosides biosynthesis, scientist can master the metabolic network comprehensively so as to make it possible to further utilize the key enzymes or improve plant quality through biological technique such as genetic transformation. On the other hand, we researched the regulation of ginsenosides biosynthesis by antisense transformation. Nowadays, antisense genetic technique is turning into an important method and efficient approach in identification of gene function during the transition from genomics to post-genomics. Our research showed an inspiring prospect of antisense technique exploited in plant improvement.
引文
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