Cloning Of Key Enzyme MVD Gene And βAS Gene For Ginsenosides Biosynthesis And Expression Research Of Antisense βAS

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.