| The active components of Chinese crude drug come from secondary metabolite. It has great significance to improve the quality of Chinese crude drug by the method of cell (somatic hybridization) and secondary metabolism engineering and molecular cloning technique.Somatic hybridization techniques (protoplast fusion) have great advantages in plant breeding. Fusing protoplast among intergeneric or inter-familial species and could transfer and increase the content of active compounds between different species. At present, some successful somatic hybridization in Chinese crude drug had been made, such as somatic hybridization between Bupleurum scorzonerifolium Willd and Swertia mussotii Franch (Xiang et al.,2003). However, it is worthy of doing further researches on the relationship between the transfser or accumulation of donor active components and the way or feature of heterogenesis chromatin introgression.Recently, the hot spot of research in plant secondary metabolic engineering has been turned into cloning and transformation of genes coding key enzymes, of which the over expression has been one of major strategies to increase the yield of target metabolite. The biosynthesis pathways of terpene were drawn generally and genes in the monoterpene pathway were cloned, however, the related genes in the pathway were still known a little in Chinese crude drug.Swertia mussotii Franch, an Tibet herb, was used as a general drug in Tibetan nationality. The whole plant could be used as a drug and is the best medicine for the therapy of hepatitis and viral hepatitis. Due to its important value and economic returns, the requirement increased quickly which induced an anthropogenic excessive exploitation. The mainly active compounds of S. mussotii are swertiamarin, mangiferin and gentiopicroside.Swertiamarin and gentiopicroside belong to iridoid monoterpenoid and their biosynthetic pathway has not been characterized in any detail. As an important composition in the iridoid pathway, geraniol could be catalyzed by a hydroxylase, geraniol 10-hydroxylase (G10H), which was an enzyme in the first rate determining step. G10H was presumed as the key enzyme in the iridoid monoterpenoid swertiamarin biosynthesis.In this research, somatic hybridization was carried out between B. scorzonerifolium and S. mussotii and the regenerated plants were obtained from the hybrids. The differences of active compounds, chromosome numbers and genome in S. mussotii and hybrids were analysized. And the differences of donator chromosome, genome DNA and SmG10H expression in hybrids were discussed. Furthermore, the relationship among donator genomic constitution, SmG10H expression and heterogenic active compounds were analyzed. Finally, the effect of ultraviolet light dosage on the growth, the elimination and gragmentation of the chromosome in the hybrids were discussed.According to the diversity of P450 gene sequences in hybrids, a P450 which was related with the biosynthesis of swertiamarin was selected and predicted as geraniol 10-hydroxylase (G10H). We firstly separated the full length SmG10H from S. mussotii. We analyzed systematically the relationship between the gene expression level and the content of swertiamarin; compared the differences of expression of the genes related with the swertiamarin metabolic pathway under methyl jasmonic acid treatments. After been heterologously expressed in both Escherichia coli and Pichia pastoris (yeast), SmG10H was demonstrated to have the catalytic activity of hydroxylating geraniol in vitro. In the SmG10H over-expression plants, the level of SmG10H transcript and the contents of 10-hydroxygeraniol and swertiamarin were increased simultaneously. These transgenic plants were established the found for the further researches of transgenic plant descendant and swertiamarin biosynthesis. These data supported the advanced research on the regulation mechanism of iridoid monoterpenoid biosynthesis.The main process and results of this research were as follows:1. Somatic hybridization between B. scorzonerifolium and S. mussotii1.1 Hybrids of B. scorzonerifolium and S. mussotiiThe protoplasts of S. mussotii irradiated with UV (380μW/cm2) for 0 s,30 s,1 min,2 min and 3 min respectively were fused by PEG method with protoplast of B. scorzonerifolium.194 clones were obtained by somatic hybridization, and 3 clones were regenerated to plants. From those clones,104 clones were verified as hybrids by chromosome counting and isozyme analysis.1.2 Nuclear genome in hybrids1.2.1Analysis of Chromosome and GISHAccording to the chromosome analysis, the chromosome number of B. scorzonerifolium calli was 11-12, that of S. mussotii calli was 17-20 and that of hybrids was 11-20.GISH results showed that the hybrids clone B24 (with regeneration capacity) and C10 had 11-13 B. scorzonerifolium chromosome,0-3/1-3 intact S. mussotii chromosomes and 0-1/5-9 recombinant chromosomes, suggested that the intact donor chromosome decreased and chromatin introgression increased as the dosage of UV enhanced. So, the dosage effect of UV on donor chromosome elimination and fragmentation is evident in this experiment.1.2.2 Analysis of RAPD200 random primers were used in the amplification of PCR. The results indicated that: Characteristic bands from both parents and new bands were contained in all hybrids;RAPD bands of 91.2-96.0% were derived from B. scorzonerifolium and that of 0.2-2.6% were derived from S. mussotii in hybrids, suggested that the contribution of receptor to the hybrid genomes was dominant in nuclear genomes. The differences of the new bands and donor characteristic bands among the hybrids were small and donor nuclear genome DNAs in these hybrids were similar except hybrid C10. Furthermore the numbers of the donor bands and the new bands had positive correlation with UV dosage.1.3 HPLC and GCMS analysis of the efficacious components and the volatile compounds in the hybridsHPLC analysis of 74 hybrid clones found that there was no gentiopicroside in the all hybrid clones. Hybrid clones B24, B27, B132, C18, C26, C47 and C124 contained 0.0074-0.0812 mg/g swertiamarin. Hybrid B6, B40, B56, C10 and C121 had 0.0863-0.8168 mg/g mangiferin, among of which B6, B56 and C10 were much higher than the donor callus of S. mussotii (0.6641 mg/g). This results demonstrated that some related gene for mangiferin and swertiamarin synthesis from S. mussotii had been transferred into B. scorzonerifolium and expressed. Analysis of GC-MS of volatile compounds in the hybrids indicated that hybrid clones had more volatile compounds of B. scorzonerifolium and fewer components of S. mussoti. 1.4 P450 genes in hybridsPCR amplification of P450 genes were carried out with degenerate primers in the parents and hybrids (A6, A67, B24, B27, B132, C18, C26, C47 and C124).11 fragments, containing 1100-1200 bp, were obtained and aligned. According to the alignments, G10H from B. scorzonerifolium had 45.7% homology with that from S. mussotii(SmG10H);While G10H from hybrids (B24, B27, B132, C18, C26, C47and C124) had 100% homology with that from S. mussot ii, suggested that it could come from S. mussoti. G10H from hybrids (A6 and A67) had 84.1% homology with that from B. scorzonerifolium and 53.1% with that from S. mussotii, suggested that it could come from B. scorzonerifolium. G10H might be involved in the synthesis of monoterpene. As the numbers of monoterpene, the synthesis of swertiamarin and gentiopicroside might need G10H. Therefore, it was necessary to research the function of G10H in this work.1.5 The relationship of swertiamarin content and SmG10H expression in hybridsAnalysis of Semi-quantitative RT-PCR suggested that SmG10H expression rank were:S. mussotii>B24> B132> C47> A6. This result indicated that the expressions of SmG10H were difference in hybrids. According to the analysis of HPLC, content of swertiamarin in S.mussotii (0.9298 mg/g) was higher than in hybrids (0.0074-0.0812 mg/g), while contents in B24 and B132 were higher than that in C47. Any swertiamarin and SmG10H expression were not detected in A6. These data suggested that SmG10H expression had positive correlation with the content of swertiamarin.2 Cloning of genes related with iridoids in S. mussotii and analysis of its function2.1 Cloning of SmG10H geneBy 5'and 3'RACE, the full length SmG10H sequence of 1488 bp (Genebank accession GU168041) was obtained. The predicted gene, with a intron in the location of 882-1030 bp, encoded a 496 residue protein (molecular mass 55.5 kDa). The amino acid sequence of this protein is 80.2% identical to CYP76B6 of Catharanthus roseus and was designated as CYP76B10.2.2 Distribution of SmG10H in genome of S. mussotiiAccording to the analysis of southern blot, SmG10H derived from a single copy in the genome of S. mussotii. 2.3 Expression analysis of genes related with iridoids pathway2.3.1 Expression analysis of SmG10H in S. mussotiiAccording to the analysis of Semi-quantitative RT-PCR and real-time PCR, SmG10H transcripts are much more abundant in the leaf than in either the root or the stem. It was 24.9 times higher than that in root and 10.2 times higher than that in stem.2.3.2 Expression of genes related and the content of swertiamarin following elicitation by MeJA.Five fragments of genes related with the synthesis of iridoids were cloned, they were SmG10H, DXS, HMGR, IPP-iso and MECS. Special primers were designed to analyze the expression under the treatment of MeJA.1) Different concentration of MeJA (0,10,20,50,100 and 150μM) were used to induce the expression of SmG10H for S. mussotii seedlings. SmG10H reached its max level under 50μM MeJA at 6h and this concentration was selected for the next experiments.2) The expression of SmG10H could be induced by the treatment with 50μM MeJA during the growth phase. The expression of other related genes, including DXS, MECS, IPP-iso and HMGR, were also up-regulated. Their mRNA levels peaked at 24 h and then decreased appreciably at 36 h after the treatment.3) As the time extended, the contents of swertiamarin, gentiopicroside and mangiferin were accumulated to a maximum at 15 d,2.62 mg/g,0.67 mg/g and 0.21 mg/g respectively. These were about 2.5,2.2 and 2.7 times higher than that in the unelicited plants. Their ratioes reached a maximum between 24-36 h. These data suggested that MeJA could promote the accumulation of active compounds in S. mussotii.4) The expression of SmG10H was increased with the the accumulation of swertiamarin which indicated that the former had positive correlation with the later.2.4 Function of SmGlOH in vitro2.4.1 Heterologous expression of the SmG10H recombinant protein in Escherichia coli1) For its heterologous expression in E. coli, full-length SmG10H was excised from the recombinant plasmid (pMD18T-SmG10H) and ligated into the sites of the pET28a expression vector The SmG10H recombinant protein in E. coli showed a protein band on SDS-PAGE with a molecular mass of 60 kDa and its expression level reached the maximum after induced by IPTG. No equivalent protein was expressed in bacteria carrying the empty vector. 2) Afer the purification of the SmG10H recombinant protein, its isoelectric point was determined at 8.63±0.3. It could catalyze geraniol to be a compound which shared the same retention time (8.3 min) with standard 10-hydroxygeraniol in LC profile. Mass spectral analysis showed that this peak had the structure of 10-hydroxygeraniol and gave a positive identification for 10-hydroxygeraniol. This result supported that SmG10H recombinant protein in transgenic E. coli could hydroxylate geraniol to 10-hydroxygeraniol.3) For the advanced research, the condition of this reaction was optimized. And its best catalyticcondition were:pH7.7±0.1 and 30℃. Its activity level was 0.36±0.02 (pkat/mg protein).2.4.2 Heterologous expression of the SmG10H recombinant protein in Pichia pastoris1) For its expression in P. pastoris yeast, the full-length SmG10H sequence was excised from the recombinant pMD18T-SmG10H and ligated into the sites of the P. pastoris expression vector pPICZα. The membrane fraction containing SmG10H protein, which was separated from transgenic yeast after induced, was used for in vitro experiments. It was also showed a protein with a molecular mass of 60.0 kDa. It could catalyze geraniol to be a compound which shared the same retention time (8.3 min) with standard 10-hydroxygeraniol in LC profile. This result supported that SmG10H recombinant protein in transgenic yeast could hydroxylate geraniol to 10-hydroxygeraniol.2) According to the extinction value, SmG10H recombinant protein was determined at 1μg/100μg membrane fraction. Km of SmG10H was estimated to be 5.2±0.2 mM at 30 min with 100μg membrane fraction which was the optimized condition.2.4.3 Functional identification of SmG10H in S. mussotiiPlant overexpression vector pBGWF7 and RNAi vector pK7GWIWG were constructed using gateway technique and transferred into 7 d grown calli (differentiation rate> 90%)of S. mussotii by particle gun. After cultured in darkness for 24 h, resistant calli were selected by 100 mg/l Kan. Then these calli differentiated into plants on IB medium. Further selection with 25 mg/l Kan was taken and 24 resistant plants were obtained (15 seedlings for over expression line,9 for RNAi line). PCR detection was used for the positive lines with primers for T35S, ccdB and SmG10H5. Transformation frequency for over expression and RNAi line were 1.6% and 2.0% respectively. Semiquantitative RT-PCR analysis showed that the expression of SmG10H in wild type was lower than that in the over expression line, but higher than that in the RNAi line. 3.3.2 Content of 10-hydroxygeraniol and active compounds in transgene linesThe ranking of 10-hydroxygeraniol, gentiopicroside and swertiamarin contents were overe expression line> wild line> RNAi line and the ranking was R3< R2< R1< WT< OS2< OS1< OS3. However, the ranking of mangiferin content was just opposite, R3> R2> R1> WT> OS2 > OS1> OS3. Contents of 10-hydroxygeraniol, gentiopicroside and swertiamarin in OS3 were 2.65,1.71 and 1.91 times higher than those in the WT respectively. These data supported that the over expression of SmGl OH promoted the accumulation of iridoids in transgenic plant, but reduced the contents of other terpenes. These results suggested that SmG10H plays an important role in the iridoid monoterpenoid swertiamarin biosynthesis in S. mussotii.
|
PDF Full Text Request