Zhihua Liao

Enhancing the scopolamine production in transgenic plants of Atropa belladonna by overexpressing pmt and h6h genes

Atropa belladonna is officially deemed as the commercial plant to produce scopolamine in China. In this study we report the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which encode the upstream key enzyme putrescine N-methyltransferase (PMT) and the downstream key enzyme hyoscyamine 6β-hydroxylase (H6H), respectively, in transgenic herbal plants Atropa belladonna. Analysis of gene expression profile indicated that both pmt and h6h were expressed at a higher level in transgenic lines, which would be favorable for biosynthesis of scopolamine. High-performance liquid chromatography result suggested that transgenic lines could produce higher accumulation of scopolamine at different levels compared with wild-type lines. Scopolamine content increased to 7.3-fold in transgenic line D9 compared with control lines. This study not only confirms that co-overexpression of pmt and h6h is an ideal method to improve the biosynthetic capacity of scopolamine but also successfully cultivates the transgenic line D9, which significantly enhanced the scopolamine accumulation. Our research can serve as an alternative choice to provide scopolamine resources for relative industry, which is more competitive than conventional market.

Effects of acetylsalicylic acid and UV-B on gene expression and tropane alkaloid biosynthesis in hairy root cultures of Anisodus luridus

Anisodus luridus hairy root cultures were established to test biological effects of acetylsalicylic acid (ASA) and ultraviolet ray-B (UV-B) on gene expression, tropane alkaloid (TA) biosynthesis and efflux. The TAs-pathway gene expression was ASA dosage dependant. The expression of PMT, TRI and CYP80F1 showed no significant difference in hairy root cultures in treatment of 0.01 and 0.1xa0mM ASA, compared with those without ASA treatment; while 0.01 or 0.1xa0mM ASA slightly upregulated H6H expression. All the four genes including PMT, TRI, CYP80F1 and H6H had a dramatic increase in 1xa0mM ASA-treated hairy root cultures compared with control. The expressing levels of all the four genes were much significantly higher in 1xa0mM ASA-treated hairy root cultures than those in 0.01 and 0.1xa0mM ASA-treated ones. As expected, hairy root cultures treated with 1xa0mM ASA had the highest capacity of TAs biosynthesis, in which the content of scopolamine and hyoscyamine reached respectively 57.2 and 14.7xa0μgxa0g−1 DW. Surprisingly, it was found that 1xa0mM ASA dramatically induced the efflux of scopolamine. In the liquid medium with 1xa0mM ASA, the content of scopolamine was 153.4xa0μgxa0flask−1, about 6.2 folds compared with that of control. At the same time, hyoscyamine was detected at trace levels in liquid medium. In the UV-B stressed hairy root cultures, all the four genes had a very strong increase of gene expression that led to more accumulation of scopolamine and lower accumulation of hyoscyamine. Only trace amounts of hyoscyamine and scopolamine were detected in the liquid medium when hairy root cultures were stressed under UV-B, and this suggested that UV-B did not affect TAs efflux.

Tryptophan decarboxylase plays an important role in ajmalicine biosynthesis in Rauvolfia verticillata

Tryptophan decarboxylase (TDC) converts tryptophan into tryptamine that is the indole moiety of ajmalicine. The full-length cDNA of Rauvolfiaverticillata (RvTDC) was 1,772 bps that contained a 1,500-bp ORF encoding a 499-amino-acid polypeptide. Recombinant 55.5xa0kDa RvTDC converted tryptophan into tryptamine. The Km of RvTDC for tryptophan was 2.89xa0mM, higher than those reported in other TIAs-producing plants. It demonstrated that RvTDC had lower affinity to tryptophan than other plant TDCs. The Km of RvTDC was also much higher than that of strictosidine synthase and strictosidine glucosidase in Rauvolfia. This suggested that TDC might be the committed-step enzyme involved in ajmalicine biosynthesis in R. verticillata. The expression of RvTDC was slightly upregulated by MeJA; the five MEP pathway genes and SGD showed no positive response to MeJA; and STR was sharply downregulated by MeJA. MeJA-treated hairy roots produced higher level of ajmalicine (0.270xa0mgxa0g−1 DW) than the EtOH control (0.183xa0mgxa0g−1 DW). Highest RvTDC expression level was detected in hairy root, about respectively 11, 19, 65, and 109-fold higher than in bark, young leaf, old leaf, and root. Highest ajmalicine content was also found in hairy root (0.249xa0mgxa0g−1 DW) followed by in bark (0.161xa0mgxa0g−1 DW) and young leaf (0.130xa0mgxa0g−1 DW), and least in root (0.014xa0mgxa0g−1 DW). Generally, the expression level of RvTDC was positively consistent with the accumulation of ajmalicine. Therefore, it could be deduced that TDC might be the key enzyme involved in ajmalicine biosynthesis in Rauvolfia.

A new isopentenyl diphosphate isomerase gene from Camptotheca acuminata: Cloning, characterization and functional expression in Escherichia coli

Isopentenyl diphosphate isomerase (EC 5.3.3.2, IPI) catalyzes the revisable conversion of 5-carbon isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), which are the essential precursors for isoprenoids, including anti-tumor camptothecin. Here we report cloning, characterization and functional expression of a new cDNA encoding IPI from Camptotheca acuminata. The full-length cDNA was 1143 bp long designated as CaIPI (GenBank® Accession Number: DQ839416), containing an open reading frame (ORF) of 930 bp which encodes a polypeptide of 309 amino acids. Bioinformatic analysis showed the cDNA sequence of CaIPI was highly homologous with other IPI gene and the deduced amino acid sequence of CaIPI was similar to known plant IPIs and contained Cys-149 and Glu-212 active sites. Phylogenic analysis indicated that all IPIs could be divided into five groups and CaIPI belonged to plant IPIs family. The tissue expression profile analysis was carried out to investigate the transcriptional level of CaIPI in different tissues. The result showed that CaIPI expression could be detected in roots, stems and tender leaves but could not in mature leaves and fruits, and the expression levels was much higher in stems than in roots and tender leaves. Finally, CaIPI was functionally expressed in engineered Escherichia coli in which the carotenoid pathway was reconstructed. In engineered E. coli, CaIPI could facilitate the metabolic flux to the carotenoids biosynthesis and made the bacteria produce the orange β-carotene. These confirmed that CaIPI had the typically function of IPI gene. In summary, cloning, characterization and functional expression of CaIPI will facilitate to understand the function of CaIPI at the level of molecular genetics and unveil the biosynthetic mechanism of camptothecin precursors.

Reference Gene Selection for Gene Expression Studies Using Quantitative Real-Time PCR Normalization in Atropa belladonna

Quantitative PCR (qPCR) is a powerful tool for measuring gene expression levels. Accurate and reproducible results are dependent on the correct choice of reference genes for data normalization. Atropa belladonna is a commercial plant species from which pharmaceutical tropane alkaloids are extracted. In this study, eight candidate reference genes, namely 18S ribosomal RNA (18S), actin (ACT), cyclophilin (CYC), elongation factor 1α (EF-1α), β-fructosidase (FRU), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and beta-tubulin (TUB), were selected and their expression stabilities studied to determine their suitability for normalizing gene expression in A. belladonna. The expression stabilities of these genes were analyzed in the root, stem, and leaf under cold, heat, NaCl, UV-B, methyl jasmonate, salicylic acid, and abscisic acid treatments using geNorm, NormFinder, and BestKeeper. The statistical algorithms indicated that PGK was a reliable gene for normalizing gene expression under most of the experimental conditions. The pairwise value analysis showed that two genes were sufficient for proper expression normalization, except when analyzing gene expression in heat-treated roots. However, the choice of the second reference gene depended on specific conditions. Finally, the relative expression level of the PMT gene of A. belladonna was detected to validate the selection of PGK a reliable reference gene. In summary, our results should guide the selection of appropriate reference genes for gene expression studies in A. belladonna under different organs and abiotic stress conditions.

A new isopentenyl diphosphate isomerase gene from sweet potato: cloning, characterization and color complementation

Isopentenyl diphosphate isomerase (IDI, EC 5.3.3.2) catalyzes the revisable conversion of 5-carbon isopentenyl diphosphate and its isomer dimethylallyl diphosphate, which are the essential precursors for isoprenoids, including carotenoids. Here we report on the cloning and characterization of a novel cDNA encoding IDI from sweet potato. The full-length cDNA is 1155 bp with an ORF of 892 bp encoding a polypeptide of 296 amino acids, which was designated as IbIDI (GenBank Acc. No: DQ150100). The computational molecular weight is 33.8 kDa and the theoretical isoelectric point is 5.76. The deduced amino acid sequence of IbIDI is similar to the known plant IDIs. The tissue expression analysis revealed that IbIDI expressed at higher level in sweet-potato’s mature leaves and tender leaves than that in tubers, meanwhile, no expression signal could be detected in veins. Recombinant IbIDI was heterologously expressed in engineered Escherichia coli which led to the reconstruction of the carotenoid pathway. In the engineered E. coli, IbIDI could take the role of Arabidopsis IDI gene to produce the orange β-carotene. In summary, cloning and characterization of the novel IDI gene from sweet potato will facilitate our understanding of the molecular genetical mechanism of carotenoid biosynthesis and promote the metabolic engineering studies of carotenoid in sweet potato.

A new 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene encoding the committed-step enzyme in the MEP pathway from Rauvolfia verticillata.

Abstract 1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR; EC 1.1.1.267) catalyzes a committed step of the methylerythritol phosphate (MEP) pathway for the biosynthesis of pharmaceutical terpenoid indole alkaloid (TIA) precursors. The full-length cDNA sequence was cloned and characterized from a TIA-producing species, Rauvolfia verticillata, using rapid amplification of cDNA ends (RACE) technique. The new cDNA was named as RvDXR and submitted to GenBank® to be assigned with an accession number (DQ779286). The fulllength cDNA of RvDXR was 1804 bp containing a 1425 bp open reading frame (ORF) encoding a polypeptide of 474 amino acids with a calculated molecular mass of 51.3 kDa and an isoelectric point of 5.88. Comparative and bioinformatic analyses revealed that RvDXR showed extensive homology with DXRs from other plant species and contained a conserved transit peptide for plastids, an extended Pro-rich region and a highly conserved NADPHbinding motif in its N-terminal region owned by all plant DXRs. The phylogenetic analysis revealed that DXRs had two groups including a plant and bacterial group; RvDXR belonged to angiosperm DXRs that were obtained from Synechocystis through gene transfer according to the phylogenetic analysis. The structural modeling of RvDXR showed that RvDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that RvDXR expressed in all tissues including roots, stems, leaves, fruits and followers but at different levels. The lowest transcription level was observed in followers and the highest transcription was found in fruits of R. verticillata; the transcription level of RvDXR was a little higher in roots and stems than in leaves. The cloning and characterization of RvDXR will be helpful to understand more about the role of DXR involved in R. verticillata TIA biosynthesis at the molecular level and provides a candidate gene for metabolic engineering of the TIAs pathway in R. verticillata.

Molecular cloning and characterization of the Strictosidine synthase Gene from Rauwolfia verticillata

The strictosidine synthase (STR, EC 4.3.3.2) catalyzes the condensation of tryptamine and secologanin to form strictosidine, which is the universal precursor for a wide range of pharmaceutical terpenoid indole alkaloids (TIAs). The full-length cDNA encoding STR was cloned and characterized from Rauwolfia verticillata a Chinese native plant producing TIAs, such as reserpine and ajmalicine. The new cDNA was designed as RvSTR and submitted to GenBank to get an accession number DQ017054. The full-length cDNA of RvSTR was 1211 bp containing a 1035-bp open reading frame encoding a deduced 344-amino-acid polypeptide with a calculated mol wt of 38.2 kD and an isoelectric point of 5.19. Comparative and bioinformatic analysis revealed that RvSTR showed a higher similarity to STRs from Apocynaceae species, including Catharanthus roseus and Ophiorrhiza pumila, but a relatively lower similarity to other plant STRs. The unique essential catalytic residue Glu-309 was conserved in all alignment plant species. The phylogenetic analysis revealed that STRs were divided into two groups, including plant and bacterial enzymes. The tissue expression pattern analysis indicated that RvSTR expression could be detected in all tested organs of R. verticillata, including roots, stems, leaves, fruits, and flowers. The lowest transcription level was observed in flowers and the highest was found in fruits; subsequently, the order of transcription level decrease was stems > roots > leaves. The cloning and characterization of RvSTR give a new STR sequence involved in TIA biosynthesis of R. verticillata, and provide a candidate gene for metabolic engineering of the TIA pathway in R. verticillata.

Variation Laws of Anthocyanin Content in Roots and Their Relationships with Major Economic Traits in Purple-Fleshed Sweetpotato [Ipomoea batatas (L.) Lam]

Abstract Variation laws of anthocyanin content in root during the development and among the varieties, and their relationships with major economic traits in purple-fleshed sweetpotato [Ipomoea batatas (L.) Lam] were studied in the present article. The dynamics of 20 economic traits in 13 purple-fleshed sweetpotato varieties at 20, 40, 60, 80, 100, 120, and 140 d after their transplanting were investigated, and these traits included anthocyanin content in root, length of the longest vine, number of base branches, root number, dry matter contents in stem, foliage and root, fresh/dry weight of root, fresh/dry weight of stem, fresh/dry weight of foliage, fresh/dry weight of stem and foliage, fresh/dry weight of whole plant, and rations of photosynthate to root, stem, and foliage. The correlations between the variations of anthocyanin content and the other 19 economic traits among varieties and during the whole developing stages, and the correlations of daily increase of anthocyanin content with other 10 kinds of yields were analyzed. The results showed that: (1) During the whole development, the anthocyanin content had three variation types, i.e. a slow-increase type, a fluctuating-change type, and a deviousrising type, and had different responses to the growth of length of the longest vine, number of base branches, fresh/dry yield of root, and photosynthate allotments. (2) The anthocyanin contents among 13 varieties began to have significant difference after 20 d, and showed completed differentiation during 40–100 d, which had significantly negative correlationships with the number of base branches, fresh/dry yield of root, photosynthate allotment ratio to root, and had significant positive correlationships with dry matter content of root, length of the longest vine, fresh/dry yield of stem, dry yield of whole plant and photosynthate allotment ratio to foliage. (3) Because of the significantly negative correlation between daily increase of anthocyanin content and dry matter weight of root, the anthocyanin accumulation competed with dry matter accumulation for photosynthate in root, and the competitive relation was resolved in different ways in different purple-fleshed sweetpotato (PFSP) varieties. So, there had three variation types of anthocyanin content among PFSP varieties during their development, and had different correlations between these variations of anthocyanin content and the major economic traits.

A natural phenylpropionate derivative from Mirabilis himalaica inhibits cell proliferation and induces apoptosis in HepG2 cells

Bioactivity-guided study led to the isolation of a natural phenylpropionate derivative, (E)-3-(4-hydroxy-2-methoxyphenyl)-propenoic acid 4-hydroxy-3-methoxyphenyl ester from the roots of Mirabilis himalaica. Cellular analysis showed that compound 1 specifically inhibited the cancer cell growth through the S phase arrest. Mechanistically, compound 1 was able to induce the apoptosis in HepG2 cells through mitochondrial apoptosis pathway in which Bcl-2 and p53 were required. Interestingly, the cellular phenotype of compound 1 were shown specifically in cancer cells originated from hepatocellular carcinoma (HepG2) while compromised influence by compound 1 were detected within the normal human liver cells (L-02). Consistently, the in vivo inhibitory effects of compound 1 on tumor growth were validated by the in xenograft administrated with HepG2 cells. Our results provided a novel compound which might serve as a promising candidate and shed light on the therapy of the hepatocellular carcinoma.