Search

The lotus (Nelumbo Adans.) is an important aquatic plant with ornamental, medicinal and edible values and cultural connotations. It has single-, semi-double-, double- and thousand-petalled types of flower shape and is an ideal material for developmental research of flower doubling. The lotus is a basal eudicot species without a morphological difference between the sepals and petals and occupies a critical phylogenetic position in flowering plants. In order to investigate the genetic relationship between the sepals and petals in the lotus, the class E genes which affect sepal formation were focused on and analyzed. Here, SEPALLATA 1(NnSEP1) and its homologous genes AGAMOUS-LIKE MADS-BOXAGL9 (NnAGL9) and MADS-BOX TRANSCRIPTION FACTOR 6-like (NnMADS6-like) of the class E gene family were isolated from the flower buds of the Asian lotus (Nelumbo nucifera Gaertn.). The protein structure, subcellular localization and expression patterns of these three genes were investigated. All three genes were verified to locate in the nucleus and had typical MADS-box characteristics. NnSEP1 and NnMADS6-like were specifically expressed in the sepals, while NnAGL9 was highly expressed in the petals, suggesting that different developmental mechanisms exist in the formation of the sepals and petals in the lotus. The significant functional differences between NnSEP1, NnMADS6-like and NnAGL9 were also confirmed by a yeast two-hybrid assay. These results expand our knowledge on the class E gene family in sepal formation and will benefit fundamental research on the development of floral organs in Nelumbo.

In this study, a Fe3O4@Au-based pseudo-homogeneous electrochemical immunosensor was prepared for detection of alpha fetoprotein (AFP), a well-known hepatocellular carcinoma biomarker. The primary antibody (Ab1) was immobilized on Fe3O4@Au NPs as the capture probe. Horseradish peroxidase (HRP) and secondary antibody (Ab2) were conjugated on gold nanoparticles (GNPs) through electrostatic adsorption to form signal-amplifying labels. In the presence of AFP, a sandwich immunocomplex was formed via specific recognition of antigen-antibody in a Fe3O4@Au-basedpseudo-homogeneousreaction system. After the immunocomplex was captured to the surface of magnetic glassy carbon electrode (MGCE), the labeling HRP catalyzed the decomposition of H2O2, resulting in a substantial current for the quantitative detection of AFP. The amperometric (i-t) method was employed to record the response signal of the immunosensor based on the catalysis of the immobilized HRP toward the reduction of H2O2 with hydroquinone (HQ) as the redox mediator. Under the optimal conditions, the amperometric current response presented a linear relationship with AFP concentration over the range of 20 ng/mL-100 ng/mLwith a correlation coefficient of 0.9940, and the detection limit was 0.64 ng/mL at signal/noise [S/N] = 3. Moreover, the electrochemical immunosensor exhibited higher anti-interference ability, acceptable reproducibility and long-term stability for AFP detection.

Chalcone synthases (CHS, EC 2.3.1.74) are key enzymes that catalyze the first committed step in flavonoid biosynthesis. In this study, we isolated a chalcone synthase, named NtCHS6, from Nicotiana tabacum. This synthase shared high homology with the NSCHSL (Y14507) gene and contained most of the conserved active sites that are in CHS proteins. The phylogenetic analysis suggested that NtCHS6 protein shared a large genetic distance with other Solanaceae CHS proteins and was the most closely-related to an uncharacterized CHS from Solanum lycopersicum. The expression analysis indicated that NtCHS6 was abundantly expressed in leaves, especially in mature leaves. By scrutinizing its upstream promoter sequences, multiple cis-regulatory elements involved in light and drought responsive were detected. Furthermore, NtCHS6 expression decreased significantly under dark treatment and increased significantly under drought stress. Our results suggested that NtCHS6 expression exhibited both light responsiveness and drought responsiveness, and might play important roles in ultraviolet protection and drought tolerance.

Chalcone synthase (CHS, EC 2.3.1.74) is a member of the plant polyketide synthase superfamily; it catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, we identified and characterized five CHS superfamily genes from Nicotiana tabacum. Our phylogenetic analysis suggested that the five tobacco CHS genes have diverged into two subgroups, including the classical CHS genes for NtCHS1–4 and CHS-Like genes for NtCHS5. Solanaceae CHSs diverged into different subfamilies prior to the divergence of the genera. We used RNA-seq data to explore the spatial and temporal expression patterns of the NtCHS1–5 genes. NtCHS1–4 were widely expressed in pigmented or/and non-pigmented vegetative tissues and floral organs. NtCHS5 was expressed strongly in the calyx and corolla organs at the full-bloom stage. Quantitative RT-PCR analysis was performed; it verified our RNA-seq results. Further, the expression of NtCHS1 and NtCHS3 increased markedly under drought and high salt stress. NtCHS2 expression and NtCHS4 expression were obviously induced by ABA and MeJA treatment, and NtCHS4 expression was specifically induced by 2,4-D. NtCHS5 expression was not induced by any of these treatments. Our results represent new data about the differentiation of gene expression and environmental responsiveness in the expression of CHS superfamily genes in Nicotiana tabacum.

Dynamic transcriptional changes of the host early responses genes were detected in PVY-resistant tobacco varieties infected with Potato virus Y; PVY resistance is a complex process that needs series of stress responses. Potato virus Y (PVY) causes a severe viral disease in cultivated crops, especially in Solanum plants. To understand the molecular basis of plant responses to the PVY stress, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potentially important or novel genes that were involved in early stages (12 h, 1, 2, 3, 5, 8 days) of tobacco response to PVY infection. Dynamic changes of the host plant early responses to PVY infection on a transcriptional level were detected. In total, 167 different expressed ESTs were identified. The majority of genes involved in the metabolic process were found to be down-regulated at 12 h and 1 day, and then up-regulated at least one later period. Genes related to signaling and transcriptions were almost up-regulated at 12 h, 1 or 2 days, while stress response genes were almost up-regulated at a later stage. Genes involved in transcription, transport, cell wall, and several stress responses were found to have changed expression during the PVY infection stage, and numbers of these genes have not been previously reported to be associated with tobacco PVY infection. The diversity expression of these genes indicated that PVY resistance is a complex process that needs a series of stress responses. To resist the PVY infection, the tobacco plant has numerous active and silent responses.

The lotus (Nelumbo Adans.) is a perennial aquatic plant with important value in horticulture, medicine, food, religion, and culture. It is rich in germplasm and more than 2000 cultivars have been cultivated through hybridization and natural selection. Microsporogenesis and male gametogenesis in the anther are important for hybridization in flowering plants. However, little is known about the cytological events, especially related to the stamen, during the reproduction of the lotus. To better understand the mechanism controlling the male reproductive development of the lotus, we investigated the flower structure of the Asian lotus (N. nucifera). The cytological analysis of anther morphogenesis showed both the common and specialized cytological events as well as the formation of mature pollen grains via meiosis and mitosis during lotus anther development. Intriguingly, an anatomical difference in anther appendage structures was observed between the Asian lotus and the American lotus (N. lutea). To facilitate future study on lotus male reproduction, we categorized pollen development into 11 stages according to the characterized cytological events. This discovery expands our knowledge on the pollen and appendage development of the lotus as well as improving the understanding of the species differentiation of N. nucifera and N. lutea.

Superoxide dismutases (SODs) are a family of metalloproteins extensively exists in eukaryote, which plays an essential role in stress-tolerance of higher plants. A full-length cDNA encoding Cu/Zn SOD (BcCSD1) was isolated from young seedlings of non-heading Chinese cabbage (Brassica campestris ssp. chinensis) by rapid amplification of cDNA ends (RACE). Bioinformatics analysis revealed that BcCSD1 belonged to the plant SOD super family and had the closest relationship with SOD from Brassica napus. Tissue expression pattern analysis revealed that the BcCSD1 was constitutively expressed in all the tested tissues, and strongest in leaf, moderate in stem, lowest in root. The expression profiles under different stress treatments such as drought, NaCl, high temperature and ABA were also investigated, and the results revealed that BcCSD1 was a stress-responsive gene, especially to ABA. These results provide useful information for further understanding the role of BcCSD1 resistant to abiotic stress in Brassica campestris in the future.

Camptothecin (CPT) is a kind of modified monoterpene indole alkaloid firstly identified from woody plant Camptotheca acuminata, and its semisynthetic CPT analogs irinothecan and topothecan are clinically used for the treatment of various cancers throughout the world. However, the extraction of CPT from limited natural CPT-producing plant resources couldn’t meet the rapidly increasing market need. The development of plant metabolic engineering provides one alternative way to increase CPT yield by genetic manipulation, which relies on in-depth understanding of the CPT biosynthesis pathway. Several attempts have been also made to obtain CPT by biotechnological approaches such as cell suspensions, endophytic fungi, hairy roots, elicitation as well as metabolic engineering in the past decade. Here, recent advances in knowledge of biosynthesis of CPT, gene isolation, molecular regulation, production improvement and biotechnological methods are summarized and future perspectives are also discussed in this review.

Tropane alkaloids (TAs) such as anisodamine, anisodine, hyoscyamine and scopolamine are extensively used in clinical practice as anticholinergic agents. Anisodus acutangulus produces TAs in root tissue, and although several genes involved in scopolamine biosynthesis have been cloned, yet the biosynthetic pathway of TAs remains poorly understood. To further understand TAs biosynthesis mechanism, transcriptome analysis with deep RNA sequencing in A. acutangulus roots was performed in this study; 48 unigenes related to tropane, piperidine and pyridine alkaloid biosynthesis, 145 linked to the distribution of arginine to TAs biosynthesis, and 86 categorized to terpenoid backbone biosynthesis have been identified in pathway enrichment analyses with eukaryotic orthologous groups (KOG) and Kyoto encyclopedia of genes and genomes. Additionally, 82 unigenes annotated as cytochrome P450 family members seemed to be involved in secondary metabolism. Genes encoding littorine mutase/monooxygenase (CYP80F1), diamine oxidase (DAO), alcohol dehydrogenase (ADH) and aromatic amino acid aminotransferase (ArAT) may also play roles in TAs biosynthetic pathways. Furthermore, over 1,000 unigenes were identified as potential transcription factors of WRKY, AP2/ERF, MYB and bHLH families, which would be helpful to understand transcriptional regulation of secondary metabolite biosynthesis. These data enable novel insights into A. acutangulus transcriptome, updating the knowledge of TAs biosynthetic mechanism at molecular level.

Tanshinone is a group of active diterpenes, which are widely used in the treatment of cardiovascular disease. In this study, methyl jasmonate (MJ) and salicylic acid (SA) were used to investigate their effects on tanshinone accumulation and biosynthetic gene expression in the hairy roots of geranylgeranyl diphosphate synthase (SmGGPPS) overexpression line (G50) in Salvia miltiorrhiza. High-performance liquid chromatography analysis showed that total tanshinone content in G50 was obviously increased by 3.10-fold (11.33 mg/g) with MJ at 36 H and 1.63 times (5.95 mg/g) after SA treatment for 36 H in comparison with their mimic treatment control. Furthermore, quantitative reverse-transcription PCR analysis showed that the expression of isopentenyl-diphosphate delta-isomerase (SmIPPI), SmGGPPS, copalyl diphosphate synthase (SmCPS), and kaurene synthase-like (SmKSL) increased significantly with MJ treatment. However, the expression of SmIPPI reached the highest level at 144 H, whereas those of SmGGPPS, SmCPS, and SmKSL only increased slightly with SA treatment. The two elicitor treatments suggested that tanshinone accumulation positively correlated to the expression of key genes such as SmGGPPS, SmCPS, and SmKSL. Meanwhile, the study also indicated that it was a feasible strategy to combine elicitor treatment with transgenic technology for the enhancement of tanshinone, which paved the way for further metabolic engineering of tanshinone biosynthesis.

Camptotheca acuminata is a Chinese tree that produces the anti-cancer monoterpenoid indole alkaloid camptothecin (CPT). 3-Hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) catalyzes the condensation of acetyl CoA and acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl-CoA as an early step in the CPT biosynthetic pathway. A full-length cDNA encoding HMGS (designated as CaHMGS, GenBank accession no. EU677841) was successfully isolated from young leaves of C. acuminata by rapid amplification of cDNA ends (RACE). The full-length cDNA of CaHMGS was 1801 bp long and contained a 1413-bp open reading frame encoding a polypeptide of 471 amino acids. Comparative and bioinformatic analyses revealed that CaHMGS showed extensive homology with HMGSs from other plant species. Southern hybridization analysis showed that there were at least two HMGS gene members in the C. acuminata genome. CPT content was found to be much higher in cotyledons and hypocotyls as compared to roots. RT-PCR analysis revealed strong expression in hypocotyls and cotyledons, but no expression in roots, indicating good correlation between CaHMGS expression and CPT content in the tested tissues. The expression of CaHMGS could be regulated by exogenous elicitors, including salicylic acid and methyl jasmonate, suggesting that CaHMGS was elicitor-responsive. This work is a first step to acquire a better understanding on the role of HMGS in CPT biosynthesis.

Hepatocellular carcinoma (HCC) is a common clinical primary malignant tumor; however, efficient drugs for the treatment of HCC are still lacking at the present time. To develop a new approach for liver cancer therapy, we designed a chimeric gene (his-HR) encoding a single-chain variable fragment of human HAb25 (hHscFv) fused to a cytotoxic ribonuclease from Rana catesbeiana (RC-RNase) and expressed the corresponding fusion protein in transgenic tobacco (Nicotiana tabacum). Eleven positive transgenic plant lines were identified from 204 regenerated tobacco plants by PCR and Southern blot analysis, and the immunocompetence of the recombinant his-HR protein was confirmed by Western blotting. The expression levels of his-HR protein ranged from 0.75 to 1.99 µg/g in the fresh tobacco leaves. To characterize the bifunction of the expressed his-HR protein in tobacco, binding specificity and cell toxicity to several cell lines were examined by the indirect immunocytochemical streptavidin-biotin complex method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Data indicated that the his-HR protein had stronger specific binding affinity to HepG2 (human liver HCC cell line) than to the other tumor cell lines and normal liver cell line, and the capacity to kill the HCC cell lines SMMC7721 and HepG2 with an half maximal inhibiting concentration of 2.0 and 2.4 nM, respectively. The results suggest that recombinant bifunctional his-HR protein derived from transgenic plants may provide a novel strategy to treat HCC in the future.

The AP2/ERF-domain transcription factor ORCA3 of Catharanthus roseus has been recognized earlier as a regulator of the expression of some important genes involved in the terpenoid indole alkaloid (TIA) biosynthetic pathway in C. roseus cells. Therefore, this factor was postulated to play an important role for the biosynthesis of secondary metabolites in plants. To investigate the effect of ORCA3 overexpression on the TIA biosynthesis in C. roseus hairy roots, biotransformation of Orca3 gene was conducted with the disarmed Agrobacterium rhizogenes C58C1 harboring pCAMBIA1304+-Orca3, a plasmid that contains an Orca3 gene, a Gus gene, and a hygromycin-resistance (hyg) gene, all driven separately by the cauliflower mosaic virus 35S promoter (35SCaMV). Putative transgenic hairy root lines were induced and subcultured. The T-DNA fragment of pCAMBIA1304+-Orca3 was proved to be integrated into C. roseus hairy root genome by genomic-PCR analysis, real-time quantitative PCR analysis, and GUS staining assay. Metabolite analysis using HPLC established that the average content of catharanthine and vindoline in the transgenic hairy root extracts was increased 2.49-fold and 4.2-fold in comparison to the control hairy root lines, respectively. However, vinblastine content could not been detected by HPLC method in transgenic and control hairy root cultures.

The AP2/ERF-domain transcription factor ORCA2 from Catharanthus roseus was demonstrated earlier to regulate the expressions of Str gene, an important gene involved in the terpenoid indole alkaloids biosynthetic pathway in C. roseus cells. Therefore, the factor was postulated to play an important role in the production of secondary metabolites in plants. To investigate the effect of over expression of ORCA2 on the TIAs biosynthesis in C. roseus hair roots, transformation of ORCA2 gene was conducted with the disarmed Agrobacterium rhizogenes C58C1 harboring pCAMBIA1304+, a plasmid that contains the Orca2 gene, a Gus gene and an Hpt gene all under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Transgenic hairy root cultures expressing Orca2 gene were obtained and demonstrated by genomic- polymerase chain reaction (PCR) analysis for the integration of the Orca2 gene in the C. roseus genome, by real-time quantitative PCR (RT-QPCR) and β-glucuronidase (GUS) staining for the expression of the foreign genes. Metabolite analysis using high performance liquid chromatography(HPLC) analysis established that the average content of catharanthine and vindoline in the transgenic hairy root extracts was increased up to 2.03 and 3.67-fold in comparison to the control lines, respectively. However, vinblastine could not been detected in the transgenic and control hairy root cultures by HPLC. Key words: Catharanthus roseus, ORCA2, hairy root, overexpression, terpenoid indole alkaloids (TIAs), AP2/ERF-domain transcription factor.

Alpha-fetoprotein (AFP) is a typical tumor marker in early diagnosis. Highly specific detection of the AFP was demonstrated using AlGaAs/GaAs high electron mobility transistors (HEMTs). Anti-AFP antibody was immobilized to the Au-coated gate area of the HEMT by a covalent modification method. To avoid any nonspecific binding, the gate was blocked by bovine serum albumin after the reduction of the carbonyl group. A detection limit for the AFP as low as a few picograms/milliliter was achieved, and cross reactivity of the sensor was found negligible, demonstrating its high selectivity. Successful detection with minute quantity of the sample indicates that the sensor has great potential in practical screening for a wide population.

Thymosin α1 (Tα1) was widely used for the treatment of hepatitis (B and C) and several cancers. However, current production of Tα1 is difficultly meeting clinical needs. To develop a novel and safety approach for Tα1 production, we synthesized a Tα1 gene (124 bp) based on the plant codon usage bias and constructed a four-copy Tα1 gene concatemer (408 bp) by using isocaudamer technique. This 4 × Tα1 structure was cloned into plant binary expression vector pCAMBIA2300 with twin transfer deoxyribonucleic acids (T-DNAs) and integrated into lettuce genome via Agrobacterium-mediated transformation. Thirteen positive plants were identified by polymerase chain reaction and confirmed by Southern blot analysis, and 11 marker-free lettuce plants were obtained in T2 generation. The content of recombined Tα1 (rTα1) protein reached 1798.317 ± 87.312 ng/g in fresh leaves of transgenic lettuce. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay demonstrated that rTα1 protein stimulated mouse splenic lymphocyte proliferation in vitro. These data suggest that biologically active rTα1 was successfully expressed in marker-free transgenic lettuce, and this method could provide an alternative choice for large-scale production of Tα1 in the future.

Catharanthus roseus L. (Madagascar periwinkle) biosynthesizes a diverse array of secondary metabolites including anticancer dimeric alkaloids (vinblastine and vincristine) and antihypertensive alkaloids (ajmalicine and serpentine). The multi-step terpenoid indole alkaloids (TIAs) biosynthetic pathway in C. roseus is complex and is under strict molecular regulation. Many enzymes and genes involved in the TIAs biosynthesis have been studied in recent decades. Moreover, some regulatory proteins were found recently to control the production of TIAs in C. roseus. Based on mastering the rough scheme of the pathway and cloning the related genes, metabolic engineering of TIAs biosynthesis has been studied in C. roseus aiming at increasing the desired secondary metabolites in the past few years. The present article summarizes recent advances in isolation and characterization of TIAs biosynthesis genes and transcriptional regulators involved in the second metabolic control in C. roseus. Metabolic engineering applications in TIAs pathway via overexpression of these genes and regulators in C. roseus are also discussed.

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) and liquid chromatography coupled with sequential mass spectrometry (LC/MSn) were applied to identify trace-level phenanthroindolizidine alkaloids in crude extracts from Tylophora atrofolliculata. Based on the relationship between the characteristic fragmentation reactions and the structural features of related compounds of known structure from this plant, the bioactive crude extract was analyzed in detail by positive and negative ion ESI-MSn, LC/UV-MS and LC/MSn techniques. A total of nine constituents in the crude extract were identified rapidly, including several isomers; seven of these constituents are new and two are known compounds. The structures of four of these constituents were subsequently confirmed by nuclear magnetic resonance (NMR) and accurate mass measurements using high-resolution fast-atom bombardment mass spectrometry (FAB-HRMS). Copyright © 2003 John Wiley & Sons, Ltd.

Camptothecin (CPT) belongs to a group of monoterpenoidindole alkaloids (TIAs) and its derivatives such as irinothecan and topothecan have been widely used worldwide for the treatment of cancer, giving rise to rapidly increasing market demands. Genes from Catharanthus roseus encoding strictosidine synthase (STR) and geraniol 10-hydroxylase (G10H), were separately and simultaneously introduced into Ophiorrhiza pumila hairy roots. Overexpression of individual G10H (G lines) significantly improved CPT production with respect to non-transgenic hairy root cultures (NC line) and single STR overexpressing lines (S lines), indicating that G10H plays a more important role in stimulating CPT accumulation than STR in O. pumila. Furthermore, co-overexpression of G10H and STR genes (SG Lines) caused a 56% increase on the yields of CPT compared to NC line and single gene transgenic lines, showed that simultaneous introduction of G10H and STR can produce a synergistic effect on CPT biosynthesis in O. pumila. The MTT assay results indicated that CPT extracted from different lines showed similar anti-tumor activity, suggesting that transgenic O. pumila hairy root lines could be an alternative approach to obtain CPT. To our knowledge, this is the first report on the enhancement of CPT production in O. pumila employing a metabolic engineering strategy.

Tanshinone is widely used for treatment of cardio-cerebrovascular diseases with increasing demand. Herein, key enzyme genes SmHMGR (3-hydroxy-3-methylglutaryl CoA reductase) and SmDXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase) involved in the tanshinone biosynthetic pathway were introduced into Salvia miltiorrhiza (Sm) hairy roots to enhance tanshinone production. Over-expression of SmHMGR or SmDXR in hairy root lines can significantly enhance the yield of tanshinone. Transgenic hairy root lines co-expressing HMGR and DXR (HD lines) produced evidently higher levels of total tanshinone (TT) compared with the control and single gene transformed lines. The highest tanshinone production was observed in HD42 with the concentration of 3.25 mg g(-1) DW. Furthermore, the transgenic hairy roots showed higher antioxidant activity than control. In addition, transgenic hairy root harboring HMGR and DXR (HD42) exhibited higher tanshinone content after elicitation by yeast extract and/or Ag(+) than before. Tanshinone can be significantly enhanced to 5.858, 6.716, and 4.426 mg g(-1) DW by YE, Ag(+), and YE-Ag(+) treatment compared with non-induced HD42, respectively. The content of cryptotanshinone and dihydrotanshinone was effectively elevated upon elicitor treatments, whereas there was no obvious promotion effect for the other two compounds tanshinone I and tanshinone IIA. Our results provide a useful strategy to improve tanshinone content as well as other natural active products by combination of genetic engineering with elicitors.

Vitamin E has been found to be associated with an important antioxidant property in mammals and plants. In photosynthetic organisms, the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD; E.C. 1.13.11.27) plays an important role in the vitamin E biosynthetic pathway. The full-length cDNA encoding HPPD was isolated from Lactuca sativa L. by rapid amplification of cDNA ends (RACE). The cDNA, designated as LsHPPD, was 1743 base pairs (bp) long containing an open reading frame (ORF) of 1338 bp encoding a protein of 446 amino acids. Sequence analysis indicated that LsHPPD shared high identity with HPPD from Medicago truncatula L. Real-time fluorescent quantitative PCR (qPCR) analysis revealed that LsHPPD was preferentially expressed in mature leaves compared with other tissues and that the LsHPPD expression was sensitive to high light and drought stress treatments. Transient expression of LsHPPD via agroinfiltration resulted in 12-fold increase in LsHPPD mRNA expression level and 4-fold enhancement in α-tocopherol content compared with the negative control. A decrease in chlorophyll content and inhibition of photosystem II were observed during stress treatments and agroinfiltration.

Talpha1 (thymosin alpha1), an immune booster, plays an important role in the maturation, differentiation and function of T-cells. It can also activate the production of cytokines in dendritic cells. Talpha1 is one of two thymosin proteins that have potential future clinical applications. In order to express Talpha1 protein in plants, we designed and synthesized the Talpha1 gene according to the plant codon usage bias and created a novel 4 x Talpha1 concatemer (four copies of the Talpha1 gene arranged end-to-end in tandem, designated 4 x Talpha1). Subsequently, a plant binary expression vector, PG-pRD12-4 x Talpha1, was constructed and introduced into tomato via Agrobacterium tumefaciens-mediated transformation. Through selection, 54 regenerated tomato plants resistant to kanamycin were obtained, and four transgenic tomato plants were further confirmed by PCR and Southern blotting. RT-PCR (reverse transcription-PCR) analysis showed that the 4 x Talpha1 gene was transcribed specifically in tomato [Solanum lycopersicum (formerly Lycopersicon esculentum)] fruits. ELISA analysis showed that the content of the 4 x Talpha1 protein reached a maximum of 6.098 microg/g fresh weight in mature tomato fruit. Western-blot analysis further confirmed the expression of 4xTalpha1 protein in transgenic tomato fruits. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay showed the 4 x Talpha1 protein derived from transgenic tomatoes exhibited bioactivity that can stimulate the proliferation of mice splenic lymphocytes in vitro, and the specific activity of Talpha1 protein from the artificial system was higher than that from the synthetic Escherichia coli system. This study is the first to report successful expression of bioactive Talpha1 in plants, and also it will provide the basis for further development of the plant system to produce Talpha1.

Talpha1 (thymosin alpha 1) is important in treating immunodeficiency and other diseases. In order to study the feasibility of expressing Talpha1 in plants, as the first attempt, we designed and synthesized the Talpha1 gene according to the plant codon usage preference and constructed the 4xTalpha1 concatemer (four copies of a DNA sequence arranged end-to-end in tandem). The latter was inserted into Escherichia coli expression vector pQE30, resulting in a recombinant plasmid that was subsequently transformed into E. coli M15. The 4xTalpha1 concatemer protein was successfully expressed in E. coli in a soluble form. The expressed protein was purified and its bioactivity was analysed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. Preliminary results showed that the 4xTalpha1 concatemer protein could stimulate the mice spleen lymphocyte proliferation. This is the first report on the expression of 4xTalpha1 concatemer that was synthesized according to plant codon usage preference in an E. coli expression system. The present study provides the basis for expressing the synthesized active Talpha1 gene in plants in the future.

An HPLC method for the chiral analysis of the four regioisomeric epoxyeicosatrienoic acids (EETs) is described. The cytochrome P450 arachidonic acid epoxygenase metabolites are resolved, without the need for derivatization, by chiral-phase HPLC on a Chiralcel OJ column. Application of this methodology to the analysis of the liver endogenous EETs demonstrates stereospecific biosynthesis and corroborates the role of cytochrome P450 as the endogenous arachidonic acid epoxygenase.

The aphid is one of the most serious pests that causes damage to crops worldwide. Lectins from Araceae plant had been proved useful to control the aphid. Herein, the full-length cDNA of Monstera deliciosa agglutinin (mda) gene was cloned and then introduced into tobacco and the influence of the expression of mda in transgenic tobacco against peach-potato aphids (Myzus persicae) was investigated. Among 92 regenerated plants, 59 positive tobacco lines were obtained. Real-time PCR assays and aphid bioassay test revealed that there is a positive correlation between the expression level of mda and the inhibitory effect on peach-potato aphids. The average anti-pests ability of mda transgenic tobacco was 74%, which was higher than that of other reported lectins from Araceae plant. These results indicated that MDA is one of promising insect resistance proteins selected for the control of peach-potato aphids.

Tropane alkaloids (TA) including hyoscyamine, anisodamine, scopolamine and anisodine, are used medicinally as anticholinergic agents with increasing market demand, so it is very important to improve TA production by metabolic engineering strategy. Here, we report the simultaneous introduction of genes encoding the branch-controlling enzyme tropinone reductase I (TRI, EU424321) and the downstream rate-limiting enzyme hyoscyamine-6β-hydroxylase (H6H, EF187826) involved in TA biosynthesis into Anisodus acutangulus hairy roots by Agrobacterium-mediated gene transfer technology. Transgenic hairy root lines expressing both TRI and H6H (TH lines) produced significantly higher (P0.05) levels of TA compared with the control and single gene transformed lines (T or H lines). The best double gene transformed line (TH53) produced 4.293 mg g(-1) TA, which was about 4.49-fold higher than that of the control lines (0.96 mg g(-1)). As far as it is known, this is the first report on simultaneous introduction of TRI and H6H genes into TA-producing plant by biotechnological approaches. Besides, the content of anisodine was also greatly improved in A. acutangulus by over-expression of AaTRI and AaH6H genes. The average content of anisodine in TH lines was 0.984 mg g(-1) dw, about 18.57-fold of BC lines (0.053 mg g(-1) dw). This is the first time that this phenomenon has been found in TA-producing plants.

In the present study, a plant binary expression vector PG-pRD12-hFIX (where PG is polygalacturonase) harbouring the hFIX (human coagulation Factor IX) gene was constructed and introduced into tomato (Lycopersicon esculentum) via Agrobacterium tumefaciens-mediated transformation. After kanamycin selection, 32 putative independent transgenic tomato plants were regenerated. PCR and Southern-blot analyses confirmed the transgenic status of some plants. RT (reverse transcription)-PCR analysis for the expression of the introduced gene (hFIX) demonstrated that the hFIX gene was expressed specifically in fruits of the tomato. Western-blot analysis confirmed the presence of a 56 kDa band specific to hFIX in the transformed tomatoes. ELISA results showed that the expression of hFIX protein reached a maximum of 15.84 ng/g fresh weight in mature fruit. A blood-clotting assay demonstrated the clotting activity of the expressed hFIX protein in transgenic tomato fruits. This is the first report on the expression of hFIX in plants, and our research provides potentially valuable knowledge for further development of the plant-derived therapeutic proteins.

Vitamin E is a group of lipid soluble compounds, which are important antioxidants and play a crucial role in mammals and plants. They can protect the membrane from photooxidation, and they are involved in signal transduction and transcription regulation as well. The enzyme homogentisate phytyltransferase (HPT) has been demonstrated to be a key enzyme limiting tocopherol biosynthesis. In this study, HPT gene isolated from Lactuca sativa L. (LsHPT) was transferred into L. sativa (lettuce) via Agrobacterium-mediated method and 15 transgenic plants were obtained. The expression of LsHPT gene and the total content of and -tocopherol in transgenic plants were increased up to 4 and 2.61 folds, respectively, demonstrating that overexpression of HPT gene is an effective way to improve vitamin E content in lettuce.Key words: Lactuca sativa, tocopherol, homogentisate phytyltransferase (HPT), Agrobacterium-mediated transformation, real-time fluorescent quantitative-polymerase chain reaction (qRT-PCR).