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Tumour necrosis factor (TNF) receptor-associated factor (TRAF) is a receptor protein that has important functions in the immune system. Nonetheless, there have been few reports of traf genes in teleost fishes. The present study aimed to identify the traf genes from the genomic information of yellow catfish (Pelteobagrus fulvidraco). Eight traf genes were identified and named, which are distributed on different chromosomes but have similar conserved protein domains. Phylogenetic and syntenic analyses demonstrated conservation of traf genes during evolution. In addition, yellow catfish has the relatively rare traf1 and traf5 genes. Gene structure and motif analysis revealed the homology and distribution diversity of the traf genes. Quantitative real-time reverse transcription PCR was used to study the expression patterns of traf genes in healthy fish tissues and after infection by Aeromonas hydrophila. The results demonstrated significant changes in traf gene expression, indicating a potential role in innate immunity.

Capsaicin (Cap) is a promising bioactive compound having many health-promoting benefits. However, it is difficult to be applied in food due to its poor aqueous solubility, low stability and bioavailability. Besides, its strong spicy taste and irritation to the gastrointestinal tract further limit the application of Cap in food. To solve this problem, Cap was loaded into a self-assembled nanocarrier formed by partial hydrolysis of α-lactalbumin (α-lac). The Cap was successfully loaded into the 21.2 nm micelles with a loading capacity of 123.4 ± 6.1 mg g

Orsellinic acid (OA) derivatives are produced by filamentous fungi using nonreducing polyketide synthases (nrPKSs). The chain-releasing thioesterase (TE) domains of such nrPKSs were proposed to also catalyze dimerization to yield didepsides, such as lecanoric acid. Here, we use combinatorial domain exchanges, domain dissections and reconstitutions to reveal that the TE domain of the lecanoric acid synthase Preu6 of Preussia isomera must collaborate with the starter acyl transferase (SAT) domain from the same nrPKS. We show that artificial SAT-TE fusion proteins are highly effective catalysts and reprogram the ketide homologation chassis to form didepsides. We also demonstrate that dissected SAT and TE domains of Preu6 physically interact, and SAT and TE domains of OA-synthesizing nrPKSs may co-evolve. Our work highlights an unexpected domain–domain interaction in nrPKSs that must be considered for the combinatorial biosynthesis of unnatural didepsides, depsidones, and diphenyl ethers.

Geochemical characteristics of aeolian sand are beneficial for understanding sand dune formation and evolution. Few studies in the Dinggye area, Southern Tibet, have focused on the geochemical characteristics of aeolian sand. Thus, we present new geochemical data that provide insights into the geochemical characteristics and environmental implications of aeolian sands in the Dinggye area. The results show that mobile dunes, climbing sand sheets, and nebkhas show heterogeneity in elemental concentrations and UCC-normalized distribution; MgO, TiO2, Ni, Pb, and Nb are higher in mobile dunes; SiO2, CaO, K2O, Na2O, P2O5, V, Cr, Co, Cu, Ba, and Ce are higher in climbing sand sheets; and Al2O3, Fe2O3, La, Zn, As, Sr, Y, Zr, Rb, and Ga are higher in nebkhas. Principal component analysis (PCA) and correlation analysis indicate that the main factor affecting elemental content is grain size sorting, followed by provenance, while chemical weathering and regional precipitation are less influential. The CIA and A-CN-K triangle indicate that the different dune types are at a lower chemical weathering stage, with plagioclase weathering and decomposition first. The combination of grain size characteristics, elemental ratios, multidimensional scale (MDS), PCA, and geomorphological conditions suggest that the flood plain and the lakeshore are the main sand sources of aeolian sands in the Dinggye area.

The microstructure features, martensitic transformation behavior and mechanical/functional properties of Ti–V–Al alloy were tailored by changing rare element Y content in the present investigation. The results showed that Y doping resulted in the grain refinement and formation of Y-rich phase mainly distributing along grain boundary in Ti–V–Al alloys. The martensitic transformation temperatures of Ti–V–Al alloys slightly increased due to the variation of matrix composition induced by the presence of Y-rich phase. The mechanical and functional properties of Ti–V–Al alloys doped moderate Y addition were significantly improved, which can be ascribed to grain refinement, solution strengthening and precipitation strengthening. The 1.0 ​at.%Y-doped Ti–V–Al alloy exhibited the highest ultimate tensile stress of 912 ​MPa and largest elongation of 17.68%. In addition, it was found that the maximum recoverable strain of 5.42% can be obtained in Ti–V–Al alloy with adding 1.0 ​at.%Y, under the pre-strain of 6% condition, which is enhanced by approximate 0.6% than that of Ti–V–Al alloy without Y addition.

As serine/threonine protein kinases, mitogen-activated protein kinases (MAPK) take part in cellular metabolism. This work found 14 MAPK genes in the yellow catfish (Pelteobagrus fulviadraco) genome and evaluated their taxonomy, conserved domains and evolutionary linkages for a better understanding of the MAPK gene family's evolutionary relationship and antibacterial immune response. The findings revealed that several MAPK genes are activated in response to immunological and inflammatory responses. Collinearity research revealed that in yellow catfish and zebrafish, there are six pairs of highly similar MAPK genes, indicating that these genes have been more conserved throughout evolution. The MAPK gene quantification findings revealed that JNK1a, JNK1b, p38delta and p38alpha b expression levels were considerably upregulated, indicating that they act in fish innate immunity. The findings implied that MAPK genes may involve in defence against detrimental microbe in yellow catfish, which will help researchers better understand how MAPK genes work in the innate immune system.

The big video data size today imposes huge pressure on storage. The variation and aging induced memory failures significantly influence the video output quality. Recently, researchers have developed different memory designs for videos, deep learning, and other data-intensive applications, which enables better energy-quality tradeoffs with design constraints. Unfortunately, designing memory has been proven to be a very challenging problem due to: 1) various design constraints; 2) multiple memory bitcell design options; and 3) challenging layout integration and cost analysis using different memory technologies. In this paper, we develop novel mathematical models for optimizing embedded video memory design without applying a time-consuming and laborious ASIC design process. The problems are formulated as nonlinear programs and integer linear programs. Different SRAM designs and hybrid SRAM and DRAM designs are considered in our models. The results of the numerical studies show that by applying our proposed method the average mean square error of the video storage can be greatly reduced, even by more than 90% in many cases.

Plant U-box (PUB) proteins are ubiquitin ligases (E3) involved in multiple biological processes and in response to plant stress. However, the various aspects of the genome and the differences in functions between the U-box E3 (UBE3) ubiquitin ligases remain quite obscure in

Fungal polyketides (PKs) are one of the largest families of structurally diverse bioactive natural products biosynthesized by multidomain megasynthases, in which thioesterase (TE) domains act as nonequivalent decision gates determining both the shape and the yield of the polyketide intermediate. The endophytic fungus

Mobile devices are becoming ever more popular for streaming videos, which account for the majority of all the data traffic on the Internet. Memory is a critical component in mobile video processing systems, increasingly dominating the power consumption. Today, memory designers are still focusing on hardware-level power optimization techniques, which usually come with significant implementation cost (e.g., silicon area overhead or performance penalty). In this paper, we propose a video content-aware memory technique for power-quality tradeoff from viewer's perspectives. Based on the influence of video macroblock characteristics on the viewer's experience, we develop two simple and effective models-decision tree and logistic regression to enable hardware adaptation. We have also implemented a novel viewer-aware bit-truncation technique which minimizes the impact on the viewer's experience, while introducing energy-quality adaptation to the video storage.

In plants, the first committed step in the pathway for biosynthesis of isoprenoids is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34). Here we report for the first time the cloning of a full-length cDNA encoding HMGR (Tm–HMGR) from a taxol-producing gymnosperm, Taxus media Rehder. The full-length cDNA of Tm–HMGR (GenBank accession number: AY277740) was 2307 base pairs (bp), with a 1791-bp open reading frame (ORF) encoding a 596-amino-acid polypeptide. Bioinformatic analysis revealed that Tm–HMGR contained two trans-membrane domains and a catalytic domain, and showed high homology to other plant HMGRs. Phylogenetic analysis indicated that Tm–HMGR was more ancient than other plant HMGRs. The structural modelling showed that Tm–HMGR had the typical spatial structure of HMGRs whose catalytic domains could be folded and divided into three spatial domains, L-domain, N-domain and S-domain. Southern blot analysis revealed that Tm–HMGR belonged to a small HMGR gene family. Northern blot analysis showed that Tm–HMGR was expressed in roots, stems and needles, with higher expression in stems and needles than in roots. Functional complementation of Tm–HMGR in a HMGR-deficient mutant yeast demonstrated that Tm–HMGR mediated the biosynthesis of mevalonate and provided the general precursor for taxol biosynthesis.

Embedded memory is a critical component in today’s mobile video processing systems, increasingly dominating power consumption and shortening battery life of mobile devices. Traditional hardware-level power optimization techniques usually come with significant implementation overhead to solve the memory failure problem during low-voltage operations. This paper presents a novel mobile video memory to exploit the power saving opportunities provided by a viewer experience under environmental visual interference. The viewing contexts, in particular the ambient luminance, significantly influence the quality of the viewer experience, and in the context with higher luminance levels, mobile users have higher tolerance to the video degradation. Accordingly, the memory failures can be introduced adaptively to achieve power savings without influencing the viewer experience. To meet the silicon area constraint in mobile devices, a simple but an efficient hardware implementation scheme is developed to minimize area overhead. The experimental results based on a 45-nm CMOS technology show that, as compared with the conventional memory design, the proposed technique can achieve up to 48% power savings with good perceivable quality and negligible implementation overhead.

Arabidopsis Mediator complex subunit 19a (MED19a), which mediates interactions between transcriptional regulators and RNA polymerase II, plays a critical role in plant response to infection by pathogens. However, the roles of MED19a in other signaling pathways are unknown. Here, we report that MED19a plays an important role in regulation of abscisic acid (ABA)-mediated transcriptional regulation in Arabidopsis. Plants deficient in MED19a showed reduced sensitivity to ABA inhibition of seed germination, cotyledon greening, root growth, and stomatal opening. MED19a-deficient mutants also had reduced resistance to drought stress, evidenced by high water-loss rates and low survival rates. Molecular genetic analysis revealed that MED19a mutants had down-regulated ABA-induced genes, including Em1, Em6, and RD29B, and MED19a could occupy the promoters of Em1 and Em6 in an ABA-dependent manner. Furthermore, MED19a interacted with the transcription factor ABA-insensitive 5 (ABI5) in split-luciferase complementation assays and co-immunoprecipitation assays. An analysis of double mutants (med19a-2 and abi5-7) suggested that the action of MED19a in ABA signaling was dependent upon ABI5. Furthermore, MED19a and ABI5 influenced each other in recruiting the promoters of the target genes Em1 and Em6, which are involved in embryonic development. Altogether, these results indicate that MED19a acts as a positive regulator in ABI5-mediated ABA responses.

This study was conducted to determine the mechanism by which di-2-ethylhexyl phthalate (DEHP) exposure influences lipid metabolism of juvenile yellow catfish Tachysurus fulvidraco. Fish were exposed to three DEHP concentrations (0, 0·1 and 0·5 mg l-1 DEHP) for 8 weeks. Fatty acid synthase (FAS) activity significantly decreased with increasing DEHP concentrations, the highest value was in the Tween control group, whereas the lowest activities of carnitine palmitoyltransferase (CPT) and lipoprotein lipase (LPL) were in this group. The messenger (m)RNA levels of 6-phospho-gluconate dehydrogenase (6PGD), FAS and acetyl-CoA carboxylase a (ACCa) significantly increased with increasing DEHP concentration, the highest values were in the 0·5 mg l-1 DEHP group. The mRNA level of peroxisome proliferator-activated receptor γ (PPARγ) was lower in Tween control than in fish exposed to 0·1 and 0·5 mg l-1 DEHP. The highest mRNA level of ACCb was in the 0·1 mg l-1 DEHP group. These results indicate that DEHP exposure can disturb lipid metabolism at the enzymatic and mRNA levels in Pelteobagrus fulvidraco.

Triplicate groups of juvenile yellow catfish Pelteobagrus fulvidraco were exposed to three levels of DEHP (0, 0.1 and 0.5mgL-1) for 56days. Fish survival (100%) was not affected by different levels of ambient DEHP. Final body weight, weight gain, specific growth rate and feed intake of fish exposed to 0.5mgL-1 DEHP were the highest. On the contrary, hepatosomatic index of fish exposed to 0.1 and 0.5mgL-1 DEHP were the lowest. Serum total protein, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase, glucose and triglycerides increased with the increasing concentrations of DEHP exposure. Superoxide dismutase and glutathione peroxidase activities of fish exposed to 0.5mgL-1 DEHP were the lowest, but malondialdehyde contents of fish exposed to 0.1 and 0.5mgL-1 DEHP were higher than that of control fish. Phagocytic indices of the control group were the highest. After being intraperitoneally injected with Aeromonas hydrophila, fish in the control group had the highest expression of toll like receptor 5, and the expression of myeloid differentiation factor 88 of fish exposed to 0.5mgL-1 DEHP was the lowest. This study indicates that DEHP exerts its toxic effects by interfering with hepatic metabolism, inducing ROS generation and malondialdehyde accumulation, leading to blood deterioration and immunosuppression.

Reduction of osa-miR171b contributes to rice stripe virus symptoms by regulating its targets, while overexpression attenuates symptoms, providing direct experimental evidence that a plant miRNA affects the development of virus symptoms., Viral infection affects the pattern of plant miRNA expression. It has been presumed that reduction of miR171 and several other miRNAs influences viral symptoms in plants. We here experimentally demonstrate the association of osa-miR171b with rice stripe virus (RSV) symptoms in rice. Inhibition of osa-miR171b caused stunting with reduced chlorophyll content in leaves similar to viral symptoms. Overexpression of osa-miR171b by an artificial miRNA extended vegetative growth and enhanced chlorophyll accumulation in leaves. Tillers were thicker, and panicles were longer with more spikelets in plants overexpressing osa-miR171b than in controls, but there were no differences in tiller numbers. Targets of osa-miR171b, OsSCL6-IIa, OsSCL6-IIb, and OsSCL6-IIc, were respectively up- and down-regulated in plants where osa-miR171b was inhibited or overexpressed. In plants overexpressing osa-miR171b, five positive regulators for heading development, Ehd1, Ehd2, Ehd3, Ehd4, and Hd3a were up-regulated, while the negative regulator Ghd7 was down-regulated. Plants overexpressing osa-miR171b were less susceptible to RSV and virus symptoms were attenuated. Taken together, the results reveal that a reduction of osa-miR171b in RSV-infected rice contributes to RSV symptoms, and provide more insight into the roles of osa-miR171b in rice.

In the present work, the mechanical and strain recovery characteristics of Ti–V–Al–B shape memory alloy were tailored by changing annealing temperatures. Moreover, effect of annealing temperatures on microstructure and martensitic transformation behavior were investigated systematically. At the annealing temperatures of 650 °C and 700 °C, both α" martensite phase and hard α phase coexisted in the annealed Ti–V–Al–B alloys. With the annealing temperatures increasing, amount of α phase gradually decreased and α" martensite phase was dominated. The reduction of α phase resulted in the compositional variation, which further triggered the rising of transformation temperatures. The elongation of annealed Ti–V–Al–B alloys continuously increased with the annealing temperatures increasing; whereas the yield strength gradually decreased. The mechanical properties were closely related to the amount of α precipitates and dislocations. Under optimum annealing condition of 700 °C, the maximum yield strength of 489 MPa can be achieved in Ti–V–Al–B alloy. Meanwhile, it showed a largest recoverable strain of 4.6% in correspondence to 6% applied pre-strain.

In the present paper, the mechanical and functional properties of Ti–V–Al alloy were tailored by adjusting the boron (B) content. In addition, the effect of B content on the microstructure and martensitic transformation behaviors of Ti–V–Al alloys was also explored. The results indicated that the microstructure of Ti–V–Al alloys was sole α'' martensite phase, irrespective of the B content. B was completely soluble in Ti–V–Al alloy, as B content was not more than 0.01 at.%. Further increased B content not only promoted to the formation of TiB phase, but also resulted in the remarkable grain refinement. During heating process, the orthorhombic structure α'' martensite transformed into β phase with a body-centered cubic structure in one stage manner for all Ti–V–Al alloys containing different B contents. With the increased B content, the transformation temperature firstly decreased and then increased. Moderate B addition can improve significantly the mechanical properties and strain recovery characteristics of Ti–V–Al alloys owing to the perfect combination of solution strengthening, grain refinement, and precipitation strengthening of in-situ TiB phase. The maximum yield strength of 711 MPa can be obtained in 0.1 at.%B doped Ti–V–Al alloy with superior elongation of 18.5%. Moreover, it showed the largest recoverable strain of 5.1% under the condition of 6% pre-strain.

The effect of postharvest heat treatment (37 °C, 3 days) on ascorbic acid (AsA) metabolism related to chilling resistance in peach fruit stored at 5 °C has been studied. Chilling injury (CI) index and hydrogen peroxide (H2O2) levels as well as gene expression levels of mitochondrial L-galactono-1, 4-lactone-dehydrogenase (GalLDH) related to AsA synthetic metabolism, cytosolic ascorbate peroxidase (APX), chloroplastic APX, peroxisomal APX1-3, cytosolic monodehydroascorbate reductase (MDAR), chloroplastic MDAR, chloroplastic dehydroascorbate reductase (DHAR), and chloroplastic glutathione reductase (GR) related to AsA regeneration cycle metabolism were measured. Treated fruit maintained its initial H2O2 levels after heat conditioning, showing significantly higher levels when compared with non-heated fruit. However, during cold storage, heat-treated fruit exhibited lower H2O2 levels than control fruit; heated fruit had the lowest CI index at the end of storage. The transcription levels of all of genes (GalLDH, APX, MDAR, and DHAR), except for GR, sharply increased, and were higher than those of control fruit, immediately after heat treatment. Then, transcription levels sharply declined to levels lower than control fruit at 7–14 days. Little difference of gene expression was observed in the end of storage. These results suggest that AsA metabolism gene expression was temporarily and sharply induced by higher H2O2 levels after heat treatment, which might inhibit the elevation of H2O2 level at the initial period of cold storage, and followed by lower genes expression of AsA metabolism accompanied by lower H2O2 levels following transfer to cold storage.

Semi-custom design flow is an efficient way to design digital integrated circuit (IC). It realizes physical IC design through translating hardware description languages (HDL) into a GDS layout file. By running scripts, the HDL code is synthesized into a cell level schematic. Then, the placing and routing procedures are performed, violations are fixed, optimizations are achieved, and verifications are finished. Synopsys and Cadence are two leading companies in the Electronic Design Automation (EDA) industry. But most of time, foundries fail to provide two separate design kits for two companies. If the foundry only provides a design kit for tools from one leading EDA company, it will be difficult to use the same kit for the other one. In this paper, the main difference between design kits of different companies are shown, and a solution to make one design kits compatible with the other one is provided.

This paper presents a MTJ Based non-volatile SRAM in 45 nm technology. It combines fast and low power partners with time-division satisfaction of high performance and low leakage energy requirements. It can store and restore the data in memory cells, inputs, outputs, clock, and address signals. Simulations show that the maximum frequency can reach 4.5 GHz. When the sleep time is more than 105 seconds, there are great power saving.

In order to elucidate the metabolism of soluble carbohydrates related to chilling injury (CI) in peach fruit (Prunus persica L. Batsch cv. Yulu) exposed to cold storage, we systematically investigated the changes in soluble carbohydrate content, transcript levels for genes encoding related enzymes, and the ascorbic acid (AsA) regeneration cycle in fruit stored at 0 °C and 5 °C for 28 days. At 0 °C, sucrose levels gradually increased up to day 21 before declining, but no significant changes of reducing-sugar levels (glucose and fructose) were observed during this time. However, the levels of sucrose decreased sharply and reducing-sugar levels increased during storage at 5 °C. Thus, fruit stored at 0 °C showed significantly higher levels of sucrose and lower levels of glucose and fructose than fruit stored at 5 °C. This difference was primarily due to lower transcription levels for neutral invertase 2 (NI/2), acid invertase (AI) and sucrose synthase (SS) and lower activities of NI and AI, and higher levels for sucrose phosphate synthase 1 (SPS/1). Both groups of fruit displayed an early, dramatic increase in the transcription levels of sorbitol and sorbitol dehydrogenase (SDH). Additionally, stable membrane permeability, a lack of CI symptoms and a regular AsA cycle were observed throughout the test period in fruit stored at 0 °C. On the other hand, membrane permeability increased sharply in fruit stored at 5 °C, which caused severe internal browning and stimulated AsA cycle activity later in storage. These results suggest that the higher levels of sucrose, resulting from the balance between its degradation and biosynthesis, contribute to membrane stability and enhanced chilling tolerance in peach fruit. Additionally, higher levels of glucose may have provided more reducing power and served as a substrate for AsA biosynthesis, which resulted in enhanced AsA cycle activity in chilling-sensitive fruit.

Flavonoid 3'-hydroxylase (F3'H: EC 1.14.13.21) is an important enzyme which determines the hydroxylation pattern of anthocyanins. In this study, the full-length cDNA and genomic DNA of F3'H were isolated and characterized from the purple-fleshed sweet potato (Ipomoea batatas). IbF3''H was 1,789 bp containing a 1,554 bp open reading frame (ORF) encoding 518 amino acids. Comparative and bioinformatic analysis revealed that IbF3'H was highly homologous with F3'Hs from other plant species. Conserved domain search revealed that IbF3'H was a cytochrome P450 dependent enzyme. Three F3'H-specific motifs (V75VVAAS80, G427GEK430 and V433DVKG437) were conserved in IbF3'H. Phylogenetic analysis revealed that IbF3'H was clustered into the same subgroup with the homologues from I. purpurea, I. tricolor and I. nil. There were multiple copies of the IbF3'H gene in the genome of I. batatas. IbF3'H was constitutively expressed in all tested tissues including fibrous roots, thick roots, storage roots, stems and leaves. During storage root formation, IbF3'H was expressed most abundantly in the storage roots, suggesting that the anthocyanin biosynthesis is also active in the under-ground organs. IbF3'H expression was associated with anthocyanin accumulation in five different sweet potato cultivars tested. Complementative analysis implied that the full-length cDNA of IbF3'H could encode a functional protein and had a special catalytic activity of flavonoid 3'-hydroxylase.

Anthocyanidin synthase (ANS), a 2-oxoglutarate (2OG) iron-dependent oxygenase, catalyzes the penultimate step in the biosynthesis of anthocyanin. This reaction is responsible for the formation of the colored anthocyanidins from the colorless leucoanthocyanidins. A full-length cDNA was isolated from purple-fleshed sweet potato (Ipomoea batatas (L.) Lam) cv. Yamakawamurasaki, designated IbANS, containing a 1,086-bp open reading frame encoding a 362-amino-acid polypeptide. Multiple alignments revealed that the deduced IbANS protein had high identity to ANS proteins of other plants such as Ipomoea nil (90.8% identities), Ipomoea purpurea (91.4% identities), and Brassica juncea (72.7% identities). Structural analysis showed that the IbANS protein might belong to the 2OG and Fe(II)-dependent oxygenase, containing three binding sites of 2OG (H236, D238, and H292) and three binding sites of Fe(II) (Y221, R302, and S304). Phylogenetic tree analysis revealed that IbANS shared the close relationships with I. nil and I. purpurea. Southern blotting showed that there were two copies of the IbANS gene in this genome. Real-time quantitative polymerase chain reaction revealed that expression of the IbANS gene was highest in storage roots and lowest in leaves. IbANS was expressed most abundantly during the formation of storage roots. In five cultivars of sweet potato, IbANS expression was strongly associated with anthocyanin accumulation, suggesting that ANS gene expression was associated with activation of anthocyanin biosynthesis.

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.

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.

A novel gene encoding a MDR-like ABC transporter protein was cloned from Catharanthus roseus, a medicinal plant with more than 120 kinds of secondary metabolites, through rapid amplification of cDNA ends (RACE). This gene (named as Crmdr1; GenBank accession no.: DQ660356) had a total length of 4395 bp with an open reading frame of 3801 bp, and encoded a predicted polypeptide of 1266 amino acids with a molecular weight of 137.1 kDa. The CrMDR1 protein shared 59.8, 62.5, 60.0 and 58.2% identity with other MDR proteins isolated from Arabidopsis thaliana (AAD31576), Coptis japonica (CjMDR), Gossypium hirsutum (GhMDR) and Triticum aestivum (TaMDR) at amino acid level, respectively. Southern blot analysis showed that Crmdr1 was a low-copy gene. Expression pattern analysis revealed that Crmdr1 constitutively expressed in the root, stem and leaf, but with lower expression in leaf. The domains analysis showed that CrMDR1 protein possessed two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) arranging in "TMD1-NBD1-TMD2-NBD2" direction, which is consistent with other MDR transporters. Within NBDs three characteristic motifs common to all ABC transporters, "Walker A", "Walker B" and C motif, were found. These results indicate that CrMDR1 is a MDR-like ABC transporter protein that may be involved in the transport and accumulation of secondary metabolites.

2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC) synthase (MECS, EC: 4.6.1.12) is the fifth enzyme of the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and further Taxol biosynthesis. The full-length MECS cDNA sequence (GenBank accession number DQ286391) was cloned and characterized for the first time from Taxus media, using the Rapid Amplification of cDNA Ends (RACE) technique. The full-length cDNA of Tmmecs was 1081 bp containing a 741 bp open reading frame (ORF) encoding a peptide of 247 amino acids with a calculated molecular mass of 26.1 kDa and an isoelectric point of 8.97. Comparative and bioinformatic analyses revealed that TmMECS had extensive homology with MECSs from other plant species. Phylogenetic analysis indicated that TmMECS was more ancient than other plant MECSs. Southern blot analysis revealed that Tmmecs belonged to a small gene family. Tissue expression pattern analysis indicated that Tmmecs expressed constitutively in all tissues including roots, stems and leaves. The cloning and characterization of Tmmecs will be helpful to understand more about the role of MECS involved in the Taxol biosynthesis at the molecular level.

Farnesyl diphosphate synthase (FPS; EC 2.5.1.10) catalyzes the production of 15-carbon farnesyl diphosphate which is a branch-point intermediate for many terpenoids. This reaction is considered to be a rate-limiting step in terpenoid biosynthesis. Here we report for the first time the cloning of a new full-length cDNA encoding farnesyl diphosphate synthase from a gymnosperm plant species, Taxus media Rehder, designated as TmFPS1. The full-length cDNA of TmFPS1 (GenBank accession number: AY461811) was 1 464 bp with a 1 056-bp open reading frame encoding a 351-amino acid polypeptide with a calculated molecular weight of 40.3 kDa and a theoretical pI of 5.07. Bioinformatic analysis revealed that TmFPS1 contained all five conserved domains of prenyltransferases, and showed homology to other FPSs of plant origin. Phylogenetic analysis showed that farnesyl diphosphate synthases can be divided into two groups: one of prokaryotic origin and the other of eukaryotic origin. TmFPS1 was grouped with FPSs of plant origin. Homology-based structural modeling showed that TmFPS1 had the typical spatial structure of FPS, whose most prominent structural feature is the arrangement of 13 core helices around a large central cavity in which the catalytic reaction takes place. Our bioinformatic analysis strongly suggests that TmFPS1 is a functional gene. Southern blot analysis revealed that TmFPS1 belongs to a small FPS gene family in T. media. Northern blot analysis indicated that TmFPS1 is expressed in all tested tissues, including the needles, stems and roots of T. media. Subsequently, functional complementation with TmFPS1 in a FPS-deficient mutant yeast demonstrated that TmFPS1 did encode farnesyl diphosphate synthase, which rescued the yeast mutant. This study will be helpful in future investigations aiming at understanding the detailed role of FPS in terpenoid biosynthesis flux control at the molecular genetic level. (Managing editor: Wei Wang)

Plant diterpenes such as ginkgolides are biosynthesized via the recently discovered 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The initial step of the MEP pathway is the formation of 1-deoxy-D-xylulose 5-phosphate (DXP) catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS, EC: 4.1.3.37), which may thus be considered the first committed step of the MEP pathway for ginkgolides biosynthesis. The full-length cDNA of DXS was isolated and characterized from the gymnosperm plant species, Ginkgo biloba. The full-length cDNA of GbDXS was 2795 bp containing a 2154 bp open reading frame (ORF) encoding 717 amino acids. Comparative and bioinformatic analyses revealed that GbDXS has extensive homology with DXSs from other plant species and, like these, contains a conserved transit peptide for plastid import, histidine residue, a putative thiamine diphosphate-binding site and a transketolase motif. Phylogenetic analysis indicates that GbDXS belongs to the plant DXS1 cluster and suggests it to be more ancient than other plant DXSs. GbDXS was found to be expressed in all tested tissues including roots, stems, leaves, pericarps and seeds. Expression profiling analyses revealed that GbDXS expression was induced by exogenous elicitors including methyl jasmonate, arachidonic acid, acetylsalicylic acid and ceric ammonium sulfate, and showed that the transcription levels were correlated with ginkgolide accumulation, suggesting that DXS might play a regulatory role in ginkgolide biosynthesis in cell culture of G. biloba at the transcriptional level.

A full-length cDNA encoding taxadienol acetyl transferase (designated as TmTAT), that catalyzes the first acylation step in Taxol biosynthesis, was cloned from young leaves of Taxus x media by rapid amplification of cDNA ends (RACE). The full-length cDNA of TmTAT was 1459 bp and contained a 1317-bp open reading frame (ORF) encoding a protein of 439 amino acids. The deduced protein had a calculated molecular weight of about 49 kDa and an isoelectric point (pI) of 5.3, similar to acetyl transferases from Taxus cuspidata and Taxus chinensis. Sequence comparison analysis revealed that the TmTAT had high similarity with other members of plant transferase family. Phylogenetic tree analysis showed that TmTAT had close relationship with acetyl transferases from T. chinenisis and T. cuspidata. Tissue expression pattern analysis revealed that TmTAT expressed strongly in leaves, weak in stems and no expression could be detected in fruits, indicating that TmTAT was a tissue-specific gene.

An easy and efficient protocol was developed for isolating good-quality total RNA from various tissues including fruits, leaves, stems, and roots of ancient gymnosperm species, taxus and ginkgo. The protocol was developed based on the CTAB method with modifications, including higher-strength CTAB to help the lysis of plant cells, more PVP, and beta-mercaptoethanol to prevent oxidation of phenolic complexes, and higher-centrifugation force to get rid of most cell debris and to ensure RNA quality. In RNA isolation, chloroform/isoamyl alcohol was used to remove proteins, genomic DNA, and secondary metabolites and lithium chloride was subsequently adopted to concentrate total RNA away from most of the cytoplasmic components. Good-quality total RNA from various tissues of native taxus and ginkgo could be easily isolated within 24 hr by this protocol which avoided the limitation of plant materials and the usage of dangerous chemicals, such as phenol, and could provide total RNA for all kinds of further molecular studies.