Molecular cloning and functional characterization of the gene encoding hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase gene from Artemisia annua L / 药学学报

Artemisinin is the first choice for malaria treatment. The plastidial MEP pathway provides 5-carbon precursors (IPP and its isomer DMAPP) for the biosynthesis of isoprenoid (including artemisinin). Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) is the last enzyme involved in the MEP pathway, which catalyzes HMBPP to form IPP and DMAPP. In this study, we isolated the full-length cDNA of HDR from Artemisia annua L. (AaHDR2) and performed functional analysis. According to gene expression analysis of AaHDR2 (GenBank:KX058541) and AaHDR1 reported ever (GenBank:ADC84348.1) by qPCR, we found that AaHDR1 and AaHDR2 had much higher expression level in trichomes than that in roots, stems, leaves and flowers. AaHDR2 had much higher expression level in flowers than that in leaves. Further, the plant hormones such as MeJA and ABA respectively up-regulated the expression level of AaHDR1 and AaHDR2 significantly, but GA3 up-regulated the expression level of AaHDR2 only. The gene expression analysis of AaHDR1 and AaHDR2 showed that AaHDR2 had a greater contribution than AaHDR1 to isoprenoid biosynthesis (including artemisinin). We used AaHDR2 for the following experiments. Bioinformatic analysis indicated that AaHDR2 belonged to the HDR family and the functional complementation assay showed that AaHDR2 did have the enzymatic function of HDR, using E. coli mutant MG1655araHDR as host cell. The subcellular localization assay showed that AaHDR2 fused with GFP at its N-terminal specifically targeted in chloroplasts. Finally, AaHDR2 was overexpressed in Arabidopsis thaliana. The AaHDR2-overexpressing plants produced the isoprenoids including chlorophyll a, chlorophyll b and carotenoids at significantly higher levels than the wild-type Arabidopsis plants. In summary, AaHDR2 might be a candidate gene for genetic improvement of the isoprenoid biosynthesis.

Cloning and bioinformatics analysis of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase（PcHDR1）gene in Phlegmarirus carinatus / 中国中药杂志

The open reading frame of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) was cloned from Phlegmarirus carinatus by RT-PCR method and the sequence was analyzed by bioinformatics tools. After searching the transcriptome dataset of P. carinatus, one unique sequence encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase was discovered. The primers were designed according to the cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from the dataset. And then, the open reading frame (ORF) of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, named as PcHDR1 (GenBank Accession number：JQ957845), was cloned by RT-PCR strategy with the template of mixed RNA extracted from roots, stem and leaf of P. carinatus. The bioinformatic analysis of this gene and its corresponding protein was performed. The ORF of PcHDR1 consisted of 1 437 base pairs (bp), encoding one polypeptide with 478 amino acids. The sequence comparison showed that PcHDR1 is closest with GbHDR (Ginkgo biloba),and the sequence homology was up to 78%. Bioinformatics prediction and analysis indicated that PcHDR1 protein contained a conserved domain of LytB, without transmembrane region and signal peptides. This study cloned and analyzed 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from P. carinatus. The result will provide a foundation for exploring the function of PcHDR1 involved in terpene biosynthesis in P. carinatus plants.

Cloning and expression analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diph- osphatereductase gene in Dendrobium officinale / 中草药

Objective: To clone the full-length cDNA encoding 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphatereductase (HDR) gene from Dendrobium officinale (DoHDR), then to analyze the expression difference in different tissues and expression patterns of DoHDR induced by signal molecule. Methods: RT-PCR and RACE technologies were used to clone the full length cDNA of DoHDR. The analyses of homologous comparison and phylogenetic tree were performed using DNAMAN and MEGA6.0 softwares, then the expression patterns of DoHDR were studied by real-time PCR. Results: The DoHDR gene was successfully obtained (GenBank accession number KC344827), and the full-length cDNA was 1 658 bp, coding the protein containing 460 amino acids. DoHDR had high homology (≥ 80%) with HDR proteins from other plants. Tissue expression analysis showed that DoHDR had the highest expression in the leaves, followed by roots, stems, and protocorm. Quantitative PCR results showed that DoHDR could be induced by signal molecule such as abscisic acid (ABA) and salicylic acid (SA). Conclusion: The cDNA encoding DoHDR is cloned. It is helpful for the future research on the mechanism of terpenoid biosynthesis in D. officinale.

Cloning and analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase genes HsHDR1 and HsHDR2 in Huperzia serrate

We cloned and analyzed the two genes of the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) gene family from Huperzia serrate. The two transcripts coding HDR, named HsHDR1 and HsHDR2, were discovered in the transcriptome dataset of H. serrate and were cloned by reverse transcription-polymerase chain reaction (RT-PCR). The physicochemical properties, protein domains, protein secondary structure, and 3D structure of the putative HsHDR1 and HsHDR2 proteins were analyzed. The full-length cDNA of the HsHDR1 gene contained 1431 bp encoding a putative protein with 476 amino acids, whereas the HsHDR2 gene contained 1428 bp encoding a putative protein of 475 amino acids. These two proteins contained the conserved domain of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (PF02401), but without the transmembrane region and signal peptide. The most abundant expression of HsHDR1 and HsHDR2 was detected in H. serrate roots, followed by the stems and leaves. Our results provide a foundation for exploring the function of HsHDR1 and HsHDR2 in terpenoid and sterol biosynthesis in Huperziaceae plants.

Stimulatory male volatiles for the Neotropical brown stink bug, Euschistus heros (F. ) (Heteroptera: Pentatomidae) / Semioquímicos produzidos pelo macho do percevejo Neotropical Euschistus heros (Fabr. ) (Heteroptera: Pentatomidae)

Volatiles compounds collected from the male Neotropical brown stink bug, Euschistus heros (F.), a serious Central and South American soybean pest, have been reevaluated. The proportion of three methyl esters is found to be quite different from previous study. A new blend is proposed as the male-produced stimulatory volatiles based on gas chromatographic-electroantennographic detection (GC-EAD) techniques. The three GC-EAD-active components reproducibly found in volatiles collected from males are methyl (2E,4Z)-decadienoate (53 percent), methyl 2,6,10-trimethyldodecanoate (3 percent), and methyl 2,6,10-trimethyltridecanoate (44 percent) respectively. Males of this hemipteran species needed to reach an age of ~15 days to produce enough male-specific compounds to be detected by capillary column GC equipped with flame ionization detector (FID). The amounts of these three stimulatory volatiles increased with age and appeared to reach a maximum at ~35 days old, with the release rate of ~2.5 mug/day/male and the ratio of these three components seemed not to be affected by aging.

Compostos voláteis obtidos de machos do percevejo marrom Neotropical Euschistus heros (F.), uma das principais pragas da cultura da soja na região da América Central e Sul, foram reavaliados. A proporção dos três ésteres já identificados mostrou-se bem diferente da determinada em estudos anteriores. Uma nova combinação de ésteres metílicos é proposta como voláteis estimulatórios produzidos pelos machos, baseando-se nos resultados obtidos das análises por detecção eletroantenográfica acoplada ao cromatógrafo gasoso (GC-EAD). Os três componentes ativos detectados por CG-EAD nos voláteis coletados da aeração de machos foram os (2E,4Z)-decadienoato de metila (53 por cento), 2,6,10-trimetildodecanoato de metila (3 por cento), e 2,6,10-trimetiltridecanoato de metila (44 por cento). A liberação dos voláteis pelos machos adultos alcança níveis detectáveis para análise por cromatografia gasosa capilar com detector de ionização de chamas quando os insetos estão com aproximadamente 15 dias na fase adulta. A quantidade dos três compostos estimulatórios aumentou com a idade dos percevejos e atingiu um nível máximo de produção com aproximadamente 35 dias de fase adulta. A taxa de liberação desses compostos é aproximadamente de 2,5 mig/macho/dia e a proporção desses três componentes parecem não ser afetada com a idade após a maturidade sexual.

Adult diapause morph of the brown stink bug, Euschistus servus (Say) (Heteroptera: Pentatomidae) / Diapausa em adultos do percevejo marrom neártico, Euschistus servus (Say) (Heteroptera: Pentatomidae)

The brown stink bug, Euschistus servus (Say), is abundant throughout most of eastern North America and is commonly found feeding on soybean, mullein, beans, tomatoes, peas, cotton, wheat, corn, tobacco and peach. Color change in E. servus from green to reddish-brown was shown to be an indicator of reproductive diapause. Reddish-brown insects lived longer than green individuals, females laid no eggs, and males did not produce pheromone. The high mortality registered for the green colony of E. servus adults was associated with the physiological cost associated with reproduction. The main pheromone component of this species is methyl 2E,4Z-decadienoate, in agreement with previous work. The first generation of this species develops on noncrop hosts and the second generation often migrates to crops where they may then exceed economic damage thresholds. Traps or trap crops baited with pheromone to catch or concentrate females for destruction, or even a pheromone-based disruption of orientation behavior to decrease the mating success, are possible semiochemical techniques to suppress populations of second generation of E. servus.

O percevejo marrom neártico, Euschistus servus (Say), é abundante em toda a Região Leste da América Norte e é encontrado geralmente alimentando-se em culturas de soja, mullein, feijão, tomate, ervilha, algodão, trigo, milho, tabaco e pêssego. A mudança da coloração de verde para marrom demonstrou ser um indicativo de diapausa reprodutiva em E. servus. Os insetos de coloração marrom viveram por mais tempo que os indivíduos de coloração verde, as fêmeas não colocaram nenhum ovo, e os machos não produziram feromônio sexual. A alta taxa de mortalidade registrada para a colônia de indivíduos verdes de adultos de E. servus foi interpretada como custo fisiológico associado à reprodução. O componente principal do feromônio dessa espécie foi confirmado como 2E, 4Z-decadienoato de metila, de acordo com trabalho realizado anteriormente. A primeira geração dessa espécie se desenvolve em plantas silvestres e a segunda geração migra frequentemente para culturas onde podem exceder os níveis de dano econômico. Porém, técnicas semioquímicas podem ser empregadas para suprimir populações da segunda geração de E. servus. Armadilhas ou culturas armadilhas iscadas com feromônio podem capturar ou concentrar as fêmeas da espécie para serem destruídas, ou mesmo a técnica do confundimento, pode ser utilizada para diminuir a possibilidade de acasalamentos.