Overexpression of Coptis japonica norcoclaurine 6-O-methyltransferase overcomes the rate-limiting step in Benzylisoquinoline alkaloid biosynthesis in cultured Eschscholzia californica

Benzylisoquinoline alkaloids are one of the most important secondary metabolite groups, and include the economically important analgesic morphine and the antimicrobial agent berberine. To improve the production of these alkaloids, we investigated the effect of the overexpression of putative rate-limiting step enzymes in benzylisoquinoline alkaloid biosynthesis. We introduced two O-methyltransferase [Coptis japonica norcoclaurine 6-O-methyltransferase (6OMT) and 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT)] expression vectors into cultured California poppy cells to avoid the gene silencing effect of endogenous genes. We established 20 independent lines for 6OMT transformants and 15 independent lines for 4'OMT transformants. HPLC/liquid chromatography-mass spectrometry (LC-MS) analysis revealed that the overexpression of C. japonica 6OMT was associated with an average alkaloid content 7.5 times greater than that in the wild type, whereas the overexpression of C. japonica 4'OMT had only a marginal effect. Further characterization of 6OMT in California poppy cells indicated that a 6OMT-specific gene is missing and 4OMT catalyzes the 6OMT reaction with low activity in California poppy, which supports the notion that the 6OMT reaction is important for alkaloid biosynthesis in this plant species. We discuss the importance of 6OMT in benzylisoquinoline alkaloid biosynthesis and the potential for using a rate-limiting step gene to improve alkaloid production.     

Interaction of sanguinarine with double stranded RNA structures

Interaction of sanguinarine with A-form RNA structures of poly(rI)poly(rC) and poly(rA).poly(rU) has been studied by spectrophotometric, spectrofluorimetric, UV melting profiles, circular dichroism and viscometric analysis. The binding of sanguinarine to A-form duplex RNA structures is characterised by the typical bathochromic and hypochromic effects in the absorption spectrum, increasing steady state fluorescence intensity, an increase in fluorescence quantum yield of sanguinarine, an increase in fluorescence polarization anisotropy, an increase of thermal transition temperature, an increase in the contour length of sonicated rod-like RNA structure and perturbation in circular dichroic spectrum. Scatchard analysis indicates that sanguinarine binds to each polymer in a non-cooperative manner. Comparative binding parameters determined from absorbance titration by Scatchard analysis, employing the excluded site model, indicate a higher binding affinity of sanguinarine to poly(rI).poly(rC) structure than to poly(rA).poly(rU) structure. On the basis of these observations, it is concluded that the alkaloid binds to both the RNA structures by a mechanism of intercalation.     

Transient gene expression in electroporated Solanum protoplasts

Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts –250 V/cm; Désirée mesophyll protoplasts –225 V/cm; Désirée suspension culture protoplasts –225 V/cm; and Désirée tuber protoplasts –150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36–48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the -glucuronidase (gus) gene, showed expression (at DNA concentrations between 0–10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20–30 pmol/ml) the patatin promoter directed 4–5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.     

A rapid method for isolation of double stranded RNA

A rapid and simple method for the isolation and purification of dsRNA is presented. The crucial step of this method is the extraction of proteins and DNA with acid phenol. After the extraction, only RNA is left in the aqueous phase. ssRNA contamination of the RNA preparation can be greatly reduced when ammonium sulfate is present during the extraction.     

Transient gene expression in electroporated bean cotyledon protoplasts

A protocol has been developed for the introduction of foreign DNA into cell protoplasts from immature bean cotyledons. The method yields high amounts of the reporter enzymes β-glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) when cognate genes are driven by the promoter and upstream sequences of a bean β-phas gene. Comparisons with expression in stable tobacco transformants indicate that transient assays can be used to investigate enhancer function. This techniques should be applicable to other gene systems as well.     

Transient gene expression in electroporated Picea glauca protoplasts

The reporter gene for chloramphenicol acetyltransferase (CAT) was introduced into white spruce (Picea glauca (Moench) Voss.) protoplasts by electroporation. CAT transient gene expression was increased by increasing the concentration of pCaMVCN plasmid and was affected by the level of the applied voltage. Highest CAT activities were obtained after electroporation with a pulse of 350V.cm^–1 having an exponential decay constant of approximately 105ms. Linearized plasmid constructs gave much higher levels of CAT activity than circular plasmid. Attempts to use the Escherichia coli -glucuronidase gene (-GUS) as a marker gene revealed very high levels of -GUS-like activity in electroporated protoplasts. This activity was mainly due to a small molecule and may mask successful transformation since -GUS-like activity increased when plasmid DNA was present during electroporation.Abbreviations CAT chloramphenicol acetyltransferase - -GUS -glucuronidase - MUG 4-methyl umbelliferyl glucuronide - F microfarads NRCC No. 29150     

Transient expression in leaf mesophyll protoplasts of Arabidopsis thaliana

Conditions for maximising transient expression of GUS in leaf mesophyll protoplasts of Arabidopsis thaliana ecotype C24 were investigated. It was found that the factors most influencing expression levels, with optimum levels in parenthesis, were plasmid DNA quantity (100 g per 5 × 10⁵ protoplasts), inclusion of carrier DNA (50 g), PEG pH and amount (pH above 6, and total PEG concentration at least 9% w/w) and the topological form of the DNA. Linearised plasmid DNA with long flanking sequences 3 and 5 to the marker gene yielded the highest levels of GUS expression.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MU methylumbelliferone - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid     

In vitro synthesis of double stranded RNA by Drosophila X virus purified virions.

Purified Drosophila X virus (DXV) presents a virion associated RNA polymerase activity. The RNA product is not spontaneously released from the virion and behaves like the virion-borne RNA in velocity gradients. Both product and template are RNase resistant at high ionic strength all over the synthesis. Reannealing of the denatured purified RNAs is faster when an excess template is added. This may be the first published case of a virion-associated replicase.     

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Transient gene expression in electroporated protoplasts of Eucalyptus citriodora Hook

Protoplasts isolated from cotyledons of Eucalyptus citriodora were electroporated using a rectangular pulse, with plasmid carrying the cat gene. The levels of transient expression and protoplast viability were influenced by the voltage and pulse duration. At a field strength of 800 V cm^-1 (1000 s), a protoplast viability of 57%, and 47% conversion of ¹⁴C-chloramphenicol to its acetylated forms, were obtained. Expression levels were improved by an increase in plasmid concentration (up to 60 g ml^-1), and also by the addition of carrier DNA. Gene expression was further enhanced by the addition of 40% (w/v) PEG, in the presence of the carrier DNA, to the protoplasts after electroporation.Abbreviations BAP 6-benzylaminopurine - CAT chloramphenicol acetyltransferase - CPW 13M CPW salts medium with 13% (w/v) mannitol - DC direct current - FDA fluorescein diacetate - f. wt fresh weight - GUS -glucuronidase - K Kao (1977) - MES 2-N-morpholinoethane sulfonic acid - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid - PVP-10 polyvinylpyrrolidone (Av MW 10,000) - TLC thin layer chromatography     

基于细菌体系生产双链RNA的条件优化

化学杀虫剂是农业害虫防治的主要手段,然而农业害虫已对化学杀虫剂产生了抗药性。RNA农药的应用将是减少使用化学杀虫剂的一种途径。RNA农药具有专一、高效、易降解和对环境友好等优点而受到了关注,但在靶标分子、靶标分子的双链RNA（dsRNA）生产工艺和应用制剂等方面仍需进一步研究。本研究以褐飞虱Nilaparvata lugens蛋白激酶B基因的dsRNA（dsNlAKT）为例,对利用细菌体系生产dsRNA的方法进行了探究。将NlAKT构建进L4440载体后,将其转化到缺乏核酸酶（RNase Ⅲ）的大肠杆菌Escherichia coli HT115（DE3）中,进行了dsRNA生产条件的测试。结果表明：（1）当异丙基-β-D-硫代半乳糖苷（IPTG）工作浓度为0.1 mM或0.5 mM时dsRNA的产量没有明显变化,而当其工作浓度增加至1 mM时dsRNA产量明显减少;（2）在IPTG诱导时间为2~8 h范围内,添加IPTG后诱导6 h获得的dsRNA产量最多;（3）在实验室常规提取RNA方法中,增加低剂量溶菌酶预处理这一步骤可增加提取产物中dsRNA的含量。试验结果证明了采用工作浓度为0.1 mM的IPTG诱导6 h的生产条件,并在提取过程中增加溶菌酶的预处理步骤有助于获得较高的dsRNA含量。研究结果为RNA农药的生产条件提供了实验依据。