药用植物转基因研究进展

随着医疗事业的发展,药用植物的遗传转化越来越成为人们关注的焦点.近年来药用植物的遗传转化取得了很大进展,已成功培育了多种转基因药用植物.从遗传转化方法、转化受体和转化的目的基因等方面来论述了近年来药用植物转基因的研究进展,并对以后的发展提出了展望.     

Human dihydrofolate reductase and thymidylate synthase form a complex in vitro and co-localize in normal and cancer cells

Enzymes involved in thymidylate biosynthesis, thymidylate synthase (TS), and dihydrofolate reductase (DHFR) are well-known targets in cancer chemotherapy. In this study, we demonstrated for the first time, that human TS and DHFR form a strong complex in vitro and co-localize in human normal and colon cancer cell cytoplasm and nucleus. Treatment of cancer cells with methotrexate or 5-fluorouracil did not affect the distribution of either enzyme within the cells. However, 5-FU, but not MTX, lowered the presence of DHFR-TS complex in the nucleus by 2.5-fold. The results may suggest the sequestering of TS by FdUMP in the cytoplasm and thereby affecting the translocation of DHFR-TS complex to the nucleus. Providing a strong likelihood of DHFR-TS complex formation in vivo, the latter complex is a potential new drug target in cancer therapy. In this paper, known 3D structures of human TS and human DHFR, and some protozoan bifunctional DHFR-TS structures as templates, are used to build an in silico model of human DHFR–TS complex structure, consisting of one TS dimer and two DHFR monomers. This complex structure may serve as an initial 3D drug target model for prospective inhibitors targeting interfaces between the DHFR and TS enzymes.     

转苏云金杆菌毒蛋白基因玉米植株的获得及其抗虫性分析

玉米 (ZeamaysL .)转化成功与否与基因型密切相关。在转化过程中 ,除少数模式品种能够形成再生频率较高且易转化的Ⅱ型愈伤组织外 ,大多数栽培品种往往只能够形成再生频率较低且不易转化的Ⅰ型愈伤组织。因此探索Ⅰ型愈伤组织的诱导及其转化条件 ,提高转化效率 ,对直接改良玉米优良自交系具有重要意义。应用基因枪转化技术将苏云金杆菌 (Bacillusthuringiensis)cry1Ac3基因导入玉米优良自交系E2 8及 34 0的Ⅰ型胚性愈伤组织中 ,经过膦丝菌素 (PPT)或潮霉素 (HygB)筛选 ,获得了再生植株。经PCR检测、Southernblot分析及Bt毒蛋白ELISA检测证实 ,外源基因已整合到玉米基因组中 ,并已获得表达。抗虫性分析结果表明 ,部分转基因玉米植株对玉米螟虫有较强的抗性。还比较了PPT和HygB两种筛选剂的筛选效果 ,表明PPT筛选的抗性愈伤组织的再生频率要高于HygB筛选的再生频率。     

转苏云金杆菌毒蛋白基因玉米植株的获得及其抗虫性分析

玉米( Zea mays L.)转化成功与否与基因型密切相关.在转化过程中,除少数模式品种能够形成再生频率较高且易转化的Ⅱ型愈伤组织外,大多数栽培品种往往只能够形成再生频率较低且不易转化的Ⅰ型愈伤组织.因此探索Ⅰ型愈伤组织的诱导及其转化条件,提高转化效率,对直接改良玉米优良自交系具有重要意义.应用基因枪转化技术将苏云金杆菌( Bacillus thuringiensis ) cry1Ac3基因导入玉米优良自交系E28及340的Ⅰ型胚性愈伤组织中,经过膦丝菌素(PPT)或潮霉素(HygB)筛选,获得了再生植株.经PCR检测、Southern blot分析及Bt毒蛋白ELISA检测证实,外源基因已整合到玉米基因组中,并已获得表达.抗虫性分析结果表明,部分转基因玉米植株对玉米螟虫有较强的抗性.还比较了PPT和HygB两种筛选剂的筛选效果,表明PPT筛选的抗性愈伤组织的再生频率要高于HygB筛选的再生频率.     

Superfluous Transgene Integration in Plants

Referee: Dr. Paul Hooykass, Institut of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333, Al Leiden, Netherlands Recent reports suggest the transfer of superfluous DNA sequences to plant genomes during transformation processes. This review investigates the evidence from the published literature for the prevalence of this phenomenon and highlights methods to limit or prevent DNA transfer and subsequent potentially detrimental evolutionary consequences. Evidence for superfluous foreign DNA transfer using both Agrobacterium-mediated transformation and direct DNA transfer methods such as microprojectile bombardment and PEG-mediated transformation of protoplasts is reported. In the case of Agrobacterium-mediated transformation, the lack of information on the integration of sequences from outside of the T-DNA borders has been due to the general belief by researchers that T-DNA processing is precise. This assumption was based on analysis of T-DNA in tumors and as a result the majority of T-DNA integration events have been identified exclusively using DNA probes, which are homologous only to DNA from within the T-DNA borders. Where direct gene transfer protocols are employed, any part of the transforming plasmid and indeed accompanying carrier DNA may become integrated into the plant genome. The main body of evidence proving that superfluous vector DNA sequences are present in plant genomes transformed using direct transfer methods is confined to the identification of plasmid concatamers integrated into plant genomes. The limited amount of recorded evidence pertaining to superfluous vector DNA integration in transgenic plants and transformed tissues makes it impossible to draw definitive conclusions as to the factors involved in promoting this phenomenon. However, there are methods available for removing superfluous sequences from transgenic plants. These have been developed for the removal of selectable marker genes, whose presence in transgenic plants has been a source of much controversy, but can equally be applied to other DNA sequences. Suggestions have been made in the review that might limit or prevent the integration of superfluous vector sequences during transformation procedures; however, these are not proven and further research is required.     

Multiscale simulations of large complexes in conjunction with cryo-EM analysis

The cellular environment is highly crowded with most proteins and RNA/DNA forming homomeric and heteromeric complexes. Essential questions regarding how these complexes switch between functional, rest, and abnormal states with regulators or modifications remain challenging and complicated. Here, we review the recent progress integrating cryoelectron microscopy and multiscale molecular modeling to understand the dynamics and function-related mechanism in protein–RNA/DNA complexes, protein–protein complexes/assemblies, and membrane protein complexes. One future direction of multiscale simulations will be to interpret the large complex multibody regulation in assembly-induced function enhancement in conjunction with advanced atomic resolution structural-biology techniques and specialized computing architectures.     

A gas phase fractionation acquisition scheme integrating ion mobility for rapid diaPASEF library generation

Data independent acquisition (DIA/SWATH) MS is a primary strategy in quantitative proteomics. diaPASEF is a recent adaptation using trapped ion mobility spectrometry (TIMS) to improve selectivity/sensitivity. Complex DIA spectra are typically analyzed with reference to spectral libraries. The best-established method for generating libraries uses offline fractionation to increase depth of coverage. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow DIA windows that cover different mass ranges of the complete precursor space, have been introduced that performed comparably to deep offline fractionation-based libraries. We investigated whether an analogous GPF-based approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data. We developed a rapid library generation approach using an IM-GPF acquisition scheme in the m/z versus 1/K0 space requiring seven injections of a representative sample and compared this with libraries generated by direct deconvolution-based analysis of diaPASEF data or by deep offline fractionation. We found that library generation by IM-GPF outperformed direct library generation from diaPASEF and had performance approaching that of the deep library. This establishes the IM-GPF scheme as a pragmatic approach to rapid library generation for analysis of diaPASEF data.     

ModuleSearch: finding functional modules in a protein–protein interaction network

Many biological processes are performed by a group of proteins rather than by individual proteins. Proteins involved in the same biological process often form a densely connected sub-graph in a protein–protein interaction network. Therefore, finding a dense sub-graph provides useful information to predict the function or protein complex of uncharacterised proteins in the sub-graph. We developed a heuristic algorithm that finds functional modules in a protein–protein interaction network and visualises the modules. The algorithm has been implemented in a platform-independent, standalone program called ModuleSearch. In an interaction network of yeast proteins, ModuleSearch found 366 overlapping modules. Of the modules, 71% have a function shared by more than half the proteins in the module and 58% have a function shared by all proteins in the module. Comparison of ModuleSearch with other programs shows that ModuleSearch finds more sub-graphs than most other programs, yet a higher proportion of the sub-graphs correspond to known functional modules. ModuleSearch and sample data are freely available to academics at http://bclab.inha.ac.kr/ModuleSearch.     

Simultaneous Measurement of DAPI-Sulforhodamine 101 Stained Nuclear DNA and Protein in Higher Plants by Flow Cytometry

A 2-step staining procedure is presented for simultaneous measurement of nuclear DNA and protein content in higher plants by flow cytometry. To release nuclei, plant tissues were chopped and stirred in the presence of the DNA specific fluorochrome 4', 6-diamidino-2-phenylindole (DAPI) and the nonionic detergent Triton X-100. Plant protoplasts were stirred in the DAPI dye solution with the detergent. After a short incubation period a second dye solution containing DAPI and the protein fluorochrome sulforhodamine 101 (SR 101) without detergent was added. Following another incubation, and after filtration through nylon gauze, the highly fluorescent nuclei were analyzed with an impulse cytophotometer. Accurate bivariate DNA-protein histograms were obtained with CV-values of about 2% or less for the 2C-peak of the univariate DNA parameter. The method presented here can be used for basic and applied cytogenetic studies of higher plants, for characterization of subcompartments of the cell cycle phases, or for examination of heterogeneity in plant tissues.     

The interaction networks of the budding yeast and human DNA replication-initiation proteins

DNA replication is a stringently regulated cellular process. In proliferating cells, DNA replication-initiation proteins (RIPs) are sequentially loaded onto replication origins during the M-to-G₁ transition to form the pre-replicative complex (pre-RC), a process known as replication licensing. Subsequently, additional RIPs are recruited to form the pre-initiation complex (pre-IC). RIPs and their regulators ensure that chromosomal DNA is replicated exactly once per cell cycle. Origin recognition complex (ORC) binds to, and marks replication origins throughout the cell cycle and recruits other RIPs including Noc3p, Ipi1-3p, Cdt1p, Cdc6p and Mcm2-7p to form the pre-RC. The detailed mechanisms and regulation of the pre-RC and its exact architecture still remain unclear. In this study, pairwise protein-protein interactions among 23 budding yeast and 16 human RIPs were systematically and comprehensively examined by yeast two-hybrid analysis. This study tested 470 pairs of yeast and 196 pairs of human RIPs, from which 113 and 96 positive interactions, respectively, were identified. While many of these interactions were previously reported, some were novel, including various ORC and MCM subunit interactions, ORC self-interactions, and the interactions of IPI3 and NOC3 with several pre-RC and pre-IC proteins. Ten of the novel interactions were further confirmed by co-immunoprecipitation assays. Furthermore, we identified the conserved interaction networks between the yeast and human RIPs. This study provides a foundation and framework for further understanding the architectures, interactions and functions of the yeast and human pre-RC and pre-IC.     

Genetically engineered bananas—From laboratory to deployment

Since the first two successful transformation events in banana were reported in 1995, considerable effort has been invested to develop new cultivars with improved tolerance to biotic and abiotic stresses and with enhanced nutrient levels, primarily using Agrobacterium‐mediated transformation and particle bombardment. In addition to many promising laboratory‐based studies, several genetically engineered banana cultivars have been trialled in the field. However, the deployment of genetically engineered varieties of bananas lags behind that of other major crops and there has been no commercial plantation. This article provides a review of advances in the genetic engineering of banana with an overview of noteworthy developments in several programmes that are being conducted worldwide. We identify the main challenges to translating the full potential of genetically engineered bananas for human consumption as the intellectual property issues surrounding the technology, public perceptions towards the adoption of the transformed bananas as well as various regulatory hurdles that hold the technology development from moving forward.     

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.     

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.     

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.     

The Arg Fingers of Key DnaA Protomers Are Oriented Inward within the Replication Origin oriC and Stimulate DnaA Subcomplexes in the Initiation Complex

ATP-DnaA binds to multiple DnaA boxes in the Escherichia coli replication origin (oriC) and forms left-half and right-half subcomplexes that promote DNA unwinding and DnaB helicase loading. DnaA forms homo-oligomers in a head-to-tail manner via interactions between the bound ATP and Arg-285 of the adjacent protomer. DnaA boxes R1 and R4 reside at the outer edges of the DnaA-binding region and have opposite orientations. In this study, roles for the protomers bound at R1 and R4 were elucidated using chimeric DnaA molecules that had alternative DNA binding sequence specificity and chimeric oriC molecules bearing the alternative DnaA binding sequence at R1 or R4. In vitro, protomers at R1 and R4 promoted initiation regardless of whether the bound nucleotide was ADP or ATP. Arg-285 was shown to play an important role in the formation of subcomplexes that were active in oriC unwinding and DnaB loading. The results of in vivo analysis using the chimeric molecules were consistent with the in vitro data. Taken together, the data suggest a model in which DnaA subcomplexes form in symmetrically opposed orientations and in which the Arg-285 fingers face inward to mediate interactions with adjacent protomers. This mode is consistent with initiation regulation by ATP-DnaA and bidirectional loading of DnaB helicases.