A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time‐consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co‐introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic‐resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high‐throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant‐specific DNA herbicides.     

Antisense gene inhibition by phosphorothioate antisense oligonucleotide in Arabidopsis pollen tubes

Sexual reproduction is an essential biological event for proliferation of plants. The pollen tube (PT) that contained male gametes elongates and penetrates into the pistils for successful fertilization. However, the molecular mechanisms of plant fertilization remain largely unknown. Here, we report a transient inhibition of gene function using phosphorothioate antisense oligodeoxynucleotides (AS‐ODNs) without cytofectin, which is a simple way to study gene function in Arabidopsis thaliana PTs. The PTs treated with AS‐ODNs against both ANX1 and ANX2 showed short, knotted, and ruptured morphology in vitro/semi‐in vitro, whereas normal PT growth was shown in its sense control in vitro/semi‐in vitro. PT growth was impaired in a manner dependent on the dose of AS‐ODNs against both ANX1 and ANX2 above 10 μm . The treatment with AS‐ODNs against ROP1 and CalS5 resulted in waving PTs and in short PTs with a few callose plugs, respectively. The expression levels of the target genes in PTs treated with their AS‐ODNs were lower than or similar to those in the sense control, indicating that the inhibition was directly or indirectly related to the expression of each mRNA. The AS‐ODN against fluorescent protein (sGFP) led to reduced sGFP expression, suggesting that the AS‐ODN suppressed protein expression. This method will enable the identification of reproductively important genes in Arabidopsis PTs.     

银杏优良单株繁殖体系的建立与技术要点

论述了银杏优良单株繁殖体系的建立过程以及各个阶段的技术要点 ,对促进广西银杏生产具有指导意义     

鼓翅蝇科(双翅目:沼蝇总科)的分类研究进展

对双翅目鼓翅蝇科分类沿革,分类地位,分类系统,系统发育的研究进展作了详细的介绍,同时提出了在该科昆虫研究中存在的一些主要问题。     

In vivo homopropargylglycine incorporation enables sampling,isolation and characterization of nascent proteins from Arabidopsis thaliana

Determining which proteins are actively synthesized at a given point in time and extracting a representative sample for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) of a small sample of the proteins being synthesized in Arabidopsis plants or cell cultures, facilitating their click-chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met sites throughout protein amino acid sequences and correlation with independent studies of protein labelling with ¹⁵N verified the data. We provide evidence that HPG-based BONCAT tags a better sample of nascent plant proteins than azidohomoalanine (AHA)-based BONCAT in Arabidopsis and show that the AHA induction of Met metabolism and greater inhibition of cell growth rate than HPG probably limits AHA incorporation at Met sites in Arabidopsis. We show HPG-based BONCAT provides a verifiable method for sampling, which plant proteins are being synthesized at a given time point and enriches a small portion of new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides samples were found to contain significantly fewer common post-translationally modified residues than the same proteins from whole plant extracts, providing evidence for age-related accumulation of post-translational modifications in plants.     

Subdiffraction‐resolution live‐cell imaging for visualizing thylakoid membranes

The chloroplast is the chlorophyll‐containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live‐cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three‐dimensional structured illumination microscopy (3D‐SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D‐SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild‐type and mutant strains. Using 3D‐SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D‐SIM. This study demonstrates that 3D‐SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.     

RNA‐directed DNA methylation and seed development: an unexpected difference between Arabidopsis thaliana and Brassica rapa

Biotechnology provides a means for the rapid genetic improvement of plants. Although single genes have been important in engineering herbicide and pest tolerance traits in crops, future improvements of complex traits like yield and nutritional quality will likely require the introduction of multiple genes. This research reports a system (GAANTRY; Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) for the flexible, in vivo stacking of multiple genes within an Agrobacterium virulence plasmid Transfer‐DNA (T‐DNA). The GAANTRY system utilizes in vivo transient expression of unidirectional site‐specific recombinases and an alternating selection scheme to sequentially assemble multiple genes into a single transformation construct. To demonstrate GAANTRY's capabilities, 10 cargo sequences were sequentially stacked together to produce a 28.5‐kbp T‐DNA, which was used to generate hundreds of transgenic events. Approximately 90% of the events identified using a dual antibiotic selection screen exhibited all of the introduced traits. A total of 68% of the tested lines carried a single copy of the selection marker transgene located near the T‐DNA left border, and only 8% contained sequence from outside the T‐DNA. The GAANTRY system can be modified to easily accommodate any method of DNA assembly and generate high‐quality transgenic plants, making it a powerful, yet simple to use tool for plant genetic engineering.     

高等级病原微生物实验室建设科技进展

高等级病原微生物实验室是生物安全级别最高的防护实验室,是从事高致病性病原微生物检测和科学研究的重要技术平台,也是保护实验室工作人员不被感染、外界环境不受污染的防护屏障。近年来,强致病性微生物引发的烈性传染病在全球范围内逐步扩散。世界各国为满足应急控制传染病突发事件以及提升生物国防实力的重大需求,纷纷开始加紧建设高等级病原微生物实验室。阐述了世界生物安全实验室的发展历程与等级划分,各国高等级病原微生物实验室的建设进展与成果以及我国高等级病原微生物实验室在关键技术、装备研发以及国产化四级模式实验室建设等方面所取得的科技进展,并对未来科技发展方向进行了展望。     

预警系统模型

本文利用物元、可拓集合、经典域、节域、关联函数等概念以及其它数学概念,建立预警系统模型,应用于缺血性中风预报,效果较好．     

油用向日葵主要农艺性状的遗传分析

对10个油用向日葵品种的主要农艺性状进行遗传分析的结果表明,株高、单株粒数、百粒重、盘径和单株粒重等农艺性状具有较大的遗传率,可以在早代单株选择或直接选择;出苗-现蕾、现蕾-开花、开花-成熟、皮壳率等性状的遗传率较低,应根据各性状间相关关系进行间接选择或将选择推迟到晚期世代进行。遗传变异幅度较大的性状有皮壳率、单株粒数、小区产量、单株粒重等。由于性状的遗传改良效果主要由该性状的遗传率和变异幅度及选择强度所决定,因此,在选择率确定为5%情况下,遗传改良效果较好的性状为单株粒数和单株粒重。 Abstract:The genetic analysis of main agronomic characters was conducted with 10 varieties of oil sunflower(Helianthus annuus L.).The results showed that direct selection can be carried out at early generation for characters with higher heritability such as plant height,grain number pere plant,100-grain weight,diameter of disc and grain weight per plant.For maturity and percentage of shell with lower heritability,indirect selection correlated with other characters or late generation selection can be utilized.The characters showing wide genetic variation were those as percentage of shell,grain number per plant,grain yield per plot and grain weight.Since the successful improvement of a character mainly depends on its heritability,variation of heritability and intensity of selection,we suggest that the characters which may give better genetic improvement are grain number and grain weight per plant under condition of 5% selection pressure.