Manipulating volatile emission in tobacco leaves by expressing Aspergillus nigerbeta-glucosidase in different subcellular compartments

Expression of the Aspergillus nigerbeta-glucosidase gene, BGL1, in Nicotiana tabacum plants (cv. Xanthi) had a profound effect on the volatile emissions of intact and crushed leaves. BGL1 was expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter and targeted to the cytoplasm, cell wall, lytic vacuole (LV), chloroplast or endoplasmic reticulum (ER). Subcellular localization was confirmed by gold immunolabelling, followed by transmission electron microscopy (TEM). Significant beta-glucosidase activity was observed in transgenic plants expressing BGL1 in the cell wall, LV and ER. Compared with controls, all intact transgenic leaves were found to emit increased levels of 2-ethylhexanol, as determined by gas chromatography-mass spectrometry (GC-MS) analysis of the headspace volatiles. Plants expressing BGL1 in the cell wall (Tcw) emitted more trans-caryophyllene than did non-transgenic controls, whereas plants expressing BGL1 in the ER (Ter) and LV (Tvc) emitted more cembrene than did non-transgenic controls. Volatiles released from crushed transgenic leaves and collected with solid-phase microextraction (SPME) polydimethylsiloxane fibre were distinctly enhanced. Significant increases in linalool, nerol, furanoid cis-linalool oxide, 4-methyl-1-pentanol, 6-methyl-hept-5-en-2-ol and 2-ethylhexanol were detected in transgenic plants when compared with wild-type controls. 3-Hydroxyl-beta-ionone levels were increased in crushed Tcw and Ter leaves, but were undetectable in Tvc leaves. The addition of glucoimidazole, a beta-glucosidase inhibitor, abolished the increased emission of these volatiles. These results indicate that the expression of a fungal beta-glucosidase gene in different subcellular compartments has the potential to affect the emission of plant volatiles, and thereby to modify plant-environment communication and aroma of agricultural products.     

黑曲霉水解京尼平苷β-葡萄糖苷酶的分离纯化及其酶学性质

黑曲霉Aspergillus niger发酵豆渣产β-葡萄糖苷酶(BGL)。粗酶液依次经过乙醇沉淀和DEAE-Sepharose Fast Flow离子交换层析,获得了两种电泳纯的β-葡萄糖苷酶BGL1和BGL2,它们的相对分子质量分别为102kDa、97kDa,总酶活回收率达到48%。在pH2.5及温度为55℃时,两种BGL水解京尼平苷的速度均达到最大;BGL水解活性受到Na+的激活但不受K+的影响,但Mg2+、Ba2+、Cu2+、Fe2+、Zn2+和Hg+等离子对BGL活性有不同程度的抑制作用(其中对BGL1的抑制普遍强于BGL2),而且同一种离子对不同BGL活性的影响性质不同,Ca2+显著激活BGL1而对BGL2无影响,Fe3+显著抑制BGL1而激活BGL2。在同样条件下,BGL对对硝基苯-β-D吡喃葡萄糖苷(pNPGlu)、对硝基苯-β-D吡喃半乳糖苷(pNPGal)和水杨苷均有催化活性,其中对pNPGlu的Km值最小,BGL1、BGL2分别为0.764和1.934mmol/L;而BGL对京尼平苷水解的催化效率最高,BGL1、BGL2的Kcat/Km值分别为4.28×104和1.04×105L/mol·s。BGL1、BGL2活性的pH稳定范围相近,分别为pH2.0-7.0、pH2.0-8.5,但它们的热稳定性差异较大,55℃时BGL2保温60min,酶活保留了60%,而BGL1的酶活半衰期只有10min。     

hpt与bar基因作为水稻转基因筛选标记的比较研究

标记基因的选择是影响植物遗传转化和转基因后代筛选成败的关键因素。hpt与bar作为两种常用的水稻转化筛选标记被广泛应用于水稻的转化。为比较两者在实际应用中的效果, 文章首先对比了在潮霉素和除草剂(Bialaphos)两种筛选剂下水稻遗传转化的情况。研究表明, hpt基因的转化筛选体系相对于bar基因在转化效率上提高近两倍, 转化周期提前至少10 d, 且插入基因拷贝数更低。随后, 文章分析了利用潮霉素浸种法在田间筛选转基因水稻的可行性, 研究显示当潮霉素浓度大于167 mg/L时, 可以对以水稻品种kitaake为亲本的转基因材料进行有效筛选, 达到常规除草剂的筛选效果。但与除草剂相比较, 潮霉素的田间筛选成本却处于劣势。文章研究和讨论了hpt和bar基因在遗传转化和后代田间筛选中的优缺点, 并提供了一种利用潮霉素浸种法筛选转基因后代阳性植株的手段, 为将来在水稻转基因研究工作中根据实际需求选择合适的遗传转化、筛选体系提供参考。     

A non-destructive screenable marker,OsFAST, for identifying transgenic rice seeds

The production of transgenic plants has contributed greatly to plant research. Previously, an improved method for screening transgenic Arabidopsis thaliana seeds using the FAST (Fluorescence-Accumulating-Seed Technology) method and FAST marker was reported. Arabidopsis seeds containing the FAST marker may be visually screened using a fluorescence stereomicroscope or blue LED handy-type instrument. Although the FAST method was originally designed for Arabidopsis screens, this study endeavors to adapt this method for the screening of other plants. Here, an optimized technology, designated the OsFAST method, is presented as a useful tool for screening transgenic rice seeds. The OsFAST method is based on the expression of the OsFAST-G marker under the control of a seed-embryo-specific promoter, similar to the Arabidopsis FAST-G marker. The OsFAST method provides a simple and non-destructive method for identifying transgenic rice seeds. It is proposed that the FAST method is adaptable to various plant species and will enable a deeper analysis of the floral-dip method.Key words: Oryza sativa, oleosin, seed, green fluorescent protein, transformation, screenable markerThe production of transgenic plants has significantly enhanced many areas of plant science research. Antibiotic/herbicide-resistance genes are traditionally used as screenable markers for the selection of transgenic plants. However, this approach does have disadvantages. First, antibiotics or herbicides occasionally inhibit the growth of transgenic plants, regardless of the incorporation of antibiotic- or herbicide-resistance genes¹ into the transgenic plants. Second, the identification of resistant transgenic plants requires that the seed population be sown onto plates containing antibiotics or herbicides. Third, the selection process is slow and labor intensive, often involving the screening of vast numbers of potentially transgenic seeds on selective plates.To overcome these disadvantages, an improved approach for selecting transgenic Arabidopsis thaliana, designated the FAST (Fluorescence-Accumulating-Seed Technology) method, was developed. This method employs the use of a fluorescent protein that is expressed in seeds and used as a visual screenable marker for the identification of transgenic seeds. The seed-specific protein oleosin, a family of oil-body-membrane proteins,² has an important role as a size regulator of oil bodies.³ AtOLE1, the most abundant oleosin, functions in the freezing tolerance of Arabidopsis seeds.⁴ A plasmid containing an AtOLE1-GFP fusion gene controlled by the AtOLE1 promoter was constructed and designated the FAST-G (Fluorescence-Accumulating-Seed Technology with OLE1-GFP) marker. Interestingly, Arabidopsis seeds containing the FAST-G marker emitted clear fluorescence under a fluorescence stereomicroscope or blue LED handy-type instrument. The transgenic seeds were visually identified by the seed fluorescence without the use of antibiotics or herbicides, thus indicating that the FAST method offers a nondestructive approach. The FAST marker permits the identification of homozygous seeds among the T2 population with a false discovery rate of less than 1% as a co-dominant screenable marker. In contrast to conventional methods using antibiotics or herbicides, the FAST method reduces the amount of time required to acquire homozygous transgenic plants from 7.5 months to 4 months. The fluorescence of the FAST-G marker was limited to a specific organ (i.e., in seeds) and a specific time (i.e., during dormancy), desirable characteristics of selectable and/or screenable markers. Furthermore, the FAST marker does not require sterile seeding and the handling of large numbers of plants.     

Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens

The expression of infectious bursal disease virus (IBDV) host-protective immunogen VP2 protein in rice seeds, its immunogenicity and protective capability in chickens were investigated. The VP2 cDNA of IBDV strain ZJ2000 was cloned downstream of the Gt1 promoter of the rice glutelin GluA-2 gene in the binary expression vector, pCambia1301-Gt1. Agrobacterium tumefaciens containing the recombinant vector was used to transform rice embryogenic calli, and 121 transgenic lines were obtained and grown to maturity in a greenhouse. The expression level of VP2 protein in transgenic rice seeds varied from 0.678% to 4.521% µg/mg of the total soluble seed protein. Specific pathogen-free chickens orally vaccinated with transgenic rice seeds expressing VP2 protein produced neutralizing antibodies against IBDV and were protected when challenged with a highly virulent IBDV strain, BC6/85. These results demonstrate that transgenic rice seeds expressing IBDV VP2 can be used as an effective, safe and inexpensive vaccine against IBDV.     

转HAL1基因番茄的耐盐性

利用农杆菌介导的叶盘法,把HAL1 基因转入番茄,Southern杂交检测得到转基因植株.耐盐实验表明, T1代转基因番茄在150 mmol/L的NaCl胁迫下仍有43%的发芽率,200 mmol/L的NaCl胁迫下发芽率为6%,而对照种子在100和150 mmol/L的NaCl胁迫下发芽率分别为11.0%和0.转基因番茄的电解质相对外渗率小于对照,而根冠比和叶绿素含量大于对照,转HAL1基因显著提高了番茄的耐盐性.盐胁迫下Na 、K 的累积状况表明,转基因番茄根、茎、叶的K /Na 均有所提高,根系的SK/Na增大,茎、叶的RSK/Na和RLK/Na减小,说明根系对K /Na 离子的选择吸收和运输能力加强.不但选择吸收K /Na ,而且表现出整株水平上的有利于耐盐的K /Na 区域化分配.     

New C-band protocol by heat denaturation in the presence of formamide

C-banding techniques detect the presence of constitutive heterochromatin, which is usually located in centromeric regions of chromosomes in the majority of analysed species. The common method for C-banding used over the last 30 years involves treatment with a mild alkali barium hydroxide 5% Ba(OH)2 at 50 degrees C for 5-15 min and subsequent incubation in salt solution (2 x SSC at 60 degrees C for 1 h). We here present a new, easy and reliable technique for C-banding, which basically involves heat denaturation of chromosomal DNA in the presence of formamide and incubation in 2 x SSC at room temperature.     

A Rapid DNA Extraction Method for RFLP and PCR Analysis from a Single Dry Seed

A single-seed DNA extraction method was developed for rapid identification of plant genotype. The method was applied to 12 plant species, including the oil seeds sesame and soybean. The results were comparable to those obtained for oil-less seeds such as rice. This method will be useful for genotypic selection which requires rapid screening of large populations. It can also be used to identify varietal purity of seed stocks by PCR and RFLP analysis. The method includes two major steps, (i) treatment by proteinase K in an SDS extraction buffer, and (ii) grinding of a single half seed in the buffer after incubation. About 1.5–2 µg of DNA per half seed (the endosperm part) of rice was obtained and more than 200 half seed samples could be handled by one person in a day. The DNA could be used for fingerprinting and detection of target genes in a transgenic plant by PCR. The amplified PCR products from the half seed DNA exhibited the same banding patterns as those from leaf DNA. Yield and quality of DNA extracted from half seeds of rice was also sufficient for RFLP analysis. The remnant half seeds containing the embryo can be maintained for later germination of selected genotypes.     

Improvement of protein quality in transgenic soybean plants

Glycinin is one of the abundant storage proteins in soybean seeds. A modified Gy1 (A1aB1b) proglycinin gene with a synthetic DNA encoding four continuous methionines (V3-1) was connected between the hpt gene and the modified green fluorescent protein sGFP(S65T) gene, and a resultant plasmid was introduced into soybean by particle bombardment in order to improve nutritional value of its seeds. After the selection with hygromycin, the efficiency of gene introduction was evaluated. More than 60 % of the regenerated plants tolerant to hygromycin yielded the hpt and V3-1 fragment by polymerase chain reaction (PCR) analysis, and the expression of sGFP was detected in about 50 % of putative transgenic soybeans. Southern hybridization confirmed the presence of transgenes in T0 plants and the transgenic soybeans hybridized with the hpt and V3-1 genes were analyzed showed different banding patterns. Most of the transgenic plants were growing, flowering normally and produced seeds. Analysis of seed obtained from transgenic soybean plants expressing hpt and V3-1 genes showed higher accumulation of glycinin compared with non-transgenic plants. In addition, protein expression in transgenic soybean plants was observed by using 2D-electrophoresis.     

Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus produced from mature seeds

Peanut, one of the world's most important oilseed crops, has a narrow germplasm base and lacks sources of resistance to several major diseases. The species is considered recalcitrant to transformation, with few confirmed transgenic plants upon particle bombardment or Agrobacterium treatment. Reported transformation methods are limited by low efficiency, cultivar specificity, chimeric or infertile transformants, or availability of explants. Here we present a method to efficiently transform cultivars in both botanical types of peanut, by (1) particle bombardment into embryogenic callus derived from mature seeds, (2) escape-free (not stepwise) selection for hygromycin B resistance, (3) brief osmotic desiccation followed by sequential incubation on charcoal and cytokinin-containing media; resulting in efficient conversion of transformed somatic embryos into fertile, non-chimeric, transgenic plants. The method produces three to six independent transformants per bombardment of 10 cm2 embryogenic callus. Potted, transgenic plant lines can be regenerated within 9 months of callus initiation, or 6 months after bombardment. Transgene copy number ranged from one to 20 with multiple integration sites. There was ca. 50% coexpression of hph and luc or uidA genes coprecipitated on separate plasmids. Reporter gene (luc) expression was confirmed in T1 progeny from each of six tested independent transformants. Insufficient seeds were produced under containment conditions to determine segregation ratios. The practicality of the technique for efficient cotransformation with selected and unselected genes is demonstrated using major commercial peanut varieties in Australia (cv. NC-7, a virginia market type) and Indonesia (cv. Gajah, a spanish market type).     

不同处理对海甘蓝种子发芽和幼苗生长的影响

由于受种子生理休眠作用的影响和硬而厚的种皮所产生的抑制作用,使海甘蓝（ＣｒａｍｂｅｍａｒｉｔｉｍａＬ．）种子发芽慢,发芽率低。为探索加快海甘蓝种子发芽和提高种子发芽率的有效方法,我们先后进行了８种不同的种子预处理试验和６种不同次氯酸钠溶液浸种的发芽试验。结果发现：（１）种子剥皮处理可以很大程度地促进发芽和提高发芽率;（２）用浓度为０．０５％的赤霉酸溶液浸种１８ｈ对海甘蓝种子发芽也有很好的促进作用;（３）用０．２０％的代森锰锌４５Ｍ（Ｄｉｉｔｈａｎｅ４５Ｍ）溶液浸种２０ｍｉｎ的消毒处理对海甘蓝种子发芽产生一定程度的抑制作用,但可减少海甘蓝幼苗死亡率;（４）适宜浓度的次氯酸钠漂白水（法文名１＇ＥａｕｄｅＪａｖｅｌ）的溶液（１０％）浸种５ｍｉｎ对促进海甘蓝种子发芽和减少幼苗死亡均有良好效果;浓度低于１０％时．不足以腐蚀种子硬而厚的种皮而促进种子发芽,也不足以杀死种子携带的病菌而减少幼苗死亡率;浓度大于１０％时,对种子的胚和种子内的酶活性产生不良影响,从而抑制种子发芽和影响幼苗的正常生长。     

快速打破结缕草种子休眠方法的比较

用水和30%NaOH对结缕草(Zoysia japonica Steud.)种子进行浸种处理,筛选能快速打破结缕草种子休眠的方法.结果表明:用水浸种6d,8d内发芽率仅为14.70%;用30%NaOH浸种120min,8d内发芽率也仅达28.00%;而用水浸泡2d后再用30%NaOH处理40min,8d内结缕草种子的发芽率达到了82.00%.说明用水和30%NaOH综合处理的方法可以快速有效打破结缕草种子休眠,缩短发芽周期,达到快速出苗的目的.     

A modification in technology improves the specificity of the Du Pont de Nemours immunoenzyme kit for the detection of anti-HIV antibodies

Du Pont De Nemours perfected a new enzyme immunoassay for the screening of anti-HIV 1 antibodies, characterized by its rapidity: 2 hours of incubation, when the normal procedure needed 3 hours 30 minutes of incubation. We tested with the normal procedure and the shortened one 4,025 randomly selected blood donors. 55 samples of the SNTS panel and 10 sera representing the Western-Blot reference panel of the "Retrovirus" study group of the SNTS. We can say that shortening the incubation time results in certain advantages opposite the previous procedure: time saving (1 hour 30 minutes); improved specificity: the rate of false positive, 0.37% with the normal method, drops to 0.07% with the short one; good sensitivity, since all the individual positive samples were detected but it seems slightly less sensitive than the normal method for the diluted positive samples.     

Effect of glycogen synthase overexpression on insulin-stimulated muscle glucose uptake and storage

Insulin-stimulated muscle glucose uptake is inversely associated with the muscle glycogen concentration. To investigate whether this association is a cause and effect relationship, we compared insulin-stimulated muscle glucose uptake in noncontracted and postcontracted muscle of GSL3-transgenic and wild-type mice. GSL3-transgenic mice overexpress a constitutively active form of glycogen synthase, which results in an abundant storage of muscle glycogen. Muscle contraction was elicited by in situ electrical stimulation of the sciatic nerve. Right gastrocnemii from GSL3-transgenic and wild-type mice were subjected to 30 min of electrical stimulation followed by hindlimb perfusion of both hindlimbs. Thirty minutes of contraction significantly reduced muscle glycogen concentration in wild-type (49%) and transgenic (27%) mice, although transgenic mice retained 168.8 +/- 20.5 micromol/g glycogen compared with 17.7 +/- 2.6 micromol/g glycogen for wild-type mice. Muscle of transgenic and wild-type mice demonstrated similar pre- (3.6 +/- 0.3 and 3.9 +/- 0.6 micromol.g(-1).h(-1) for transgenic and wild-type, respectively) and postcontraction (7.9 +/- 0.4 and 7.0 +/- 0.4 micromol.g(-1).h(-1) for transgenic and wild-type, respectively) insulin-stimulated glucose uptakes. However, the [14C]glucose incorporated into glycogen was greater in noncontracted (151%) and postcontracted (157%) transgenic muscle vs. muscle of corresponding wild-type mice. These results indicate that glycogen synthase activity is not rate limiting for insulin-stimulated glucose uptake in skeletal muscle and that the inverse relationship between muscle glycogen and insulin-stimulated glucose uptake is an association, not a cause and effect relationship.     

Development of transgenic pigeonpea using high throughput plumular meristem transformation method

Tissue culture based poor regeneration along with restricted rooting responses are considered to be major hindrances for in vitro transgenic pigeonpea development. Present study was designed to establish a novel method of Agrobacterium tumefaciens mediated plumular meristem transformation in pigeonpea for improvement of transgenic development frequency. Three days old decapitated seedlings of pigeonpea cultivar ICPL 87119 were pricked at plumular meristem region under in vitro conditions. After infecting with Agrobacterium binary vector pBI121, the explants were co-cultivated in 6-benzylaminopurine and α-naphthaleneacetic acid supplemented modified- Murashige and Skoog medium. Transformed seedling with well-developed tap root system were established in soil. GUS activity as well as PCR based confirmation of transgene presence was demonstrated in transgenic events. Transformation frequency of 72% was achieved for the first time in pigeonpea. Further, kanamycin mediated stringent selection was used for the screening of T₁ seeds. Established T₁ progenies were analysed by PCR and Southern blot, to confirm transgene integration and copy number, respectively. This is the first report of transgenic pigeonpea development, where the combination of culture based Agrobacterium-infection and culture independent plant establishment, coupled with PCR based selection method was found to be most preferable for faster and frequent establishment of transgenic plants. This method will contribute to large scale transgenic pigeonpea development for its improvement and satisfy the requirement of routine transformation experiments for T-DNA insertion mutagenesis.     

Expression of transgenic stilbene synthases in wheat causes the accumulation of unknown stilbene derivatives with antifungal activity

The expression of foreign phytoalexins in a new host is thought to increase fungal resistance, since host-specific pathogens have not experienced selection for detoxifying or metabolising the novel antifungal compounds. Two resveratrol synthase genes vst1 and vst2 from grapevine (Vitis vinifera L.) and the pinosylvin synthase gene pss from pine (Pinus sylvestris L.) were stably transformed into bread wheat. The expression of the target genes is regulated by stress-inducible grapevine promoters. The vst1 and vst2 promoters were functional in wheat and retained their expression profiles described for grapevine. ALL vst and pss transgenic lines accumulated stilbene derivatives upon induction by UV light. The detected stilbenes showed a remarkable similarity to resveratrol and pinosylvin, however were found to be more hydrophilic than resveratrol and pinosylvin. Upon inoculation with the biotrophic pathogen Puccinia recondita f.sp. tritici several vst expressing wheat lines showed a significant reduction of disease symptoms (19 +/- 9% to 27 +/- 8%) compared to wild-type plants. The reduction of disease symptoms was even more obvious after inoculation with the facultative biotrophic pathogen Septoria nodorum Berk. and ranged from 42 +/- 13% to 71 +/- 4%. None of the four tested pss expressing lines showed a reduction in disease incidence.     

Overexpression of sinapine esterase BnSCE3 in oilseed rape seeds triggers global changes in seed metabolism

Sinapine (O-sinapoylcholine) is the predominant phenolic compound in a complex group of sinapate esters in seeds of oilseed rape (Brassica napus). Sinapine has antinutritive activity and prevents the use of seed protein for food and feed. A strategy was developed to lower its content in seeds by expressing an enzyme that hydrolyzes sinapine in developing rape seeds. During early stages of seedling development, a sinapine esterase (BnSCE3) hydrolyzes sinapine, releasing choline and sinapate. A portion of choline enters the phospholipid metabolism, and sinapate is routed via 1-O-sinapoyl-β-glucose into sinapoylmalate. Transgenic oilseed rape lines were generated expressing BnSCE3 under the control of a seed-specific promoter. Two distinct single-copy transgene insertion lines were isolated and propagated to generate homozygous lines, which were subjected to comprehensive phenotyping. Sinapine levels of transgenic seeds were less than 5% of wild-type levels, whereas choline levels were increased. Weight, size, and water content of transgenic seeds were significantly higher than those of wild-type seeds. Seed quality parameters, such as fiber and glucosinolate levels, and agronomically important traits, such as oil and protein contents, differed only slightly, except that amounts of hemicellulose and cellulose were about 30% higher in transgenic compared with wild-type seeds. Electron microscopic examination revealed that a fraction of the transgenic seeds had morphological alterations, characterized by large cavities near the embryonic tissue. Transgenic seedlings were larger than wild-type seedlings, and young seedlings exhibited longer hypocotyls. Examination of metabolic profiles of transgenic seeds indicated that besides suppression of sinapine accumulation, there were other dramatic differences in primary and secondary metabolism. Mapping of these changes onto metabolic pathways revealed global effects of the transgenic BnSCE3 expression on seed metabolism.     

A modified semipermeable membrane technique for carbonic anhydrase histochemistry of the nervous system

We present a modification of Hansson's method for the demonstration of carbonic anhydrase activity. Using a semipermeable membrane together with a fluid incubation medium, frozen sections of aldehyde-fixed tissue were incubated without floating or dipping. Thin sections (thickness, 20-40 microns) were mounted on the outer surface of a tubular-shaped, semipermeable cellophane dialysis membrane containing the incubation fluid. After incubation for 25-30 min at room temperature, the sections were rinsed in buffer and treated with 0.5% (NH4)2S solution. The histochemical reaction was fully inhibited by 10(-4) M acetazolamide.