Effect of pea proteins extraction and vicilin/legumin fractionation on the phase behavior in admixture with alginate

Soluble and natural mixed pea proteins (PP) were extracted from defatted pea seeds according to acidic precipitation (PPP) or ultrafiltration/diafiltration (PPDF) procedures. The isolates contained proteins with a low level of denaturation. Mixed pea globulins isolates presented quite similar solubility and thermal profiles, also a similar polypeptide composition. Vicilin/convicilin 7S (Vic) and legumin 11S (Leg) fractions were obtained by batch chromatography using a salt gradient for the elution. Several incompatible systems were built by mixing the pea proteins with an anionic polysaccharide (sodium alginate, SA), when biopolymers were both negatively charged. Most of mixtures exhibited a phase separation phenomenon. From phase diagrams, experimental binodal curves obtained with either mixed globulins or legumin fraction were apparently very close. However phase boundary was better-defined with the Leg fraction. No macroscopic phase separation was evidenced for mixtures with the vicilin fraction. Microstructure of the PP-SA mixtures was investigated by confocal microscopy (CLSM) according to PP composition and biopolymer initial composition. The Leg-SA and most of PPP-SA mixtures exhibited a droplet-like structure, while structure of PPDF-SA mixtures was aggregated-like. With mixed PP, an alginate entrapment within the PP-enriched phase would disturb phase separation. Also density and shape of the protein-enriched microdomains influenced kinetics of demixing. Polydispersity within the PP-SA mixtures, in terms of wide range molecular weights distribution and charge heterogeneity would explain such differences.     

Studies on the thermal behaviour of pea (Pisum sativum)vicilin

The effect of heat on pea (Pisum sativum L) vicilin has been studied using differential scanning calorimetry (DSC), gel filtrtion chromatography and turbidimetry. By adjusting the variables of pII, ionic strength and protein concentration, two processes could be identified from the DSC thermograms: protein denaturation and protein aggregation. The results are discussed in terms of electrostatic and hydrophobic interactions. A mechanism for the thermal aggregation of pea vicilin is proposed.     

转基因植物中标记基因定性PCR检测方法研究

为了建立以标记基因为检测靶标的高效定性检测方法,本研究系统地分析了日前申请商业化的转基因植物中标记基因的应用频率,选取了应用频率高或在末来应用潜力大的标记基因neomycin phosphotrarsferaseII （NPTll） ,phosphinothricin acetyltransferase （ PAT）, 5 - enolpyrul - shikimate - 3 - phosphate synthase （ CP4 - EPSPS ） ,phosphlnothricin acetyltransferase （bar） ,hygromycin phosphotransferase H（HPTI1） ,β - glucuronidase （GUS） 和 phospho-mannose isomerase （PMI）作为检测靶标,建立了这七个基因的普通PCR和实时荧光PCR检测方法。该研究建立的普通PCR方法和实时荧光PCR方法特异性强、灵敏度高、重现性好,适用于农产品中转基因成分的大规模筛查检测,而且应用实时荧光PCR方法可大大提高检测效率,降低检测过程中样品交叉污染和环境污染的几率。     

农杆菌介导快速、高效获得转基因烟草纯合株系

利用植物表达载体PBI121空载体,对农杆菌介导法转化烟草技术体系进行了综合改进,探索激素配比、侵染时间、分化和生根培养阶段卡那霉素的筛选压对烟草(Nicotiana tabacum cv.Petit Havana SR1)植株再生和转化效率的影响;同时根据孟德尔显性性状杂合子自交后代遗传分离规律,在转基因株系后代扩繁时用卡那霉素筛选分离,可以在T2代较快得到纯合株系.本研究通过使用整个叶片浸染、高的卡那霉素筛选剂浓度、控制生长条件以及强化管理,极大地缩短了获得纯合株系的时间,确立了该烟草品种快速、高效获得大量转基因阳性植株及快速获得转基因纯合株系的转化体系,其转化周期可减少至5-6个月,转基因植株阳性率可达到90％,其中60％的转基因植株后代卡那霉素抗性性状分离比例接近3:1.     

Thermodynamic approach to the comparative analysis of integral hydrophobicity of legumin and legumin-T in native and denaturated forms. 1. Broad bean legumin

The specific integral hydrophobicity of denatured forms of legumin and legumin-T from broad beans was evaluated through their hydration heat capacities. These were calculated as differences between partial heat capacities obtained from differential scanning microcalorimetry data and heat capacities in the gas phase, which were calculated by the use of the method of additive group contributions, taking into account the expansion of protein molecule in the gas phase. It is shown that there is no significant difference between the data for denatured forms of both proteins. The comparative evaluation of the hydration heat capacities of native forms of legumin and legumin-T was carried out. It is shown that the native legumin-T has a greater value of the hydration heat capacity and respectively is more hydrophobic than the native legumin. On the basis of the values of differences between the heat capacities in denatured and native states it is shown that the accessible hydrophobic surface of the native legumin-T related to a mean amino acid residue is 8-13 Ų greater than that of the native legumin.     

Improved polyhydroxybutyrate (PHB) production in transgenic tobacco by enhancing translation efficiency of bacterial PHB biosynthetic genes

Polyhydroxybutyrate [P(3HB)] was produced in the transgenic tobacco harboring the genes encoding acetoacetyl-CoA reductase (PhaB) and polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha (Cupriavidus necator) with optimized codon usage for expression in tobacco. P(3HB) contents in the transformants (0.2mg/g dry cell weight in average) harboring the codon-optimized phaB gene was twofold higher than the control transformants harboring the wild-type phaB gene. The immunodetection revealed an increased production of PhaB in leaves, indicating that the enhanced expression of PhaB was effective to increase P(3HB) production in tobacco. In contrast, codon-optimization of the phaC gene exhibited no apparent effect on P(3HB) production. This result suggests that the efficiency of PhaB-catalyzed reaction contributed to the flux toward P(3HB) biosynthesis in tobacco leaves.     

耐热菌D-乳酸脱氢酶突变体的可视化建模和大肠杆菌中突变体对产苯乳酸的影响

以生物信息学为基础,分析D-乳酸脱氢酶（D-LDH）的保守氨基酸残基、活性中心氨基酸残基、蛋白质三维结构和同源建模,可视化比较建模突变体空间构象,优选最佳突变体模型。结果显示,在D-LDH的20个保守氨基酸中,4个与酶活性中心有关。比较突变体模型发现,49和297位的phe或try的苯环形成空间位阻,F49A或Y279A及F49A和Y279A双突变体可解除位阻。对已构建的三个突变体初步发酵显示,IPTG和乳糖都能诱导突变体酶在大肠杆菌中产生苯乳酸,静置培养比摇振培养产量高,用乳糖诱导时,突变体F49A（A.a D-LDH-F49A株）苯乳酸的量比野生型（A.a.D-LDH株）的高。优选可视化突变体可作为高效构建工程菌的方法之一。     

Improvement in pulping and bleaching properties of xylem from transgenic tobacco plants

In an attempt to improve the pulping properties of xylem pieces and the bleaching properties of the resultant pulp, we compared two lines of transgenic tobacco in which the biosynthesis of lignin had been altered by suppression of the activity of 4‐coumarate: CoA ligase (4CL). The results of Kraft pulping on a laboratory scale indicated that the xylem of transgenic tobacco exhibited a higher delignification efficiency than that of corresponding controls subjected to the same conditions. This facilitated delignification was accompanied by a high pulp yield, but there was no excessive degradation of cellulose in the pulp fibres. The unbleached pulps from the 4CL‐suppressed tobacco plants were easily bleached with a lower consumption of chemicals than was required for the controls. Furthermore, the strength and optical properties of handsheets prepared from the 4CL‐suppressed tobacco plants were similar to those of handsheets prepared from the controls. Our observations suggest that modification of lignin by genetic engineering of the suppression of 4CL activity should result in both an increased pulping yield and a saving of bleaching reagent. © 2002 Society of Chemical Industry     

牛乳汁中葡萄糖转运基因的表达

以牛初乳和末乳为研究对象,利用乳汁体细胞的分子生物学技术平台,对牛乳汁中葡萄糖转运基因的表达进行研究.结果表明,初乳和末乳中均有GAT-(1,4)和SGLT-1表达,且初乳中的表达高于末乳;GLUT-1只在末乳中有表达,初乳中不表达.提示乳腺细胞葡萄糖的摄取方式与泌乳阶段相关,初乳期乳腺合成乳糖的作用高于末乳期乳腺.     

Expression and bioactivity of recombinant human lysozyme in the milk of transgenic mice

Human milk lysozyme is an important protein for innate immunity, but human breast milk is a fairly poor source for commercial production of this enzyme. Research on the expression of recombinant human lysozyme (rHlys) is therefore potentially valuable to the dairy industry. In this study, 2 different kinds of transgenic mice, PBC-hLY and PBC-sighLY, were generated and used as system models to express rHlys. Six lines of PBC-hLY transgenic mice with human lysozyme genomic DNA-based constructs were generated, and a maximum expression level of rHlys approaching 0.154 mg/mL was achieved. Antibacterial activity of the whey from PBC-hLY female transgenic mice was determined by a turbidimetric assay. Results showed that antibacterial activity of the whey was strongly enhanced, and confirmed that rHlys retained full activity. For rHlys to be secreted efficiently into the milk of transgenic mice, 5 lines of mice were also generated, in which the signal peptide DNA of bovine β-casein was substituted for that of lysozyme in PBC-hLY transgenic mice. Compared with PBC-hLY transgenic mice, both the expression levels of rHlys and the antibacterial activity of the whey were much higher in the PBC-sighLY transgenic mice. The concentration of rHlys in one of these mice amounted to 1.405 mg/mL—3 times higher than the level in human whey. The antibacterial activity of the whey was also 3 times higher than that of human whey. The rHlys from both PBC-hLY and PBC-sighLY transgenic mice had the same antibacterial activity as human milk lysozyme. The effect of the signal peptide and copy numbers of the transgene on expression of rHlys was also evaluated. This work will certainly permit a better understanding of how mammary gland bioreactor systems can be applied to produce rHlys in other mammals, such as cattle.     

Protein from pea mutants as a co-product in starch separation--isolates from wet and dry separation: yield, composition and solubility.

The dry separation behaviour of cotyledon material of certain pea genotypes (wild type and 5 r mutants) with different metabolic background in starch biosynthesis allowed satisfying protein recovery only with smooth seeded genotypes. Structural effects and protein distribution (starch granule size distribution, protein in non-starch part) play an important role. According to expectations protein composition remained unaltered during dry separation procedures. Independent of genotypes used the applied conditions in wet separation allowed to recover just 50% of seed proteins while approximately 10% got lost with fibres and 40% with unspecified process water streams. Globular vicilins remained dissolved during iso-electric precipitation and were discarded with waste water streams. The solubility of isolates was rather low under alkaline conditions, even for the round seeded cv. Odalett (0.4 to 0.7% dry substance).     

Toxic factors in pea haulm silage effluent: The factor affecting germination and growth in plants

The liquor expressed from decomposing pea haulm has been shown to be capable both of inhibiting the germination and retarding the growth of ryegrass (Lolium perenne), wheat (Triticum vulgare) and cress (Lepidum sativum). The toxic factor responsible is a mixture of the volatile fatty acids: acetic, propionic, butyric, valeric and caproic.     

Xanthan gum production of Xanthomonas spp. Isolated from different plants

Xanthan gum were produced from the following Xanthomonas strains; standard strain Xanthomonas campestris NRRL B-1459 and isolated strains Xanthomonas arbicola pv. juglandis, Xanthomonas axonopodis pv. vesicatoria, Xanthomonas axonopodis pv. begonia, Xanthomonas axonopodis pv. dieffenbachia. The viscosity features of the xanthan gums obtained were determined at 25–80°C with different pH values and were compared to commercial xanthan gum. Our results indicate that X. arbicola pv. juglandis showed the highest productivity (8.22±1.52 g/L gum). This was followed by X. axonopodis pv. begonia (7.74±1.30 g/L gum), and the control bacterial strain X. campestris NRRL B-1459 (7.46±0.28 g/L gum). X. axonopodis pv. vesicatoria showed the lowest productivity (6.40±0.55 g/L gum). No xanthan gum could be obtained from X. axonopodis pv. dieffenbachia. Xanthan gum produced by X. axonopodis pv. vesicatoria showed the highest viscosity value (428 mPa·sec at 1% solution) in all Xanthomonas strains isolated from plants.     

Vicilin and the basic subunit of legumin are putative chickpea allergens

IgE-mediated reactions to food allergens constitute a major health problem in industrialized countries. Chickpea is consumed in Mediterranean countries, and reportedly associated with IgE-mediated hypersensitivity reactions. However, the nature of allergic reactions to chickpea has not been characterized.     

Metabolic engineering strategies for the production of beneficial carotenoids in plants

Adequate consumption of carotenoids including lycopene, β-carotene, lutein, zeaxanthin, and astaxanthin have many benefits for human health. In plants, carotenoids are derived from isoprenoid precursors from the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway located in plastids. The MEP pathway is also required for the biosynthesis of chlorophyll, terpenoids, plant hormones, and other metabolites. Despite its complexity and difficulty, various strategies have been successfully used to improve the carotenoid biosynthesis in plants through metabolic engineering. Here, these metabolic engineering strategies are reviewed. In addition, the development of gene stacking technologies for carotenoid biosynthesis is evaluated. These technologies will expedite our efforts to bring the health benefits of carotenoids and other nutritional compounds to our diet.