Expression of legumin and vicilin genes in pea mutants and the production of legumin in transgenic plants

Pea seeds contain two major storage proteins, legumin and vicilin, in proportions that are genetically and environmentally determined. They are synthesized from at least 40 genes and at least 10 different genetic loci. Mutant alleles at loci involved in starch synthesis, which result in perturbations in starch accumulation, also affect the expression of legumin genes, thereby influencing the legumin : vicilin ratio within the total seed protein. Examples of such alleles includer(starch‐branching enzyme) and rb(ADP‐glucose pyrophosphorylase( both of which result in a reduction in legumin synthesis; double mutants (rrb) show a particularly severe reduction in the amount of legumin. The effects of such mutations are specific to legumins. The amounts of vicilin are unaffected by mutations at rorrb. One of the consequences of the production of legumin from many genes is structural heterogeneity that is believed to preclude the purification of homogeneous legumin for crystallization and 3D‐structure determination. Expression of cloned legumin cDNA in E. coli can result in sequence homogeneity, butE. coli is unable to carry out the normal proteolytic processing of legumin precursors and consequently such material is different from that produced in pea seeds. This paper describes the high‐level synthesis, processing and assembly of pea legumin in transgenic wheat seeds, leading to the spontaneous in vitroformation of paracrystalline arrays of legumin, which may be attributed to the fact that the legumin consists of a single type of subunit. Such material might be used as a source of single‐sequence, processed and assembled pea legumin for structural investigation.     

Agronomical factors influencing the legumin/vicilin ratio in pea (Pisum sativum L.) seeds

BACKGROUND: Many research studies have investigated the impact of agronomical factors on the protein content of pea ( Pisum sativum ). This study aimed to establish if a correlation exists between protein content and legumin/vicilin (L/V) ratio in pea seeds and to identify agronomical factors that have an impact on this ratio. RESULTS: The L/V ratio was positively correlated with protein content (r = 0.58, P?0.01), but no linear regression was applicable (adjusted R² = 0.31). Both variety and cultivation year had a highly significant effect on the ratio (P < 0.001). The interaction between these two factors was also highly significant (P < 0.001), some varieties being less sensitive to climatic conditions than others. Cultivation location had a highly significant effect (P < 0.01). There was no interaction between variety and location. Seeding density had a highly significant effect on the ratio (P < 0.01), with a saturation effect above 60 seeds m^?2. CONCLUSION: Further studies should establish if a linear regression model can be applied to pea varieties independently. Varieties with a stable L/V ratio can prove to be useful in the food industry. Other agronomical factors (soil type and seeding density) should be considered for the production of pea seeds with a specific L/V ratio. Copyright © 2011 Society of Chemical Industry     

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.     

药用/工业用转基因植物数据库的构建

药用/工业用转基因植物已经成为转基因生物新的增长点。本研究收集了以植物作为生物反应器生产药用/工业用产品的数据信息300多份,转化事件近200条,并完成信息上传108条。每个转化事件收集了包括外源蛋白表达量、毒性、致敏性、临床数据、安全等级在内的20多种信息。利用Linux Apache Mysql PHP （LAMP）架构首次设计了药用/工业用转基因植物数据库,面向用户不同需求设计了初级检索和高级检索两种检索方式。该数据库具有综合性、专业性、实用性和首创性的特点,为药用/工业用转基因植物新品种的研发提供信息帮助,为政府机构审批和监管以及制定其安全评价标准提供技术支撑。     

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。     

保护性耕作下小麦和豌豆投入产出能值分析--测试文章--测试文章

应用能值分析的基本理论和方法,探讨了传统耕作(T)、免耕(NT)、传统耕作+秸秆还田(TS)、免耕+秸秆覆盖(NTS)、传统耕作+地膜覆盖(TP)、免耕+地膜覆盖(NTP)等不同耕作模式下小麦和豌豆的投入产出能值,测算了其净能值产出率、能值投入率、宏观经济价值。结果表明：同一耕作方式下,种植小麦能值投入大于豌豆,豌豆能值产出是小麦的5倍以上,且豌豆宏观经济价值远远大于小麦;不同耕作方式下,两种作物净能值产出率均为免耕处理大于相应的耕作处理;由于传统耕作的三种模式人工和机械费用的投入远高于免耕三种模式,使得耕作处理下能值总投入大于相应的免耕处理;免耕+秸秆覆盖处理下,小麦和豌豆净能值产出率和宏观经济价值均达到最大,分别达到了0.64、3.93和9.26、53.1。说明免耕+秸秆覆盖不仅能够实现节约投入,而且可以增产增收,能够达到生态经济效益的最优。     

浅析肉制品中植物源性转基因成分风险

肉制品种类繁多, 是人们日常饮食中重要的组成部分。在现代肉类产品加工过程中, 为改善肉制品的品质、优化产品质量、确保营养均衡, 植物源的成份如植物蛋白、淀粉、膳食纤维、食用胶、植物油以及各种调味品等被广泛运用。伴随着当前全球范围内转基因作物的商品化, 导致肉制品中不可避免地面临植物源性转基因成分风险。本文主要对我国肉制品的现状、肉制品中植物源性成分的使用、肉制品中植物源转基因成分风险以及转基因成分检测进行了概述。旨在引起社会对来自于转基因农作物成分在肉制品下游加工环节中使用情况加以关注, 从而推动转基因食品追溯体系的建立, 进一步规范标识。     

Novel electromagnetic separation technology for the production of pea protein concentrate

Use of plant protein is of growing interest to address consumer demands and sustainability of future food supply. In this study, Lorentz force-assisted charge carrier separation enhancement strategy was employed to separate pea protein (Pisum sativum L.). The magnetic field is generated by parallel magnets, 20 cm long, 10 cm wide and 10 cm apart, and perpendicular to the electric field formed between parallel electrodes. The charged particles would cut the magnetic induction lines during the movement and were subjected to a Lorentz force towards the negative electrode, which improves the highest purity and yield of the protein concentrate by 3.6% (db) and 1.8% respectively, over exclusively triboelectric separation. After the ultrafine milling, air pre-classification and optimized electromagnetic separation, the purity of the resulting pea protein concentrate was increased from 27.8 ± 0.6% to 72.1 ± 0.8% (db) with a yield of 9.2 ± 1.2%. This study pointed out the value of magnetic field used as an auxiliary means in the electrostatic separation.     

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.     

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).     

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.