干旱和复水条件下转基因甘薯的光合特性

以转铜锌超氧化物歧化酶(Cu/Zn SOD)和抗坏血酸过氧化物酶(APX)基因甘薯(TS)及未转基因甘薯(NT)为实验材料,研究在旱后复水条件下转基因甘薯及未转基因甘薯抗氧化酶活性和光合特性变化。结果显示,连续36 h胁迫条件下,TS和NT的SOD活性都先降低后升高,但TS的SOD活性始终高于NT。胁迫至24 h时,TS的SOD活性约为NT的1.2倍,复水后二者SOD活性都下降。持续胁迫,TS的APX活性先升高后降低,NT与之相反,复水后TS和NT的APX活性都是先升高后降低,复水12 h,TS的APX活性是NT的1.5倍。水分胁迫条件下TS的膜质受伤害程度要轻于NT,胁迫24 h,复水12 h,NT的MDA含量均约为TS的1.2倍。胁迫12 h,TS和NT净光合速率都下降,继续胁迫,TS净光合速率开始上升,NT几乎保持不变,胁迫36 h,TS的净光合速率约为NT的1.5倍。复水后二者净光合速率都开始上升,复水12 h,TS净光合速率约为NT的3倍。胁迫时TS、NT胞间CO2浓度(Ci)都逐渐增大,胁迫36 h时NT胞间CO2浓度显著高于TS,是其1.4倍。实验结果表明,同时转入SOD、APX抗氧化基因后,在...

Transforming kelp into a marine bioreactor

The past decade has seen the genetic engineering of various types of seaweed. To date, genetic transformation studies have been carried out in several seaweeds, including the red seaweeds Porphyra, Gracilaria, Grateloupia, Kappaphyclus and Ceramium and the green seaweed Ulva. A genetic transformation model system has been established in the most commonly cultivated seaweed, the brown seaweed Laminaria japonica (kelp), based on the transfer of technology used in land plant transformation and also by modulating the seaweed life cycle. This model showed the potential for application of transgenic kelp to the production of valuable products and an indoor cultivation system for transgenic kelp was proposed, taking into account necessary factors for bio-safety. In this review, the establishment at use of the kelp transformation model is introduced, highlighting the potential for transforming kelp into a marine bioreactor.

An electrochemical DNA sensor based on electrodepositing aluminum ion films on stearic acid-modified carbon paste electrode and its application for the detection of specific sequences related to bar gene and CP4 Epsps gene

An electrochemical DNA biosensor was fabricated by immobilizing DNA probe on aluminum ion films that were electrodeposited on the surface of the stearic acid-modified carbon paste electrode (CPE). DNA immobilization and hybridization were characterized with cyclic voltammetry (CV) by using methylene blue (MB) as indicator. MB has a couple of well-defined voltammetric redox peaks at the CPE. The currents of redox peaks of MB decreased after depositing aluminum ion films on the CPE (Al(III)/CPE) and increased dramatically after immobilizing DNA probe (ssDNA/Al(III)/CPE). Hybridization of DNA probe led to a marked decrease of the peak currents of MB, which can be used to detect the target single-stranded DNA. The conditions for the preparation of Al(III)/CPE, and DNA immobilization and hybridization were optimized. The specific sequences related to bar transgene in the transgenic corn and the PCR amplification of CP4 epsps gene from the sample of transgenic roundup ready soybean were detected by differential pulse voltammetry (DPV) with this new electrochemical DNA biosensor. The difference between the peak currents of MB at ssDNA/Al(III)/CPE and that at hybridization DNA modified electrode (dsDNA/Al(III)/CPE) was applied to determine the Specific sequence related to the target bar gene with the dynamic range comprised between 1.0 X 10(-7) mol/L to 1.0 x 10(-4) mol/L. A detection limit of 2.25 x.10(-8) mol/L. of oligonucleotides can be estimated.

Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.

Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the micro-particle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 mug/mg soluble proteins on average and the highest value was 2.497 mug/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR-Southern and total DNA hybridization. Prospect of kelp bio-reactor producing high value materials such as edible HBV vaccine was discussed as well.

Expression of human epidermal growth factor gene in cyanobacteria

The human epidermal growth factor (hEGF) is a small single-chain polypeptide of 53 amino acid residues. It can stimulate the proliferation of many cell types, mainly those of epidermal and epithelial tissues both in vivo and in vitro. A vector pRL-hEGF was constructed using plasmids pRL-489 and pUC-hEGF. The synthetic hEGF gene was recombined into the downstream of strong promoter psbA in plasmids pRL-489. Then, the vector was introduced into Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120 by triparental conjugative transfer. The transformation was confirmed by PCR amplification. The pRL-hEGF is thought to be retained as a plasmid form in the transgenic Anabaena sp. PCC 7120, since it can be recovered. However, it has been integrated into the chromosome of Synechococcus sp. PCC 7002 as there is no duplication origin in the pRL-hEGF in this cyanobacterium. and plasmid cannot be isolated from the Synechococcus sp. PCC 7002 either. The radioimmunoassay (RIA) proved that the hEGF gene has been expressed as the protein existed in these two strains of transgenic cyanobacteria, and the hEGF protein in Anabaena sp. PCC 7002 could be secreted into the medium.

Foreign gene transfer into Chinese shrimps (Penaeus chinensis) with gene gun

Plasmids pG DNA-RZ1 with a GFP (green fluorescent protein) reporter gene and a ribozyme gene incising penaeid white spot baculovirus (WSBV) were first introduced into the fertilized eggs of Chinese shrimps by gene gun. The treated and control samples of different development stages were observed with a fluorescent microscope. The transient expression of GFP gene was high in nauplius and zoea larvae. Results from RT-PCR and PCR for adults showed that the foreign genes had been transferred into the shrimps and had expressed the corresponding proteins. This work has established a transgenic method for penaeid shrimps, which will set base for the application of genetic engineering breeding into industry.

Expression of TNF-alpha gene in Anabaena sp PCC 7120 and purification of its product by affinity chromatography

The effects of N (NaNO3) and C (NaAc) source in medium on the expression of tumor necrosis factor-alpha (TNF-alpha) gene in transgenic Anabaena sp. PCC 7120 were compared. The data showed that N source stabilized the expression of foreign protein and C source altered the synthesis of cell walls. Comparing several methods for breaking the cells, supersonic was able to extract TNF-alpha better than others. For purification of TNF-alpha, transgenic Anabaena cells were broken, the extracts were precipitated with ammonia sulfate, and the impure TNF-alpha was eluted from DEAE ion exchange chromatography. Electrophoresis (PAGE-SDS) showed a single band at 17 kD position.

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coil has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sg PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)p labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.

Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta)

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been integrated into the genome of gametophytes of L. japonica, and the expression product showed the expected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

Genetic transformation in Kappaphycus alvarezii using micro-particle bombardment: a potential strategy for germplasm improvement

This study investigated the delivery of a SV40 promoter driving lacZ gene into cells of Kappaphycus alvarezii using particle bombardment. Thallus pieces 0.5-0.8 mm in diameter and 1 cm in length were prepared as gene recipients. Bombardment parameters of 450 psi (rupture pressures) x 6 cm (particle travel distances), 650 psi x 6 cm, 1,100 psi x 6 cm and 1,100 psi x 9 cm were used. A significant increase in transformation efficiency from about 33% under the rupture pressure of 450 psi to 87% at 650 psi was observed in transformed thalli. Most of the positive cells appeared in epidermal cells bombarded at 450 psi, whereas positive signals were seen in both epidermal and medullary cells at 650 psi. No positive transient expression was detected at a bombardment of 1,100 psi, or in negative or blank controls. For the conditions tested, the best parameter was obtained at 650 psi at a distance of 6 cm. Thus, the strategy of taking vegetative thalli as recipients, using particle bombardment, and combining this with micro-propagation, together with developing an in vivo selectable marker, is a viable way to produce stable transformants, to eliminate chimeric expression, and to achieve transgenic breeding in K. alvarezii.

Transformation of Platymonas (Tetraselmis) subcordiformis (Prasinophyceae, Chlorophyta) by agitation with glass beads

A transient transformation system for the unicellular marine green alga, Platymonas subcordiformis, was established in this study. We introduced the pEGFP-N1 vector into P. subcordiformis with a glass bead method. P. subcordiformis was incubated in cell wall lytic enzymes (abalone acetone powder and cellulase solutions) to degrade the cell wall. The applicable conditions for production of viable protoplasts were pH 6.5, 25 degrees C, and 3 h of enzyme treatment. The protoplast yield was 61.2% when P. subcordiformis cells were added to the enzyme solution at a concentration of 10(7) cell ml(-1). The protoplasts were immediately transformed with the pEGFP-N1 vector using glass-bead method. The transformation frequency was about 10(-5), and there was no GFP activity observed in either the negative or the blank controls. This study indicated that GFP was a sensitively transgenic reporter for P. subcordiformis, and the method of cell wall enzymolysis followed by glass bead agitation was applicable for the transformation of P. subcordiformis.

转基因海带配子体的制备与高效增殖

海带具有很高的营养价值和经济社会价值。自20世纪90年代以来，本实验室在借鉴高等植物基因工程原理和方法的基础上，根据海带自身特点，建立了海带遗传转化体系（海带孢子体表达系统），它的基本原理是利用基因枪法转化海带配子体，经孤雌或受精途径再生幼孢子体后，用氯霉素筛选幼孢子体获得转基因海带，然后进行海上安全栽培和转基因产品的检测与提取。目前该表达系统已成功实现报告基因（β-半乳糖苷酶基因，lacZ）和功能基因（乙肝表面抗原基因，HBsAg）的稳定表达。 由于海带孢子体表达系统需经孢子体再生和海上栽培等阶段，周期较长，而且转基因安全性问题也在一定程度上制约其研究与应用。因此，我们在海带孢子体表达系统的基础上又建立和优化了海带配子体表达系统，并成功实现了报告基因（绿色荧光蛋白基因，GFP）的瞬间表达和功能基因（瑞替普酶基因，rt-PA）的稳定表达。虽然海带配子体表达系统能避免转基因安全性问题，周期较短，但在表达量和生物量积累方面，与孢子体表达系统相比还有较大差距。 本文首先在海带配子体表达系统中成功实现了人酸性成纤维细胞生长因子基因（hafgf）和鲎素基因（tac）的稳定表达，制备了转基因海带配子体，然后将光生物反应器培养技术应用于转基因海带配子体的高效增殖，以期解决阻碍海带配子体表达系统发展的量的问题，并为转基因海带配子体的大规模培养提供试验依据和技术支持。 本文的研究结果为： 1、人酸性成纤维细胞生长因子基因和鲎素基因可以稳定整合到海带配子体基因组中，实现转基因产物的表达。 2、根据转基因海带配子体的生长特点，研制开发了一套培养体积为300 ml的鼓泡式光生物反应器，它具有操作简便、成本低廉、适合海带配子体生长等特点。随后将培养体系扩大到2.5 L，并研究了光对转基因海带配子体生长的影响，试验结果显示，转基因海带配子体在光强为30 μE m-2 s-1时即可达到光饱和生长，最优光周期为14:10 LD，而且蓝光可促进转基因海带配子体的生长。 3、在前期研究工作的基础上，为改善反应器内的传质条件，我们又设计研制了2.5 L气升式光生物反应器用于转基因海带配子体的高效增殖。研究发现，气升式光生物反应器较鼓泡式光生物反应器能明显地改善反应器内的传质状态，实现转基因海带配子体更高密度的培养（生物量可达到1,990 mg L-1），是一套高密度悬浮培养转基因海带配子体的有效装置和设备。

饲料中转基因成分检测及对水产动物安全性评价的初步研究

自转基因作物问世以来，转基因产品的安全性问题一直是人们关注的焦点。本文根据GenBank中登录的转基因大豆完整外源DNA序列设计了几对引物，对转基因大豆进行了巢式PCR检测。结果表明，巢式PCR可以扩增10-10g/μl浓度的DNA溶液，检测灵敏度高达0.01％。该巢式PCR技术具有高度特异性、灵敏度和很好的重复性。用巢式PCR对部分市售的水产饲料和豆制食品进行检测，90.6％的水产饲料和46.5％的食品能检测出外源基因片段，表明转基因大豆广泛存在于水产饲料和我们的日常食品中，为食品安全分析和管理提供了方法和依据。环介导的等温扩增技术（LAMP）依赖于能够识别靶序列上6个特异区域的引物和一种具有链置换特性的DNA聚合酶，在等温条件下可高效、快速、高特异地扩增靶序列。本研究建立了转基因大豆的LAMP扩增技术，针对豆制品以及饲料的转基因LAMP检测技术正在研究和开发中。 利用转基因和非转基因豆粕制作的饲料，喂养吉富罗非鱼，分别于4周、7周取样，对其体重和血液指标进行了检测。实验显示，投喂转基因饲料7周以后，增重率和血清指标，转基因组与非转基因饲料组相比没有显著差异。全血指标中白细胞数目、大血小板比率、平均血小板体积和血小板体积分布宽度4项指标显著高于非转基因饲料组，而且差异达到极显著水平。由以上结果可见，转基因大豆与非转基因大豆相比，对罗非鱼的一些生理过程造成了一定的影响，但是并未对其生长造成可见的影响。分别于投喂1h、4h和8h以后取罗非鱼胃内容物、肠道内容物和粪便，并分别于4周、7周和继续饥饿2周后，取罗非鱼不同组织，提取DNA，用巢式PCR法检测转基因大豆中的外源基因在各种组织中的分布，结果显示在胃内容物、肠内容物、粪便、心脏、肝脏、胃、肠、卵巢、精巢、脑、鳃丝、脾脏、胆囊、肌肉等不同部位的DNA中都能检测到外源基因的存在，说明转基因大豆中的外源DNA并不能被罗非鱼的消化道完全降解，其DNA片段可能通过消化吸收转移到鱼体的各种组织。在投喂转基因饲料7周以后以及停止投喂饥饿2周以后分离水体中的微生物，提取其DNA，进行转基因检测。结果显示在所分离纯化的各种微生物中都没有检测到转基因大豆中外源基因35S-EPSPS的存在。

The effect of supplementary feeds on the bodyweight of yaks in cold season

The present study was conducted to determine the effects of supplementary feeds, oat hay (OH), highland barley straw (HBS) and multi-nutrient blocks supplementation (UMMB) on reducing liveweight losses of both yak cows and calves grazed on low quality pastures during cold season. The trials of OH and HBS supplementation were conducted by using completely random design on 104 yak cows between 6 and 12 years of age as the following treatments: pure grazing (41 animals, body weight 230 67 kg) as control (CK); grazing+1.5 kg DM of OH per head daily (30 animals, body weight 216 28 kg); gazing. 1.5 kg DM of HBS per head daily (33 animals, body weight 221 34 kg). The trial of UMMB was conducted on three types of yaks, 1-year calves (8-12 months old, body weight 61.1 6.9 kg), 2-year calves (18-24 months old, 98.0 11.3 kg) and yak cows (164.5 27.1 (S.D.) kg) with 20 animals in control group (CK) and 20 animals in supplement group for each type by using completely random design as the following treatments: pure grazing for CK group; grazing+ 150, 250 and 500 g UMMB per day averagely for 1-year calf, 2-year calf and cow at night. The results indicate that the animals supplemented with oat hay received body weight gain (32 20.7 g day(-1)), while those supplemented with highland barley straw still suffered from body weight loss (-56.7 39.3 a day(-1)); UMMB supplementation can decrease the body weight loss by 109.7%, 86.6% and 63.4% for the 1-year calves, 2-year calves and yak cows, respectively, as compared with pure grazing. Around US$1.60 output can be achieved on the basis of US$1 input for UMMB supplementation in the farming systems of the 1-year calves, 2-year calves and yak cows, while US$1 input can produce US$1.55 and 1.14 output for OH and FIBS supplementations, respectively, in yak cows' farming system. It can be preliminary concluded that UMMB supplementation was the most economic way to alleviate body weight loss of grazing yaks over cold season, and the higher productive returns were obtained from OH supplementation for grazing yak cows during winter/spring months. © 2004 Elsevier B.V All rights reserved.