鸡溶菌酶基因3＇端MAR在同源细胞系对基因表达调控的研究

用带有鸡溶菌酶基因增强子（Ｅ）和启动子（Ｐ）的编码细菌氯霉素乙酰基转移酶（ＣＡＴ）的融合基因（ＥＰＣ），在其上游区、下游区或在上游和下游区同时插入鸡溶菌酶基因３＇端核基质附着区（ＭＡＲ）的不同表达载体ＭＥＰＣ、ＥＰＣＭ和ＭＥＰＣＭ，稳定转染鸡ＨＤ１１Ｐｒｏｍａｃｒｏｐｈａｇｅ同源细胞系，筛选不同表达载体稳定整合后的细胞株，检测ＣＡＴ表达水平，确定其表达基因拷贝数，从而分析鸡溶菌酶基因３＇端ＭＡＲ对基因表达的影响。稳定整合表达和瞬时表达实验结果表明，鸡溶菌酶基因３＇端ＭＡＲ在同源细胞系对基因表达没有激活作用，与该基因５＇端ＭＡＲ在同源或异源细胞系中能激活基因表达形成鲜明对照，说明５＇瑞ＭＡＲ和３＇端ＭＡＲ在调节鸡溶菌酶基因表达上存在一种协调关系。     

叶黄素和中草药对高温环境中罗曼蛋鸡产蛋性能与蛋品质的影响

试验旨在利用叶黄素与中草药组合缓解热应激对蛋鸡的危害。选取210只罗曼粉壳蛋鸡,在高温环境中设立6个试验组,每组30只鸡,Ⅰ组饲喂基础日粮,Ⅱ组饲喂基础日粮+0.3%中草药+维生素C(200mg/kg),Ⅲ组、Ⅳ组、Ⅴ组、Ⅵ组在饲喂基础日粮+维生素C(200mg/kg)的基础上分别添加180mg/kg、240mg/kg、300mg/kg和360mg/kg叶黄素,常温环境中设立1个试验组,有30只鸡,饲喂基础日粮,分析叶黄素、中草药、叶黄素与中草药组合对高温环境中罗曼蛋鸡产蛋性能和蛋品质的影响。结果表明:(1)高温使蛋鸡的产蛋率、平均蛋重、蛋壳厚度分布分别显著下降9.79%、7.21%和11.55%(P0.05);(2)比Ⅰ组高温对照组相比,Ⅱ组中草药组的产蛋率、平均蛋重、鸡蛋蛋白质含量分别显著提高7.66%、4.09%和11.53%(P0.05);(3)在高温环境中,与Ⅰ组高温对照组相比,Ⅳ组中240mg/kg叶黄素与中草药组的产蛋率、蛋壳厚度显著提高6.90%和12.42%(P0.05),破软壳蛋率显著降低76.69%(P0.05),罗氏等级显著提高(P0.05),鸡蛋蛋白质含量提高8.56%(P0.05);(4)与Ⅱ组中草药组相比,Ⅳ组中240mg/kg叶黄素与中草药组合的破软壳蛋率显著降低72.26%(P0.05),罗氏等级显著提高(P0.05)。试验结果显示,在本试验条件下,高温使蛋鸡的生产性能下降,0.3%中草药能提高高温环境中蛋鸡的产蛋率,蛋鸡日粮中添加240mg/kg的叶黄素+0.3%中草药可提高高温环境中蛋鸡的产蛋性能,改善蛋品质。     

Quantifying the effect of Managed Aquifer Recharge on the microbiological human health risks of irrigating crops with recycled water

Managed Aquifer Recharge (MAR) is increasingly being used for water recycling via aquifers and recovery for irrigation. Quantitative Microbial Risk Assessment (QMRA) was used to assess the human health risks from irrigation using reclaimed water and to evaluate the reduction in risk where MAR is used for irrigation management. Four MAR sites (Shafdan, Israel; Nardò, Italy; Bolivar, Australia; and Sabadell, Spain) that use reclaimed water for crop and/or park irrigation were evaluated, and the risk to human health was quantified in terms of DALYs (Disability Adjusted Life Years). The results indicated that median risks for all scenarios and pathogens evaluated were acceptable (<10⁻⁶ DALYs) with the exception of risks from accidental aerosol ingestion and bacterial pathogens at the Nardò site. MAR was found to be one of the most important treatment barriers in terms of log₁₀ inactivation credits and hence a useful tool for recycled water irrigation management. The Shafdan site relied almost completely on the MAR treatment to reduce the human health risks from irrigation to acceptable levels. For the Nardò site MAR was also an important barrier, where if MAR had not been used as part of the irrigation system the risk would be unacceptable for protozoa and viruses. The Bolivar and Sabadell sites had much larger and more technologically complex recycled water treatment systems and as such MAR was not a critical barrier in managing human health risks.     

融合杀虫基因对甘蔗的遗传转化

[目的]利用MAR序列介导转基因表达及融合杀虫基因,以期克服外源基因失活,增强抗虫基因表达能力,延缓昆虫抗性的发生,获得高抗虫转基因甘蔗植株.[方法]以甘蔗品种ROC25为材料,进行愈伤组织、芽苗分化和生根的Hyg抗性筛选试验.利用农杆菌介导,将含MAR序列介导融合杀虫基因(AVAc-CpTI)导入甘蔗基因组.[结果]愈伤组织增殖、出芽时Hyg的最佳筛选浓度为50 mg/L,生根时Hyg的最佳筛选浓度为30 mg/L.获得13个MAR序列介导转基因表达基因系,48株Southern杂交阳性甘蔗植株;获得8个无MAR序列介导转基因表达基因系,7株Southern杂交阳性甘蔗植株.MAR序列介导转基因表达使转化过程中转基因系的获得数量提高了62.5%.[结论]建立了ROC25的潮霉素抗性筛选体系;MAR序列介导融合杀虫基因进行甘蔗遗传转化,可提高甘蔗外源基因转化效率.     

甘蓝短散布元件对转基因表达效果的研究*

 为检验甘蓝短散布元件（SINE）对植物转基因表达的影响,采用PCR方法,从甘蓝基因组中克隆了一段短散布元件序列（Short Interspersed Nuclear Element,SINE）,该SINE具有核基质结合区（matrix attachment region,MAR）的结构特征,将其构建到β-葡糖醛酸酶（β-glucuronidase,GUS）基因（uidA）的两侧翼,形成SINE调控的植物表达载体,采用农杆菌介导法,将含SINE序列和不含SINE序列的植物表达载体导入烟草中。对转基因植株进行GUS活性定量测定,结果表明,SINE表现出类似MAR的功能,可以提高外源uidA基因的表达水平,与不含SINE的转化植株相比,外源基因的平均表达水平提高了2倍,但转基因植株个体间表达水平存在较大的差异。     

MAR序列介导野苋菜凝集素基因在白菜中的表达

以‘丰顺’白菜带柄子叶为转化受体, 用带有MAR (Matrix Attachment Region) 和不带MAR的两种植物表达载体进行农杆菌介导转化野苋菜凝集素基因(Amaranthus viridis L. agglutinin, AVA ) 获得转基因的抗蚜小白菜。分析MAR序列介导对转基因表达的影响。表明利用MAR序列介导AVA 基因表达,获得转基因植株的数量比对照提高29.63%; 转AVA基因白菜对桃蚜(Myzus persicae) 的群体发展有一定的抑制作用, 平均抑制率为55.8%; MAR序列介导AVA 基因表达的转基因植株中, 该基因的表达水平比对照高, 并且不同转基因单株间AVA基因表达差异比对照小。     

核基质结合区的研究进展

核基质结合区是真核生物染色质中可以与核基质结合的一段ＤＮＡ序列。它能使染色质形成环状结构,还可以作ＤＮＡ复制的起始点或调控基因的转录。在基因工程研究中发现,用ＭＡＲ构建载体能够提高外源基因的整体表达水平,并增强外源基因表达的稳定性。深入了解ＭＡＲ的性质,功能等特点对克服遗传操作中常出现的外源基因失活现象有重要的理论与应用意义。本文就ＭＡＲ的分离,结构功能特征及在植物基因工程中的应用等几个方面进行了     

拟南芥AtTTG1基因的克隆及用于转化花生的MAR调控表达载体构建

黄曲霉毒素污染对花生产业危害巨大,通过基因工程改变花生果种皮结构以提高花生抗黄曲霉能力。根据GenBank中拟南芥AtTTG1基因的cDNA编码区设计引物,通过RT-PCR克隆AtTTG1基因,结果显示,克隆获得的片段长1026bp,与基因库中数据比对相同,该片段编码341个氨基酸,预测其蛋白分子量为86.96KDa,等电点为4.85。结合实验室已获得的花生果种皮特异启动子S19和MAR序列调控的植物表达载体pLMAR,构建了植物高效表达载体pLMAR-S19-TTG1,并将其导入根癌农杆菌EHA105。为进一步对花生进行遗传转化,获得转基因高抗黄曲霉花生奠定基础。     

含有MAR序列的STI-STII-LTB融合基因在苜蓿中的表达

产肠毒素性大肠杆菌(ETEC)LT和ST是导致人和动物腹泻的主要病原菌。利用三引物PCR法和酶切实现了STI、STII、LTB基因的融合,LTB基因的5′位于STII基因的3′端,三者在同一阅读框。将融合基因STI-STII-LTB构建到带有核基质结合区序列(MARs)的植物表达载体pBI121-MARs中GUS基因的位置。同时构建不含MARs序列的重组质粒。重组质粒pBI121-MARs-STI-STII-LTB、pBI121-STI-STII-LTB通过冻融法转化根癌农杆菌,并通过农杆菌介导法转化杂花苜蓿。转基因苜蓿植株经PCR检测、Southern-blotting分析表明,转基因苜蓿植株基因组中可检测到STI-STII-LTB融合基因;提取转基因苜蓿植株总蛋白质,SDS-PAGE结果显示,转基因植株表达了重组抗原蛋白,且含MAR序列的蛋白表达量要高于不含MAR序列的表达量;Western-dotting免疫检测证实转基因植株表达的重组抗原具有免疫原性。     

Photoprotective Bioactivity Present in a Unique Marine Bacteria Collection from Portuguese Deep Sea Hydrothermal Vents

Interesting biological activities have been found for numerous marine compounds. In fact, screening of phylogenetically diverse marine microorganisms from extreme environments revealed to be a rational approach for the discovery of novel molecules with relevant bioactivities for industries such as pharmaceutical and cosmeceutical. Nevertheless, marine sources deliverables are still far from the expectations and new extreme sources of microbes should be explored. In this work, a marine prokaryotic collection from four Mid-Atlantic Ridge (MAR) deep sea hydrothermal vents near the Azores Islands, Portugal, was created, characterized and tested for its photoprotective capacity. Within 246 isolates, a polyphasic approach, using chemotaxonomic and molecular typing methods, identified 23-related clusters of phenetically similar isolates with high indexes of diversity. Interestingly, 16S rRNA gene sequencing suggested the presence of 43% new prokaryotic species. A sub-set of 139 isolates of the prokaryotic collection was selected for biotechnological exploitation with 484 bacterial extracts prepared in a sustainable upscalling manner. 22% of the extracts showed an industrially relevant photoprotective activity, with two extracts, belonging to new strains of the species Shewanella algae and Vibrio fluvialis, uniquely showing UV-A, UV-B and UV-C protective capacity. This clearly demonstrates the high potential of the bacteria MAR vents collection in natural product synthesis with market applications.