重组马立克病毒通用转移载体的构建及初步应用     

刘召明  张丽萍  王秀丽  赵冬凤  刘新文  李明义 《中国动物检疫》2008,25(1):26-28

本实验以独立启动子控制的增强型绿色荧光基因(GFP)作为报告基因,同时将CMV启动子及其多克隆位点与之连接,构成外源基因表达盒,插入到马立克病毒(MDV)复制非必需区基因(短独特区US2等)构成的同源臂中,构建成重组马立克病毒的通用载体。鉴定正确后,将转移载体与提取的MDV基因组共转染鸡胚成纤维细胞(CEF),同源重组获得具有感染性的重组病毒,待病毒蚀斑出现后,荧光显微镜下观察,可见到明显的绿色荧光病毒蚀斑,经三次筛选,初步分离到重组病毒。结果表明,转移载体与MDV基因组共转染可获得感染性病毒,US2基因可作为重组病毒构建中的外源基因插入位点,证实通用转移载体的构建是可行的,为重组马立克病毒新型疫苗的研究奠定物质基础。  相似文献   

The use of visual marker genes as cell-specific reporters of Agrobacterium-mediated T-DNA delivery to wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.).   总被引：4，自引：0，他引：4  

Alex C. McCormac  Huixia Wu  Manzhu Bao  Yibing Wang  Ruiji Xu  Malcolm C. Elliott  Dong-Fang Chen 《Euphytica》1998,99(1):17-25

Transfer of T-DNA from Agrobacterium tumefaciens and A. rhizogenes to cells of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) is demonstrated following the inoculation of immature embryos and immature embryo-derived callus. Agrobacterium T-DNA vectors containing the C1/Lc anthocyanin-biosynthesis regulatory genes, the gusA gene or a synthetic green fluorescent protein gene (sgfp-S65T) were constructed from original binary vectors. The visual T-DNA markers were used as cell-autonomous reporters of early Agrobacterium-mediated transformation events in the wheat and barley cells. This localization of the transformed cells revealed a non-random distribution throughout each embryo and callus piece.  相似文献   

In vivo infection of Bursaphelenchus xylophilus by the fungus Esteya vermicola     

Hai‐Hua Wang  Yun‐Bo Wang  Can Yin  Jie Gao  Ran Tao  Yu‐Lou Sun  Chun‐Yan Wang  Zhen Wang  Yong‐Xia Li  Chang‐Keun Sung 《Pest management science》2020,76(8):2854-2864

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Transcription‐inducing activity of natural and synthetic juvenile hormone agonists through the Drosophila Methoprene‐tolerant protein     

Taiyo Yokoi  Taku Nabe  Chiharu Ishizuka  Ken'ichiro Hayashi  Sayoko Ito‐Harashima  Takashi Yagi  Yoshiaki Nakagawa  Hisashi Miyagawa 《Pest management science》2020,76(7):2316-2323

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根结线虫生防真菌交枝顶孢原生质体的制备与再生体系构建     

姚玉荣  霍建飞  郝永娟  王万立 《植物保护》2020,46(6):149-154

本研究以根结线虫生防真菌交枝顶孢Acremonium implicatum为供试菌株, 探究了菌龄?降解酶种类?酶解温度和时间?不同渗透压稳定剂?不同pH等因素对其原生质体制备的影响, 并建立了交枝顶孢原生质体制备体系; 制备的原生质体在SR培养基上再生率达到17.78%?此外, GFP遗传转化菌株的获得充分表明制备的原生质体可作为后续遗传转化材料, 为进一步研究交枝顶孢对根结线虫的生防机理奠定基础?  相似文献   

In vivo fish models for visualizing Aeromonas hydrophila invasion pathway using GFP as a biomarker     

Wei-Hua Chu  Cheng-Ping Lu 《Aquaculture (Amsterdam, Netherlands)》2008,277(3-4):152-155

The invasion pathway of Aeromonas hydrophila in vivo was studied in Crucian carp (Carassius auratus gibelio) with a virulent strain A. hydrophila J-1 transformed with a plasmid encoding green fluorescent protein (pGFPuv) (A. h J-1^GFP), which had similar virulence characteristics (haemolysin, extracellular proteases production, toxicity for EPC cells, survival in fish serum and LD₅₀ value) as the parent strain. Fish were divided into four experimental groups: (1) normal fish; (2) bacteria bath challenged, unwounded fish; (3) skin artificially wounded by scalpel, bacterial bath challenged fish; and (4) skin mucus layer partially removed by paper towel, bacterial bath challenged fish. The number of bacteria from blood, gills, kidney, muscle, liver and intestine were detected at 2, 4, 8, 12, and 24 h post-challenge. High bacterial numbers were observed in the muscle of the artificial wound group, in the kidney of the mucus removed group and in the gills of all groups. In conclusion, the gills and damaged skin are likely to be the main routes of entry for A. hydrophila, and GFP can be used as a real-time biomarker to study intimate host–pathogen interaction in fish.  相似文献   

Isolation and characterization of the zebrafish <Emphasis Type="Italic">Danio rerio</Emphasis> insulin-like growth factor binding protein-3 promoter region     

Ming Jun Chou  Jian Chyi Chen  Jyh Yih Chen  Hong Yi Gong  Li Tzu Li  Tzou Chi Huang  Jen Leih Wu  Ching Ming Kuo 《Fisheries Science》2008,74(1):153-166

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利用Cre/lox定位重组系统获得无选择标记GFP转基因烟草   总被引：5，自引：0，他引：5  

宋洪元  任雪松  司军  李成琼  宋明 《中国农业科学》2008,41(10):2973-2982

【目的】利用Cre/lox系统具有的特异重组特性,以绿色荧光蛋白GFP为目的基因,获得无选择标记的GFP转基因烟草。【方法】将Bar基因表达盒置于两个同向lox位点之间并与GFP基因表达盒融合后获得相应植物表达载体,转化烟草后获得抗除草剂Basta的GFP转基因植株。GFP转基因植株叶盘二次转化导入重组酶Cre基因后,实现与GFP基因连锁的Bar基因表达盒剔除,剔除Bar基因的植株开花后自交使重组酶Cre基因分离,获得无选择标记的GFP转基因烟草。【结果】将含Bar基因表达盒以及GFP基因表达盒的植物表达载体转化烟草Wisconsin 38后获得了抗除草剂Basta以及GFP荧光表达的转基因植株。随机抽取5株转基因植株二次转化导入Cre基因,所获得的再生植株叶盘进行Basta的抗性检测,绝大多数单株对应叶盘在含8 mg&#8226;L-1 PPT（phosphinothricin）的筛选培养基上无法再生死亡,删除效率在76%～100%。对Bar基因删除后区域片段进行克隆测序分析显示,Bar基因表达盒已经被精确删除。Bar基因删除植株开花后自交,获得的自交后代进行NPTⅡ抗性检测,NPTⅡ敏感植株分子检测显示均只含有GFP基因。【结论】利用Cre/lox系统获得烟草无选择标记的转基因植物是稳定可行的,可广泛应用于其它无选择标记转基因植物的培育。  相似文献   

百合花粉管通道法遗传转化的研究     

李双  袁霖  贾桂霞 《黑龙江农业科学》2011,(8):15-18

为了提高百合花粉管通道法遗传转化效率,采用pBI121-GFP-FT载体,对外源基因携带形式、导入方式和导入时间进行了研究。对质粒和农杆菌两种形式携带的外源基因的研究结果表明：菌液形式携带外源基因有利于百合花粉管通道法遗传转化。采用切割柱头授粉后涂抹外源基因、常规授粉后涂抹外源基因以及子房注射外源基因3种导入方式进行百...  相似文献   

植物病毒表达载体pClYVV/CP/W的构建及GFP表达的研究     

王振东  王晓华  孟鹏  秦会权  郑新伟  刘芳普  薛立江  张敏 《安徽农业科学》2009,37(28)

[目的]研究植物病毒表达载体pC1YVV/CP/W的构建及用pC1YVV/CP/W表达绿色荧光蛋白(GFP),为植物反应器生产有用蛋白、开发有效的植物病毒载体提供参考。[方法]用三叶草黄脉病毒侵染性全长cDNA克隆pC1YVV基因组的Mb/CP基因之间的一段多克隆位点和两侧含有病毒蛋白酶NIa切割识别序列的多聚脱氧核糖核苷酸接头,构建pC1YVV/CP/W载体,并将绿色荧光蛋白基因插入pC1YVV/CP/W中构建pC1YVV/CP/W/GFP载体,用反转录PCR对重组病毒克隆转录情况进行检测,并用Western杂交对重组病毒克隆表达的目的基因产物进行测定。[结果]pC1YVV/CP/W/GFP接种的蚕豆幼苗表现出与野生型C1YVV相同的症状,发病率达100%,表明重组病毒克隆pClYVV/CP/W/GFP有侵染性,外源基因的插入没有破坏载体pClYVV/CP/W的开放阅读框;外源基因在F_4子代病毒基因组中稳定存在;重组病毒克隆pC1YVV/CP/W/GFP至少到F_4子代病毒能稳定表达GFP。[结论]该研究结果为用植物表达有用蛋白提供了有用的植物病毒载体。  相似文献