芽孢杆菌绿色荧光蛋白标记及其在小麦体表定殖的初探   总被引：17，自引：0，他引：17  

田涛  亓雪晨  王琦  梅汝鸿 《植物病理学报》2004,34(4):346-351

 将来自质粒pAD4412的启动子和绿色荧光蛋白基因gfpmut3a插入大肠杆菌-枯草芽孢杆菌穿梭载体pBE2,构建成芽孢杆菌表达载体pGF P4412,用其转化野生型生防芽孢杆菌83-6和A-47等8个菌株,均得到良好的发光表型。质粒稳定性实验表明重组质粒pG FP4412稳定性为92%。借助荧光显微镜对gfp标记的菌株A-47-gfp在小麦体表的定殖进行初步的研究。结果表明:A-47-gfp能够在小麦根际及小麦体表定殖(包括根表和茎叶表面);相对于在茎叶表面定殖的A-47-gf p在根表定殖的菌体与根的结合更为牢固;从根基到根尖A-47-gfp的定殖量有明显的减少趋势。  相似文献   

Optimizing injection time of GFP plasmid into sheep zygote     

Mohammadreza Ebrahimi  Laura Mara  Bernardo Chessa  Fabrizio Chessa  Abbas Parham  Maria Dattena 《Reproduction in domestic animals》2021,56(3):467-475

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6–8 hpi, n: 120; 8–10 hpi, n: 122; 10–12 hpi, n: 110 and 12–14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8–10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8–10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments.  相似文献   

Optimization of Agrobacterium-mediated transformation parameters for Melastomataceae spp. using green fluorescent protein (GFP) as a reporter     

Wilson Thau Lym Yong  Janna Ong Abdullah  Maziah Mahmood 《Scientia Horticulturae》2006

Agrobacterium-mediated transformation for both Melastoma malabathricum and Tibouchina semidecandra were optimized using green fluorescent protein (GFP) as a reporter. The binary vector pCAMBIA1304 harboring the modified green fluorescent protein (mgfp) gene driven by the CaMV 35S promoter was used. Parameters optimized were bacterial strain, bacterial concentration, pre-culture period, co-cultivation period, immersion time, acetosyringone concentration and wounding type. Results obtained were based on the percentage of GFP expression which was observed 3 days post-transformation. Agrobacterium tumefaciens strain LBA4404 and EHA105 at concentration 1 × 10⁷ cfu ml⁻¹ (OD_600nm 0.8) showed the highest virulence on M. malabathricum and T. semidecandra, respectively. Four days of pre-culture and 2 days of co-cultivation were optimum for M. malabathricum transformation, while 3 days of pre-culture and co-cultivation for T. semidecandra. Result also showed that 60 min of immersion and addition of 200 μM acetosyringone gave the highest percentage of positive transformants for both M. malabathricum and T. semidecandra. Mild wounding also significantly increased the efficiency of M. malabathricum transformation.  相似文献   

工程菌DH5a感受态细胞的制备及质粒pGEM的转化研究     

杜祯  马海利  陈瑞芳 《中国畜牧兽医》2007,34(5):88-90

绿色荧光蛋白（green fluorescent protein，GFP）是源于海洋生物多管水母(aequoia victoria)的一种发光蛋白，能够自身催化形成生色团并在蓝光激发下发出绿色荧光，能在活细胞内稳定表达，本试验首先从含有GFP的大肠杆菌中提取质粒，并利用胶回收试剂盒纯化质粒，采用氯化钙法将工程菌DH_5a制成感受态细胞，然后将纯化后的质粒转入已制备好的感受态细胞中，使得工程菌DH_5a转化成具有抗氨苄青霉素的大肠杆菌，并表达绿色荧光蛋白，接种于加氨苄青霉素的LB培养基上，筛选阳性克隆。  相似文献   

MrMYB1-MrbHLH1:一个有潜力的园艺植物转基因可视化报告基因     

张玺丽  殷学仁  李方  陈昆松  刘晓芬 《园艺学报》2017,44(12):2296-2304

将杨梅果实中参与花色苷生物合成的转录因子编码基因MrMYB1和MrbHLH1同时搭载到双元表达载体的多克隆位点内,重组成双价表达载体,通过农杆菌介导的叶盘法将其分别转入烟草、番茄和森林草莓,成功转化MrMYB1-MrbHLH1的烟草、番茄和森林草莓不定芽因大量积累花色苷而呈红色,与野生型或假阳性不定芽相比,表型差异明显;转化35S::MrMYB1-MrbHLH1的烟草和番茄植株中可显著积累花色苷呈红色,且该性状可稳定遗传。MrMYB1-MrbHLH1在3种园艺植物具有调控花色苷积累而使阳性转化植物部分组织器官呈现红色的功能,是1个有潜力的遗传转化可视化报告基因。  相似文献   

抗菌肽天蚕素B突变体ABP-S1基因在E.coli中的融合表达   总被引：1，自引：0，他引：1  

卢强  刘维全  赵凤芹  刘明远  江禹  殷震 《中国兽医学报》2002,22(3):241-243

利用绿色荧光蛋白 (GFP)基因与天蚕素 B突变体 ABP- S1基因的融合基因 ,构建了 2个原核表达载体 p Am GS1和 p Bm GS1,转化表达宿主菌 E.coli BL 2 1(DE3)和 E.coli BL 2 1(DE3) p L ys S。结果 ,p Am GS1未能得到转化子 ,而p Bm GS1的转化子高效表达了融合蛋白 ,经 IPTG诱导 ,SDS- PAGE检查 ,融合表达产物最高可占菌体总蛋白的49.45 %。表达产物经包涵体复性 ,柱层析初步纯化 ,通过平板抑菌试验 ,对大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌、变形杆菌以及鱼类重要的致病菌嗜水气单胞菌等多种革兰氏阳性、阴性菌表现出较强的抗菌活性  相似文献   

马铃薯损伤诱导型启动子Wun1基因的克隆及其GFP表达活性   总被引：1，自引：0，他引：1  

贾笑英  向云  张金文  王蒂 《分子植物育种》2006,4(3):333-338

通过聚合酶链式反应(PCR),以马铃薯基因组DNA为模板,根据已报道Wun1序列设计了一对特异引物,在优化的PCR反应条件下扩增出了Wun1基因片段,通过序列分析与文献报道的碱基序列有96.86%的同源性,该基因已登录到GenBank(No.AY803296)。以pBIPG(携带GFP基因)的质粒DNA为模板,通过PCR技术亚克隆到了源自水母(Aequorea)大小为756bp的绿色荧光蛋白(GFP)基因,与已知序列同源率为100%。利用GFP基因作为报告基因,构建了用于比较鉴定所克隆启动子活性的pBIG(35S-GFP)和pBIWG(Wun1-GFP)两个植物表达载体,采用基因枪法进行对洋葱表皮细胞的遗传转化,检测Wun1启动子在受体细胞中调控基因表达的活性,结果表明克隆到的Wun1启动子活性强于组成型表达的35S启动子,GFP瞬时表达的分析方法也让我们有效的筛选到用于进行马铃薯抗病育种的调控元件。  相似文献   

A melon genotype with superior competence for regeneration and transformation   总被引：7，自引：0，他引：7  

M. Galperin    L. Patlis    A. Ovadia    D. Wolf    A. Zelcer  D. Kenigsbuch 《Plant Breeding》2003,122(1):66-69

Transformation efficiency of melon is low and is still regarded as a challenge. In this paper, the regeneration and transformation response of ‘BU‐21/3′, a newly characterized melon breeding line, is described. The line seems to be superior in this regard to previously evaluated genotypes. Agrobacterium‐mediated delivery of the GUS or GFP reporter genes into cotyledon explants was used to evaluate efficiency of transient and stable transformation. Good transient expression was observed, and stable transformation frequencies of 0.4‐1.5 transgenic shoots per explant were obtained. Transgenic plantlets were transferred to a contained greenhouse as early as 8‐10 weeks after transformation. Transgenic plants are fertile and exhibit a true‐to‐type phenotype. The ‘BU‐21/3’ line may become a useful tool for the facilitation of transgenic breeding in melon.  相似文献   

甘蓝型油菜柱头特异启动子的克隆和序列分析   总被引：1，自引：0，他引：1  

柯丽萍  郑滔  吴学龙  陈锦清  祝水金 《农业生物技术学报》2007,15(2):257-262

芸薹属自交不亲和相关基因SLR1只在柱头中特异表达，根据已知的SLR1基因启动子序列设计特异引物，用PCR法从几个甘蓝型油菜品种和品系中分离得到相应的启动子片断，进行序列比较分析后，并构建它们与GFP融合的植物表达载体，转化拟南芥，验证所分离到的启动子的时空特异性。研究表明，来自自交不亲和羽衣甘蓝和来自自交亲和的甘蓝型油菜的SLR1启动子具有相似的序列结构和相同的组织特异表达功能。该结果将为下一步研究S-locus基因在甘蓝型油菜中的表达和相互作用奠定基础。  相似文献   

大豆营元对HEC-1B细胞增殖及雌激素受体 报告基因表达的影响     

刘小丽’.薛晓鸥’.唐炳华.牛建昭 《勤云标准版测试》2011,31(1):25-29

〔摘要〕目的探讨大豆葺元对子宫内膜癌细胞增殖的影响及其对雌激素受体报告基因表达的影响、方法体 外培养子宫内膜癌细胞子宫内膜癌雌、孕激素受体阴性细胞系(HEC 1B),采用四甲基偶氮哩盐法(M1"P)检测不同 浓度大豆葺元对子宫内膜癌细胞增殖的影响,同时检测大豆葺元对雌激素应答原件(ERE)调控的雌激素受体。 (ERa)和雌激素受体(3 ( ER(3)的荧光素酶报告基因表达的影响〔结果大豆葺元对HEC-1B细胞体外增殖有明显 抑制作用,差异有统计学意义(P<0.01),其抑制作用有明显量效和时效性特征〔在浓度为20-80x 1 O}mol/L的大豆葺 元作用卜,报告基因1u。的表达较正常对照组明显提高,差异有统计学意义(P<0.05 ),在80x 10-`mol/L时到达最高 值;大豆葺元通过ER日介导的报告基因表达水平的升高程度强于ERa,差异有统计学意义(P<0.01)〔结论大豆葺 元可通过调节子宫内膜癌细胞ER。和ER日介导的报告基因的表达,调整ERa/ER日比例,从而抑制子宫内膜癌细 胞的增殖、  相似文献