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为研究水稻蜡质基因(waxy)5'上游调控区中存在的顺式作用元件,我们将水稻waxy基因翻译起始声、(ATG)5'上游3.4kb(-2118~+1291bP)片段经外切核酸酶ExoⅢ部分酶解,得到一系列5'端缺失的片段。将这些缺失片段分别与gus基因编码区连接,构建成融合质粒,经PEG介导引入水稻原生质体,26℃培养48h后,定量测定GUS酶活力,并以同时导入的由35S启动子指导的荧光素酶(LUC)基因表达的酶活力作为内对照。结果表明,GUS酶活性随5’上游调控区长度的减少而逐渐减弱。由─861bp缺失至─640bP时,gus基因表达水平有较明显的降低,推测在该区域中可能存在一个顺式作用元件区。 相似文献
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Distribution and properties of pea (Pisum sativum L.) cells, competent forAgrobacterium-mediated transformation were analysed byin situ histochemical detection of GUS (-glucuronidase) activity, 4 d after inoculation with engineeredAgrobacterium tumefaciens. The vector system consisted of the hypervirulent disarmed strain EHA101 and the binary plasmid pIBGUS, carrying an intron-containing, 35S-promotor drivengusA (oruidA) gene and two selectable marker genes. Cells competent for transformation were mainly restricted to the dedifferentiating cells neighbouring the vascular system of cotyledon and epicotyl explants. A standardized assay was developed, allowing determination and quantification of factors influencing number and distribution of competent cells. In etiolated seedlings, competence for transformation decreased with the distance of the epicotyl explant from the shoot apex and was specifically induced by the exogenous application of auxins. Transient expression ofgusA afterAgrobacterium-mediated DNA transfer was dramatically reduced upon application of cell-cycle and DNA replication inhibitors aphidicolin, colchicine and nalidixic acid. GUS expression after direct DNA transfer of double-stranded plasmid DNA (via PEG into protoplasts or via particle bombardment of epicotyl segments) was independent of cell-division/DNA replication.A GUS-positive mutant of EHA101 was constructed to allowin situ analysis of attaching bacteria within the plant tissue. Attachment and invasion was inhibited by well-developed cuticula but was restored after chloroform treatment of the tissue surface. Moreover, no correlation was found between distribution of attaching bacteria and the pattern of transformation-competent cells. 相似文献
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Sonication: A new method for gene transfer to plants 总被引:9,自引:0,他引:9
Sonication is a novel method for gene transfer into plant protoplasts and intact plant cells. The mode of action of ultrasound and its chemical, biochemical and physiological effects are reviewed. The state of the art of acoustic transformation is presented and possible mechanisms are discussed. 相似文献
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Kensaku Maejima Masayoshi Hashimoto Yuka Hagiwara-Komoda Akio Miyazaki Masanobu Nishikawa Ryosuke Tokuda Kohei Kumita Noriko Maruyama Shigetou Namba Yasuyuki Yamaji 《Molecular Plant Pathology》2020,21(4):475-488
Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection. 相似文献
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植物原生质体是去除了细胞壁的裸露细胞,其具有细胞全能性,现广泛应用于植物分子细胞生物学的研究中,可以大大缩减实验周期,并有助于得到体内实验的实时检测数据。该文除了介绍植物原生质体的提取和纯化方法外,还对国内外利用各种植物的原生质体进行细胞瞬时转化、亚细胞定位、细胞融合和大分子复合物相互作用等试验进行了总结和讨论。植物原生质体还可用于基因表达模式的实时检测,并作为生物反应器的受体细胞进行代谢物的体外生产。此外,还对当前该技术所面临的瓶颈进行了分析,为植物原生质体在分子细胞生物学领域的应用提供帮助,为技术的优化和推广提供参考。 相似文献