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首页> 外文期刊>Journal of Biotechnology >A family 19 chitinase (Chit30) from Streptomyces olivaceoviridis ATCC 11238 expressed in transgenic pea affects the development of T. harzianum in vitro
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A family 19 chitinase (Chit30) from Streptomyces olivaceoviridis ATCC 11238 expressed in transgenic pea affects the development of T. harzianum in vitro

机译：转基因豌豆中表达的来自寡链链霉菌ATCC 11238的19家族几丁质酶（Chit30）影响哈茨木霉的体外发育

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摘要

Embryo axes excised from mature seeds of pea (Pisum sativum L.) cv. 'Sponsor' were used as explants for Agrobacterium-mediated transformation using pGreenII 0229 binary vectors. The vectors harbored a chimeric chitinase gene (chit30), driven by the constitutive 35S promoter or the elicitor inducible stilbene synthase (vst) promoter from grape (Vitis vinifera L.). The secretion signal of the bacterial chitinase gene from Streptomyces olivaceoviridis ATCC 11238 (DSM 41433) was replaced by the A. thaliana basic chitinase leader sequence. Functional properties of the recombinant gene were tested in tobacco as a model system before the long process of pea transformation was undertaken. Several transgenic pea clones were obtained and the transgenic nature confirmed by different molecular methods. The accumulation and activity of chitinase in stably transformed plants were examined by Western blot analysis and in-gel assays, which showed the presence of an additional 3 isoform bands. Using in vitro bioassays with Trichoderma harzanium as a model, we found an inhibition or delay of hyphal extension, which might indicate enhanced antifungal activity compared with non-transformed pea plants. Up to the 4th generation, the transgenic plants did not show any phenotypic alterations compared with non-transgenic control plants.
机译：从豌豆（Pisum sativum L.）cv的成熟种子中切除的胚轴。 “ sponsor”用作外植体，用于使用pGreenII 0229二元载体进行农杆菌介导的转化。所述载体具有嵌合的几丁质酶基因（chit30），其由来自葡萄（Vitis vinifera L.）的组成型35S启动子或激发子诱导的二苯乙烯合酶（vst）启动子驱动。来自拟南芥碱性几丁质酶前导序列代替了来自寡链链霉菌ATCC 11238（DSM 41433）的细菌几丁质酶基因的分泌信号。在进行豌豆转化的漫长过程之前，以烟草作为模型系统测试了重组基因的功能特性。获得了几个转基因豌豆克隆，并通过不同的分子方法证实了转基因性质。通过蛋白质印迹分析和凝胶内分析法检测了稳定转化植物中几丁质酶的积累和活性，结果表明还存在3个同工型条带。使用以木霉属哈氏菌为模型的体外生物测定，我们发现抑制或延缓了菌丝的延伸，与未转化的豌豆植物相比，这可能表明其抗真菌活性增强。到第四代为止，与非转基因对照植物相比，转基因植物没有表现出任何表型改变。

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