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首页> 外文期刊>In Vitro Cellular and Development Biology. Plant: Journal of the Tissue Culture Association >Electroporation-mediated transient gene expression in intact cells of sweetpotato.
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Electroporation-mediated transient gene expression in intact cells of sweetpotato.

机译：甘薯完整细胞中电穿孔介导的瞬时基因表达。

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摘要

Transient expression of the beta-glucuronidase (GUS) gene has been studied in leaf-derived embryogenic callus of sweet potato Ipomoea batatas by electroporation. The influence of several factors including electric field strength, buffer composition,time course of transient GUS gene expression, DNA concentration, enzyme, and polyethylene glycol (PEG) treatment was examined on GUS gene expression (number of blue spots). Maximum GUS gene expression (an average of 90 blue spots/fifty mg fresh weight callus tissue) was observed after 48 h when callus pieces were preincubated with electroporation (EPR) buffer for 1 h, followed by electroporation with a single electric pulse of 500 V/cm discharged from a 960-鍲 capacitor in the presence of 20 鎔 DNA/mland 8.3 鎙 NaCl (3 M). Changing the electroporation buffer conductivity (by varying the buffer composition with low-high salt concentrations), had only slight effect on the number of blue spots. Similarly, the time course study of GUS gene expression revealed that GUS activity could be detected 12 h after electroporation with a maximum activity after 72 h (112 blue spots). Increasing the amount of DNA from 5 to 50 鎔/ml in the EPR buffer had a slight effect on the expression frequency (from 20-110 bluespots, and 112 blue spots with 20 鎔/ml). The number of blue spots was increased by enzymatic wounding of callus pieces for 10 min and by addition of 200 鎙 PEG 4000 (15%) before electroporation. These results suggest that intact cell electroporation canbe used for producing transgenic sweetpotato tissue.
机译：已经通过电穿孔在甘薯番薯叶的叶片衍生胚发生愈伤组织中研究了β-葡糖醛酸糖苷酶（GUS）基因的瞬时表达。考察了电场强度，缓冲液组成，瞬时GUS基因表达的时程，DNA浓度，酶和聚乙二醇（PEG）处理等几个因素对GUS基因表达（蓝点数量）的影响。将愈伤组织块与电穿孔（EPR）缓冲液预孵育1 h，然后用500 V单电脉冲进行电穿孔后48小时后，观察到最大GUS基因表达（平均90个蓝点/每50毫克鲜重的愈伤组织）在存在20镕DNA / mland 8.3鎙NaCl（3 M）的情况下，从960鍲电容器中放电的/ cm / cm。改变电穿孔缓冲液的电导率（通过以低-高盐浓度改变缓冲液组成）对蓝点的数量影响很小。类似地，对GUS基因表达的时程研究表明，电穿孔后12 h可以检测到GUS活性，而72 h后可以检测到最大的活性（112个蓝点）。将EPR缓冲液中的DNA量从5增加到50镕/ ml对表达频率有轻微影响（从20-110个蓝点开始，而在20镕/ ml时有112个蓝点）。在电穿孔之前，酶解愈伤组织块10分钟并添加200鎙PEG 4000（15％）可增加蓝点的数量。这些结果表明完整的细胞电穿孔可用于生产转基因甘薯组织。

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