https://scholars.lib.ntu.edu.tw/handle/123456789/138125
標題: | A reliable transformation method and heterologous expression of β-glucuronidase in Lentinula edodes | 作者: | Kuo, C.-Y. CHING-TSAN HUANG |
關鍵字: | Electroporation;β-glucuronidase;Heterologous expression;Lentinula edodes | 公開日期: | 二月-2008 | 卷: | 72 | 期: | 2 | 起(迄)頁: | 111-115 | 來源出版物: | Journal of Microbiological Methods | 摘要: | A simple and reliable mushroom transformation procedure based on electroporation of basidiospores or mycelial fragments was developed. This method eliminated the problem of protoplast preparation, the transformation efficiency were 30-150 transformants per mug DNA and the hygromycin resistant marker gene and gus were expressed in Lentinula edodes successfully. No false positive antibiotic-resistant cultures were detected by PCR amplification and the beta-glucuronidase (GUS) expression was maintained stable during mitotic cell division without selection pressure for more than 6 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. Using the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter with the first intron of gpd gene, the average GUS activity in L. edodes reached 144.6+/-3.9 U mg(-1) soluble protein, while only 30.1+/-0.7 U mg(-1) soluble protein was detected for those without the intron. The percentage of GUS in total soluble protein was 5.67x10(-4) (0.06%) for the transformant with the highest GUS activity. This rapid and convenient electroporation procedure offers a new approach for the genetic manipulation and tool to tag genes of important edible mushroom species. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/64142 | ISSN: | 0167-7012 | DOI: | 10.1016/j.mimet.2007.11.006 |
顯示於: | 生化科技學系 |
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08JMM72(111).pdf | 673.14 kB | Adobe PDF | 檢視/開啟 |
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