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碳离子束诱变选育高产角鲨烯类酵母及电转化初探

肖艳 王璐 王森 丛培虎 陆栋 冯银刚 崔球 宋晓金

肖艳, 王璐, 王森, 丛培虎, 陆栋, 冯银刚, 崔球, 宋晓金. 碳离子束诱变选育高产角鲨烯类酵母及电转化初探[J]. 南方水产科学, 2022, 18(2): 98-104. doi: 10.12131/20210294
引用本文: 肖艳, 王璐, 王森, 丛培虎, 陆栋, 冯银刚, 崔球, 宋晓金. 碳离子束诱变选育高产角鲨烯类酵母及电转化初探[J]. 南方水产科学, 2022, 18(2): 98-104. doi: 10.12131/20210294
XIAO Yan, WANG Lu, WANG Sen, CONG Peihu, LU Dong, FENG Yingang, CUI Qiu, SONG Xiaojin. Mutation and selection of high squalene production yeast Pseudozyma sp. induced by carbon-ions beam irradiation and its electrotransfor-mation[J]. South China Fisheries Science, 2022, 18(2): 98-104. doi: 10.12131/20210294
Citation: XIAO Yan, WANG Lu, WANG Sen, CONG Peihu, LU Dong, FENG Yingang, CUI Qiu, SONG Xiaojin. Mutation and selection of high squalene production yeast Pseudozyma sp. induced by carbon-ions beam irradiation and its electrotransfor-mation[J]. South China Fisheries Science, 2022, 18(2): 98-104. doi: 10.12131/20210294

碳离子束诱变选育高产角鲨烯类酵母及电转化初探

doi: 10.12131/20210294
基金项目: 国家重点研发计划项目 (2019YFD0901904);山东省重点研发计划项目 (2019GHY112006)
详细信息
    作者简介:

    肖艳:肖 艳 (1982—),女,副研究员,博士,从事微生物代谢工程研究。E-mail: xiaoyan@qibebt.ac.cn

    通讯作者:

    宋晓金 (1980—),男,副研究员,博士,从事海洋微藻/微生物研究。E-mail: songxj@qibebt.ac.cn

  • 中图分类号: Q 939.9

Mutation and selection of high squalene production yeast Pseudozyma sp. induced by carbon-ions beam irradiation and its electrotransfor-mation

  • 摘要: 为进一步提高角鲨烯产量,以海洋产角鲨烯类酵母 (Pseudozyma sp.) SD301为出发菌株,采用碳离子 (12C6+) 束诱变技术选育高产角鲨烯的突变菌株,并对突变菌株的电转化条件进行优化。利用100 μmol·L−1过氧化氢 (H2O2) 作为筛选压力,在12C6+束辐照剂量为120 Gy时,获得了比出发菌株具有更高角鲨烯产量的突变株PS120,该突变株在培养3 d后,角鲨烯产量达到1.33 g·L−1,角鲨烯质量 (细胞质量) 达到41.31 mg·g−1。利用绿色荧光蛋白EGFP作为表征标记,对PS120进行电转化条件优化,酶切、电泳和测序研究结果表明900 V电压条件下可成功将编码EGFP的基因转入到PS120细胞中,通过激光共聚焦显微镜进一步证实EGFP蛋白在该细胞中高水平表达。
  • 图  1  类酵母SD301碳离子束辐照诱变后菌落存活情况

    Figure  1.  Survival colony forming units of yeast-like strain SD301 after carbon-ions mutagenesis

    图  2  不同浓度的过氧化氢对类酵母SD301生长的影响

    Figure  2.  Effects of H2O2 with different concentrations on growth of yeast-like SD301

    图  3  SD301及其突变株的角鲨烯质量稳定性

    Figure  3.  Squalene mass stability of SD301 and its mutants

    图  4  SD301及其突变株PS120和PS150的角鲨烯生产性能分析

    Figure  4.  Performance analysis of SD301 and its mutants (PS120 and PS150)

    图  5  质粒和转化子的PCR验证

    Figure  5.  PCR validation of plasmid and transformants

    图  6  激光共聚焦显微镜验证增强型荧光蛋白EGFP在突变株PS120的表达水平

    Figure  6.  Fluorescence intensity of enhanced fluorescent protein (EGFP) in mutant strain PS120 observed by laser scanning confocal microscopy

    表  1  本研究中质粒构建所用引物

    Table  1.   Oligonucleotides used in vector construction

    引物
    Primer
    序列 (5'—3')
    Sequence (5'—3')
    Hph-F CGGGATCCATGAAAAAGCCTGAACTCACCGC
    Hph-R CCGCTCGAGTTCCTTTGCCCTCGGACGAG
    P-C-F GGTCGCGTTCCTGAAACGCAG
    P-C-R CAAGCAAGGTTTTCAGTATAAT
    P-hph-C-F ATATGTCCTGCGGGTAAATAGC
    P-hph-C-R ATGCCTCCGCTCGAAGTAGCGCGTC
    P-egfp-F ACTCGTCCGAGGGCAAAGGAAATGGTGAGCAAGGGCGAGGAG
    P-egfp-R CCCTCTAGATGCATGCTCGAGCTTGTACAGCTCGTCCATGCC
    P-egfp-C-F ATCGTCCGATCCGGAGCCGGGACTGT
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-10-10
  • 修回日期:  2022-01-27
  • 录用日期:  2022-01-28
  • 网络出版日期:  2022-02-14
  • 刊出日期:  2022-04-01

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