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

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

肖艳^1,,
王璐²,
王森¹,
丛培虎³,
陆栋⁴,
冯银刚¹,
崔球¹,
宋晓金^1, ,

1.
中国科学院生物燃料重点实验室/山东省合成生物学重点实验室/山东省能源研究院/中国科学院青岛生物能源与过程研究所，山东青岛 266101
2.
山东大学澳国立联合理学院，山东威海 264209
3.
山东省科技服务发展推进中心，山东济南 250101
4.
中国科学院近代物理研究所，甘肃兰州 730000

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
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- 文章访问数: 76
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- 被引次数: 0
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

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

1.
Key Laboratory of Biofuels, Chinese Academy of Sciences/Shandong Provincial Key Laboratory of Synthetic Biology/Shandong Energy Institute/Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China
2.
SDU-ANU Joint Science College, Shandong University, Weihai 264209, China
3.
Shandong Science and Technology Service Development and Promotion Center, Jinan 250101, China
4.
Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China

摘要

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。
- 角鲨烯 /
- 类酵母 /
- 碳离子束 /
- 诱变 /
- 电转化
Abstract: In order to improve the yield of squalene, using Pseudozyma sp. SD301 as the starting strain, we selected the mutant strain with high squalene yield by carbon-ions (¹²C⁶⁺) beam irradiation technology, and optimized the electrotransformation conditions of the mutant strain. We used 100 μmol·L⁻¹ H₂O₂ as the screening pressure. When the carbon heavy ion beam irradiation dose was 120 Gy, the mutant PS120 was obtained with higher squalene yield than the original strain. After 3 days of culture, the squalene yield of the mutant reached 1.33 g·L⁻¹ and the squalene mass reached 41.31 mg·g⁻¹. The green fluorescent protein EGFP was used as the characterization marker to optimize the electrotransformation conditions of PS120. The results of enzyme digestion, electrophoresis and sequencing show that the gene encoding EGFP could be successfully transferred into PS120 cells under 900 V voltage. The high-level expression of EGFP protein in PS120 cells was further confirmed by laser confocal microscope.
- Squalene /
- Pseudozyma /
- Carbon-ions beam /
- Mutagenesis /
- Electrotransformation

HTML全文

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

下载: 全尺寸图片幻灯片

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

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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

下载: 全尺寸图片幻灯片

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)

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图 5 质粒和转化子的PCR验证

Figure 5. PCR validation of plasmid and transformants

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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

下载: 全尺寸图片幻灯片

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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