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| 基于流式细胞仪分析的欧洲云杉原胚团细胞核提取液筛选 |
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胡继文1,2, 高晗3, 王军辉1,2, 朱天擎2
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1. 中国林业科学研究院林业研究所,国家林业和草原局林木培育重点实验室,国家林木种质资源平台,北京 100091；2. 林木遗传育种全国重点实验室,中国林业科学研究院,北京 100091； 3. 三峡植物园管理处,湖北 宜昌 443111
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| 摘要: |
| 细胞核悬液是开展多种分子生物学研究的重要基础。为高效制备欧洲云杉（Picea abies）原胚团（proembryogenic masses，PEM）细胞核悬液，该研究应用流式细胞仪检测DNA含量，对LB01、Otto’s、Galbraith’s、GPB、WPB和Tris·MgCl2 6种细胞核提取液提取的欧洲云杉胚性细胞系PaVIII、PI3、PI5PEM细胞核数量和质量进行评估，筛选最适提取液，并对最适提取液稳定性、细胞核完整性进行检测。结果表明：（1）与其他提取液相比，欧洲云杉PEM经LB01提取获得的细胞核数量最多、细胞碎片少。（2）PEM经LB01提取获得的细胞核G0/G1、G2/M核变异系数均小于5%，变异系数小。（3）不同欧洲云杉细胞系PEM经LB01提取的细胞核数量及细胞碎片无显著差异，稳定性高。（4）显微观测显示，LB01提取欧洲云杉PEM获得的细胞核膜保持较高的完整性。综上认为，LB01提取欧洲云杉PEM细胞核能表现出更好的核质量，可作为欧洲云杉首选细胞核提取液。该研究为裸子植物细胞核的提取提供了参考。 |
| 关键词: 针叶树，欧洲云杉，原胚团，植物细胞核提取，流式细胞仪 |
| DOI：10.11931/guihaia.gxzw202401002 |
| 分类号: |
| 基金项目:国家重点研发计划项目（2023YFD2200104）；中国林业科学研究院中央级公益性科研院所基本科研业务费专项资金（CAFYBB2023MB041）；国家资助博士后研究人员计划（GZC20233017）；林木遗传育种全国重点实验室基本科研业务费(TGBFRF202301) |
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| Screening of the optimum nuclei extraction buffer for Picea abies proembryogenic masses based on flow cytometry analysis |
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Hu Jiwen1,2, Gao Han3, Wang Junhui1,2, Zhu Tianqing1.2
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1. State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of State Forestry and Grassland Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China; 2. State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing, 100091, China; 3. Management Office of Three Gorges Botanical Garden, Yichang 443111, Hubei, China
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| Abstract: |
| Nuclear suspension is an important foundation for a variety of molecular biology researches. To effectively prepare the nuclear suspensions of Norway spruce (Pice aabies) proembryogenic masses (PEM), PEMs of Norway spruce embryonic cell lines PaVIII, PI3, and PI5were used as the material for nuclear extraction with six lysis buffers: (LB01, Otto’s, Galbraith’s, GPB, WPB and Tris·MgCl2). Based on detecting DNA content by flow cytometry, the quantity and quality of the nuclei suspensions were evaluated to screen for the optimal buffer. Further, the integrity of nucleus and the stability of optimal buffer were measured. The results were as follows: (1) Compared with the other lysates, the number of nuclei obtained of PaVIII line PEMs from LB01 was the highest, but lower amount of cell debris. (2) In addition, coefficients of variation (CVs) of G0/G1 and G2/M nuclei, obtained from LB01, were not more than 5%. The CVs were relatively low. (3) The nuclei of different lines PEMs were extracted by LB01. There was no significant difference in the efficiency of nucleus extraction and the proportion of cell debris among the different lines. (4) The nuclear membrane of most cells maintained in a high integrity when the nuclei of Norway spruce PEMs were extracted via LB01 through morphology investigation. To sum up, compared with other buffers, LB01 is the optimal one for Norway spruce PEMs nuclear extraction, which might provide reference for nuclear extraction from gymnosperms. |
| Key words: Conifer, Picea abies, proembryogenic masses, plant cell nucleus extraction, flow cytometry |
