| 陈 凤，闫晋强，李梅兰，江 彪.冬瓜均一化酵母双杂交 cDNA 文库的构建及鉴定[J].广东农业科学,2021,48(8):58-64 |
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冬瓜均一化酵母双杂交 cDNA 文库的构建及鉴定 |
| Construction and Identification of a Homogenized Yeast Two-hybrid cDNA Library in Benincasa hispida |
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| DOI：10.16768/j.issn.1004-874X.2021.08.007 |
| 中文关键词: 冬瓜 DSN 均一化 cDNA 文库 酵母文库 |
| 英文关键词: wax gourd DSNhomogenization cDNA library yeast library |
| 基金项目:广东省重点领域研发计划项目（2020B020220003）；广东省基础与应用基础研究基金 - 粤穗联合基金（2020A1515111138）；广东省农业科学院科技创新战略专项资金（高水平农科院建设）- 人才项目（R2020PYJG003）；广东省蔬菜新技术研究重点实验室开放基金（201902） |
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| 中文摘要: |
| 【目的】通过构建冬瓜酵母双杂交 cDNA 文库，为开展冬瓜基因功能研究提供技术支撑。【方
法】以冬瓜高代自交系 B227（冬瓜参考基因组测序材料）的根、茎、叶、雄花、嫩果等不同组织为材料，利
用 CTAB 法提取 RNA、分离纯化获得 mRNA 并进行双链 cDNA 合成和 DSN 均一化处理后，与 pDONR222 载
体进行 BP 重组反应，转化大肠杆菌 DH10B 感受态细胞，构建初级文库；提取初级文库质粒，与 pGADT7-
DEST 载体通过 LR 重组反应，转化大肠杆菌 DH10B 感受态细胞，构建酵母杂交 cDNA 次级文库。利用倍比
稀释法计算文库库容量，并通过菌落 PCR 计算文库重组率和插入片段的大小。【结果】初级文库库容量为
8.24×106
CFU，冬瓜均一化酵母双杂交次级 cDNA 文库库容量为 1.03×107
CFU；次级文库中插入片段主要集
中在 850~3 000 bp，重组率为 100%，具有良好的多态性。【结论】成功构建了冬瓜均一化酵母双杂交 cDNA
文库，文库完整性较高、质量较好，符合酵母双杂交实验要求，可应用于后续相关基因产物互作蛋白的筛选
等试验，为冬瓜基因功能研究提供前提条件。 |
| 英文摘要: |
| 【Objective】The study was carried out to provide technical support for the researches on gene function
of wax gourd by constructing a wax gourd yeast two-hybrid cDNA library.【Method】The roots, stems, leaves, male
flowers and tender fruits of the wax gourd inbred line B227（wax gourd cultivar used for genome sequencing ）were used
as materials to extract RNA by using the CTAB method. Then, the extracted RNA was used to isolate and purify mRNA,
and double-stranded cDNA synthesis and DSN homogenization treatment were carried out. Later, cDNA was ligated with
pDONR222 vector through BP recombination and transformed into E.coli DH10B competent cells to construct a primary library. LR recombination reaction was performed with plasmid extracted from primary library and pGADT7-DEST vector,
and the products were transformed into E.coli DH10B competent cells to construct a secondary yeast two-hybrid cDNA
library. The library capacity was calculated by the multiple dilution method, and the recombination rate and the sizes of
the inserted fragments were calculated by colony PCR.【Result】The library quality test showed that the primary library
capacity was 8.24×106
CFU, and the homogenized secondary yeast two-hybrid cDNA library capacity of wax gourd was
1.03×107
CFU. The insert fragments mainly concentrated between 850 bp and 3 000 bp with good polymorphism and the
recombination rate was 100%.【Conclusion】A homogenized yeast two-hybrid cDNA library in wax gourd was successfully
constructed. The library is highly integrated and of good quality, which meets the requirements of yeast two-hybrid
experiments. The library can be used in related experiments such as screening of interaction proteins, laying a foundation for
the study on gene function of wax gourd. |
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