| 剑麻酵母双杂交cDNA表达文库的构建及与AhKNOX2相互作用蛋白的筛选 |
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| 引用本文： | 杨子平,孙艺桓,杨倩,鹿志伟,张燕梅,李俊峰,周文钊. 剑麻酵母双杂交cDNA表达文库的构建及与AhKNOX2相互作用蛋白的筛选[J]. 热带作物学报, 2020, 41(9): 1748-1755. DOI: 10.3969/j.issn.1000-2561.2020.09.004 |
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| 作者姓名： | 杨子平 孙艺桓 杨倩 鹿志伟 张燕梅 李俊峰 周文钊 |
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| 作者单位： | 1.中国热带农业科学院南亚热带作物研究所/湛江市热带作物遗传改良重点实验室,广东湛江 5240912.中国农业科学院蔬菜花卉研究所,北京 100081 |
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| 基金项目： | 现代农业产业技术体系建设专项资金项目(CARS-16);国家自然科学基金青年基金项目(31801679);中国热带农业科学院基本科研业务费专项资金项目(1630062018010) |
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| 摘 要： | 植物KNOX（Knotted1-like homeobox）可通过蛋白质相互作用参与调控落果、纤维细胞发育、次生细胞壁发育、开花等事件。为筛选剑麻叶中与AhKNOX2相互作用的蛋白,采用SMART同源重组技术构建剑麻叶片酵母双杂交表达文库,利用AhKNOX2为诱饵筛选与之相互作用的蛋白。结果表明：文库总容量为3.9×106 CFU,转化效率为9.15×105 CFU/μg pGADT7-Rec,插入片段大小主要分布于0.5~3.0 kb之间,重组率为92%,保存的文库细胞密度为1.35×108/mL,文库滴度为2.22×107CFU/mL。构建的pGBKT7-AhKNOX2诱饵载体存在自激活性,5 mmol/L的3-氨基- 1,2,4-三唑（3-Amino-1,2,4-triazole,3-AT）能抑制诱饵载体的自激活,通过筛选获得了16个与AhKNOX2相互作用的蛋白。本研究成功构建剑麻叶片酵母表达文库,并筛选获得与AhKNOX2相互作用的蛋白,该结果为进一步研究AhKNOX2在剑麻中的功能奠定前期基础,为培育高纤维产量的剑麻新品种提供基因资源和理论基础。
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| 关 键 词： | 剑麻 AhKNOX2 酵母双杂交 cDNA文库 蛋白质相互作用 |
| 收稿时间： | 2019-11-08 |
Construction of Yeast Two-hybrid Library of Agave hybrid No. 11648 and Screening of Proteins Interacting with AhKNOX2 |
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| YANG Ziping,SUN Yihuan,YANG Qian,LU Zhiwei,ZHANG Yanmei,LI Junfeng,ZHOU Wenzhao. Construction of Yeast Two-hybrid Library of Agave hybrid No. 11648 and Screening of Proteins Interacting with AhKNOX2[J]. Chinese Journal of Tropical Crops, 2020, 41(9): 1748-1755. DOI: 10.3969/j.issn.1000-2561.2020.09.004 |
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| Authors: | YANG Ziping SUN Yihuan YANG Qian LU Zhiwei ZHANG Yanmei LI Junfeng ZHOU Wenzhao |
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| Affiliation: | 1. South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences / Zhanjiang City Key Laboratory for Tropical Crops Genetic Improvement, Zhanjiang, Guangdong 524091, China2. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China |
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| Abstract: | To obtain the proteins interacting with AhKNOX2 in Agave hybrid No.11648, a full length-expression cDNA library was constructed in yeast strain Y187 using homologous recombination-mediated SMART technology. The capacity of the cDNA library was 3.9×10 6CFU and the transformation efficiency was about 9.15×105 CFU/μg pGADT7-Rec. The PCR amplification of 24 clones randomly selected from the cDNA library indicated that the length of most inserts was ranged from 0.5 kb to 3.0 kb, with a recombination rate of 92%. The cell density was 1.35×108/mL and the titer was up to 2.22×107CFU/mL. The bait vector (pGBKT7-AhKNOX2) was transformed into yeast strain Y2HGold, colonies appeared on SD/-Trp/-His media and was inhibited by 5 mmol/L 3-AT. After screening the library using bait plasmids,a total of 16 proteins interacting with AhKNOX2 were obtained by sequencing and homology analysis. In conclusion, a high-quality cDNA library was constructed and 16 proteins interacting with AhKNOX2 were screened from cDNA library. |
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| Keywords: | Agave hybrid AhKNOX2 yeast two-hybrid cDNA library protein-protein interaction |
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