Liang Hao, Sun Hai, Shao Cai, Lyu Bo-Chen, Cao Wei-Yu, Long Hong-Ju, Zhang Ya-Yu
Jilin Provincial Key Laboratory of Traditional Chinese Medicinal Materials Cultivation and Propagation, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences Changchun 130112, China.
Jilin Provincial Key Laboratory of Traditional Chinese Medicinal Materials Cultivation and Propagation, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences Changchun 130112, China College of Food and Biological Engineering, Chengdu University Chengdu 610106, China.
Zhongguo Zhong Yao Za Zhi. 2024 Nov;49(22):6107-6118. doi: 10.19540/j.cnki.cjcmm.20240813.102.
To construct a high-quality Panax ginseng cDNA library, transcription factors binding to the P. ginseng PgD14 gene promoter were screened by yeast one-hybrid, and proteins interacting with the P. ginseng Pgpht2-1 gene-encoded protein were screened by yeast two-hybrid. In this study, root tissues of P. ginseng were used as materials. Gateway technology was used to construct the P. ginseng yeast one-hybrid library, and duplex-specific nuclease(DSN) homogenization technology was used to construct the P. ginseng yeast two-hybrid library. The pAbAi-PgD14-Pro961 vector was used as bait to screen candidate transcription factors that might bind to the PgD14 gene promoter from the yeast one-hybrid library, and the pGBKT7-Pgpht2-1 vector was used as bait to screen candidate proteins that might interact with the Pgpht2-1 gene-encoded protein from the yeast two-hybrid library. The yeast one-hybrid library had a size of 1.20×107 CFU, a recombination rate of 100%, and an average inserted fragment length of more than 1 000 bp. The yeast two-hybrid library had a size of 1.832×105 CFU, a recombination rate of 100%, and an average inserted fragment length of about 1 000 bp. The recombinant vectors pAbAi-PgD14-Pro961 and pGBKT7-Pgpht2-1 were transformed into Y1HGold and AH109 strains, respectively, and interacting proteins were screened by yeast one-hybrid and yeast two-hybrid. As a result, 54 transcription factors that could bind to the PgD14 gene promoter of P. ginseng and 42 proteins that may interact with the protein encoded by the Pgpht2-1 gene were identified. This study successfully constructed the P. ginseng yeast one-hybrid and yeast two-hybrid cDNA libraries, laying a foundation for subsequent studies on the functions of the P. ginseng PgD14, Pgpht2-1, and other genes.
为构建高质量的人参cDNA文库，通过酵母单杂交筛选与人参PgD14基因启动子结合的转录因子，并通过酵母双杂交筛选与人参Pgpht2-1基因编码蛋白相互作用的蛋白质。本研究以人参根组织为材料，采用Gateway技术构建人参酵母单杂交文库，采用双链特异性核酸酶（DSN）均一化技术构建人参酵母双杂交文库。以pAbAi-PgD14-Pro961载体为诱饵，从酵母单杂交文库中筛选可能与人参PgD14基因启动子结合的候选转录因子；以pGBKT7-Pgpht2-1载体为诱饵，从酵母双杂交文库中筛选可能与人参Pgpht2-1基因编码蛋白相互作用的候选蛋白。酵母单杂交文库库容为1.20×10⁷ CFU，重组率为100%，平均插入片段长度大于1000 bp。酵母双杂交文库库容为1.832×10⁵ CFU，重组率为100%，平均插入片段长度约为1000 bp。将重组载体pAbAi-PgD14-Pro961和pGBKT7-Pgpht2-1分别转化到Y1HGold和AH109菌株中，通过酵母单杂交和酵母双杂交筛选相互作用蛋白。结果，鉴定出54个可能与人参PgD14基因启动子结合的转录因子和42个可能与人参Pgpht2-1基因编码蛋白相互作用的蛋白。本研究成功构建了人参酵母单杂交和酵母双杂交cDNA文库，为后续人参PgD14、Pgpht2-1等基因功能研究奠定了基础。
