摘要
为构建高质量的人参cDNA文库,并通过酵母单杂交文库筛选结合人参PgD14基因启动子的转录因子,通过酵母双杂交文库筛选与人参Pgpht2-1基因编码蛋白发生互作的蛋白。该研究以人参的根部组织为材料,利用Gateway技术构建人参酵母单杂交文库,利用特异性核酸酶(duplex-specific nuclease,DSN)均一化技术构建人参酵母双杂交文库,并以pAbAi-PgD14-Pro961为诱饵载体从酵母单杂交文库中筛选了可能结合PgD14基因启动子的候选转录因子,以pGBKT7-Pgpht2-1为诱饵载体从酵母双杂交文库中筛选了可能与Pgpht2-1基因编码蛋白发生互作的候选蛋白。通过文库鉴定,检测到酵母单杂交文库的库容为1.20×10^(7)CFU,重组率为100%,插入片段平均长度大于1000 bp;酵母双杂交文库的库容为1.832×10^(5)CFU,重组率为100%,插入片段平均长度在1000 bp左右。分别将pAbAi-PgD14-Pro961和pGBKT7-Pgpht2-1重组载体转化Y1HGold和AH109菌种,通过酵母单杂交和双杂交进行互作蛋白的筛选,最终筛选到54个可能结合到人参PgD14基因启动子的转录因子,以及42个可能与人参Pgpht2-1基因编码蛋白发生互作的蛋白。该研究同时构建了人参酵母单杂交与酵母双杂交的cDNA文库,为后续人参PgD14、Pgpht2-1等基因功能的研究奠定了基础。
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 1.20×10^(7)CFU,a recombination rate of 100%,and an average inserted fragment length of more than 1000 bp.The yeast two-hybrid library had a size of 1.832×10^(5)CFU,a recombination rate of 100%,and an average inserted fragment length of about 1000 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.
作者
梁浩
孙海
邵财
吕柏辰
曹炜玉
龙泓桔
张亚玉
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;College of Food and Biological Engineering,Chengdu University,Chengdu 610106,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2024年第22期6107-6118,共12页
China Journal of Chinese Materia Medica
基金
财政部和农业农村部国家现代农业产业技术体系项目(CARS-21)
中国农业科学院科技创新工程项目(CAAS-ASTIP-2021-ISAPS)。
关键词
人参
酵母单杂交
酵母双杂交
互作蛋白
Panax ginseng
yeast one-hybrid
yeast two-hybrid
interacting protein
