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

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。

Construction of Yeast Two-hybrid Library of Agave hybrid No. 11648 and Screening of Proteins Interacting with AhKNOX2

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.