盐胁迫下细叶百合酵母文库构建及LpNAC14互作蛋白筛选  

Construction of Yeast Library and Under Salt Stress in Lilium pumilum Screening of LpNAC14 Interacting Proteins

作　　者：刘同非 李徐斐 车海涛 张彦妮[1] LIU Tongfei;LI Xufei;CHE Haitao;ZHANG Yanni(College of Landscape Architecture,Northeast Forestry University,Harbin 150040,Heilongjiang,China)

机构地区：[1]东北林业大学园林学院,黑龙江哈尔滨150040

出　　处：《西北林学院学报》2025年第1期32-41,共10页Journal of Northwest Forestry University

基　　金：黑龙江省自然科学基金项目(LH2019C004)。

摘　　要：本研究旨在探索细叶百合NAC转录因子LpNAC14应答盐胁迫的信号通路,分析其互作蛋白,奠定相关基因功能研究基础。利用盐胁迫下细叶百合的叶片和根,通过Gateway法构建了酵母双杂交cDNA文库,使用2个无自激活活性的LpNAC14基因片段构建诱饵载体,分别与文库质粒共转化筛选互作蛋白,对筛选到的结果进行回转验证和GO富集分析,选取其中一个转录因子LpDi19-2与LpNAC14进行BiFC验证。结果表明,构建细叶百合酵母文库容量1.12×10^(7) CFU,重组率100%,插入片段平均长度1 000 bp以上。文库共转化初步筛选到19个与LpNAC14互作的蛋白。BiFC验证LpDi19-2与LpNAC14体内互作。研究表明盐胁迫下细叶百合酵母文库质量较高,符合筛选标准,筛选结果可靠性较高,为探究LpNAC14响应盐胁迫机制提供新方向。This study aimed to investigate the signaling pathway of LpNAC14,a NAC transcription factor in Lilium pumilum,in response to salt stress,to analyze the protein interaction,so as to provide a basis for the further study of the functions of the related genes.RNA collected from the leaves and roots of L.pumilum under salt stress was used to construct a yeast two-hybrid cDNA library using the Gateway method.Two LpNAC14 gene fragments without self-activating activity were used to construct bait vectors,and then they were co-transformed into the library plasmids to screen for interaction proteins.The screened results were subjected to yeast validation and GO enrichment analysis,and one of the transcription factors,LpDi19-2,was selected for BiFC validation with LpNAC14.The yeast library was constructed with a library capacity of 1.12×10^(7) CFU,with a recombination rate of 100%,and the average length of the inserted fragments was greater than 1000 bp.Nineteen proteins potentially interacting with LpNAC14 were preliminarily screened by library transformation.BiFC verification test showed that LpDi19-2 interacted with LpNAC14 in vivo.It is concluded that the quality of the constructed yeast library of L.pumilum under salt stress is high,which can meet the screening criteria,so that the screening results are reliable.This study provides a new way to explore the mechanism of LpNAC14 in response to salt stress.

关 键 词：细叶百合 cDNA文库 NAC转录因子 盐胁迫 BIFC 

分 类 号：S644.1[农业科学—蔬菜学]