盐胁迫下小偃麦酵母双杂交文库构建及TtLEA2-1互作蛋白筛选

Construction of yeast two-hybrid library and screening of proteins interacting with TtLEA2-1 under salt stress in Tritipyrum

  • 摘要: 【目的】构建小偃麦酵母双杂交(Y2H)文库,筛选胚胎发育晚期丰富蛋白(TtLEA2-1)的互作蛋白,为探究该基因的功能和表达调控机理提供理论参考。【方法】以小偃麦Y1805为材料,经提取RNA、分离mRNA、cDNA合成和均一化(DSN)处理后进行BP (attB和attP位点)重组反应,把重组产物转化大肠杆菌DH10B感受态细胞,构建cDNA初级文库。抽提初级文库质粒,以pGADT7-DEST载体进行LR (attL或attR位点)重组反应,再转化大肠杆菌DH10B感受态细胞,构建Y2H文库。【结果】对初级文库和Y2H文库进行质量分析,其滴度分别是3.98×106 CFU/mL和6.14×106CFU/mL,库容量分别为7.96×106 CFU和1.23×107 CFU,重组率均为100%。从Y2H文库中挑取的24个单克隆的插入片段长度为850~4000 bp,平均片段长度大于1000 bp。采用Y2H技术和回转验证试验,从小偃麦Y2H文库中筛选了48个可能与TtLEA2-1互作的蛋白。48个TtLEA2-1互作蛋白中,有7个互作蛋白富集在遗传信息处理通路,4个蛋白富集在蛋白质家族:遗传信息过程通路,4个蛋白富集在核苷酸代谢通路,3个蛋白富集在甘氨酸生物合成和代谢通路,2个蛋白富集在碳水化合物代谢通路;富集在蛋白质家族:代谢、细胞过程、环境信息处理、氨基酸代谢通路的蛋白各有1个。【结论】构建的Y2H文库质量高、完整性好和覆盖面广,成功用于小偃麦盐胁迫响应基因互作蛋白的筛选试验,也为新基因发掘及其表达调控机理研究提供高效、便捷途径。

     

    Abstract: 【Objective】The purpose of the study was to construct Tritipyrum yeast two-hybrid(Y2H)library and screen proteins interacting with late embryogenesis-abundant protein(TtLEA2-1),so as to provide theoretical reference for exploring the function and expression regulation mechanism of this gene.【Method】Using Tritipyrum Y1805 as the material,after RNA extraction,mRNA isolation,cDNA synthesis,and normalization(DSN)treatment,BP(attB and attP sites)recombination reaction were carried out,which was transformed into Escherichia coli DH10B competent cells to construct a cDNA primary library. The primary library plasmid was extracted,and the pGADT7-DEST vector was used for LR(attL or attR sites)recombination reaction,and transformed into E. coli DH10B competent cells to construct a Y2H library.【Result】Quality analysis of primary library and Y2H library showed that their titers were 3.98×106 CFU/mL and 6.14×106 CFU/mL repectively,and their library capacity were 7.96×106 CFU and 1.23×107 CFU respectively,and their recombination rate was 100%. The insertion fragments of 24 single clones selected from the Y2H library were 850-4000 bp in length,and the average fragment length was more than 1000 bp. Using Y2H technology and reversion verification tests,48 proteins that might interact with TtLEA2-1 were screened from Tritipyrum Y2H library. Among the 48 TtLEA2-1 interacting proteins,7 interacting proteins were enriched in the genetic information processing pathway,4 proteins were enriched in the protein family:genetic information process pathway,4 proteins were enriched in the nucleotide metabolism pathway,3 proteins were enriched in the glycine biosynthesis and metabolism pathway,2 proteins were enriched in the carbohydrate metabolism pathway;and 1 protein was enriched in each of the protein family:metabolism, cellular process,environmental information processing,and amino acid metabolism pathways.【Conclusion】The constructed Y2H library is of high quality,good integrity and wide coverage,which is successfully used for the screen test of salt stress responsive genes interacting proteins in Tritipyrum,and also provides an efficient and convenient way for the research on the discovery of new genes and their expression regulation mechanism.【Objective】The purpose of the study was to construct Tritipyrum yeast two-hybrid(Y2H)library and screen proteins interacting with late embryogenesis-abundant protein(TtLEA2-1),so as to provide theoretical reference for exploring the function and expression regulation mechanism of this gene.【Method】Using Tritipyrum Y1805 as the material,after RNA extraction,mRNA isolation,cDNA synthesis,and normalization(DSN)treatment,BP(attB and attP sites)recombination reaction were carried out,which was transformed into Escherichia coli DH10B competent cells to construct a cDNA primary library. The primary library plasmid was extracted,and the pGADT7-DEST vector was used for LR(attL or attR sites)recombination reaction,and transformed into E. coli DH10B competent cells to construct a Y2H library.【Result】Quality analysis of primary library and Y2H library showed that their titers were 3.98×106 CFU/mL and 6.14×106 CFU/mL repectively,and their library capacity were 7.96×106 CFU and 1.23×107 CFU respectively,and their recombination rate was 100%. The insertion fragments of 24 single clones selected from the Y2H library were 850-4000 bp in length,and the average fragment length was more than 1000 bp. Using Y2H technology and reversion verification tests,48 proteins that might interact with TtLEA2-1 were screened from Tritipyrum Y2H library. Among the 48 TtLEA2-1 interacting proteins,7 interacting proteins were enriched in the genetic information processing pathway,4 proteins were enriched in the protein family:genetic information process pathway,4 proteins were enriched in the nucleotide metabolism pathway,3 proteins were enriched in the glycine biosynthesis and metabolism pathway,2 proteins were enriched in the carbohydrate metabolism pathway;and 1 protein was enriched in each of the protein family:metabolism, cellular process,environmental information processing,and amino acid metabolism pathways.【Conclusion】The constructed Y2H library is of high quality,good integrity and wide coverage,which is successfully used for the screen test of salt stress responsive genes interacting proteins in Tritipyrum,and also provides an efficient and convenient way for the research on the discovery of new genes and their expression regulation mechanism.

     

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