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

【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.