Analysis Of CsTCTP Regulated Signal Pathway And Construction Of Yeast Two-Hybrid System In Cucumber

Powdery mildew of Cucumber[Sphaerotheca fuliginea(Schlecht)Poll.]is a widespread leaf disease,often causing great economic losses.At present,in addition to the application of chemical method to control powdery mildew,mainly rely on the breeding of resistant varieties,but because of long-term cultivation,plant resistance levels have gradually decreased.Therefore,it is very important to study the molecular mechanism of cucumber and find new resistance genes in the process of powdery mildew infection.The previous study indicated that translational controlled tumor protein CsTCTP1 plays an important role in response to powdery mildew infection process and may involve in ABA,TOR and ethylene pathway.But how CsTCTP1 perform its function to response to powdery mildew is not clear.Based on this,the function and structure of CsTCTP was analyzed by bioinformatics and the real-time fluorescence quantitative PCR technique was used to detect the expression of key genes in the TCTP-related signal pathway under the stress of powdery mildew.The CsTCTP bait expression vector was constructed and its toxicity and self-activation activity were detected.The experimental basis was established for the screening of CsTCTP interacting protein by yeast two-hybrid system.The main results of this study are as follows:(1)Bioinformatics analysis indicated cucumber two TCTP proteins(CsTCTP1 and CsTCTP2),CDS were 504 bp,encoding 168 amino acid and the polypeptide chains had two TCTP characteristic domains(TCTP-1 and TCTP-2),the proteins located in the cytoplasmic.CsTCTP proteins were hydrophilic acidic proteins.The alpha helix was the main structure of the secondary structure of CsTCTP proteins.The function analysis showed that CsTCTP was involed in amino acid biosynthesis,translation and energy metabolism.The phylogenetic tree of TCTP proteins from different plant was constructed,indicating that CsTCTP1 has recent relationship with cucumber melon.The TCTP amino acid sequence alignment of different plants was 85.96%,and had the same conserved domain.(2)TOR signal pathway gene TOR,SnRK1,ABA signal pathway genes PP2C,SnRK2.1,ABI5 and ethylene signal pathway genes CTR1,EIN2 were able to responds to powdery mildew infection.The trend of gene expression levels is consistent with CsTCTP1 responding to powdery mildew infection.It is speculated that CsTCTP may be involved in TOR,ABA and ET signals under the biotic stress in cucumis sativus.(3)The CDS region of CsTCTP1 and CsTCTP2 were cloned,and were recombined into the bait expression vector pGBKT7,produced PGBKT7-CsTCTP1 and pGBKT7-CsTCTP2.The recombinant vectors were successfully transformed into the yeast cells(Y2HGold).The results showed that the fusion protein have no cytotoxicity and no self-activating activity,which will lay the foundation for further study of the interaction mechanism of cucumber TCTP.(4)Overexpression vector was constructed.The CsTCTP coding region was constructed into the overexpression vector pB7FWG2 by Gateway technique.The sequencing results showed that the coding vector was identical with the known sequence,and the overexpression vector contained GFP tag,which could be used for the subsequent experimental materials of subcellular localization,transient and stable expression.