Construction Of Expression Vector, Purification And Identification The Target Genes Of NAC Transcription Factor In Tomato

The NAC transcription factor comprise a large plant specific transcriptional factors family,considerable progress has been made to reveal the biological function and regulatory network of NAC family in current studies,it is of significance to separate the NAC protein from tomato and study the regulation network of NAC transcription factor.SlNAC1-74 had been isolated in the previous study,and SNAC4-9 proteins may relate to tomato development and ripening.In this study,the prokaryotic expression vector pET-SNAC4/5/7/8/9 were constructed to express proteins SNAC4-9,and then proteins SNAC4/9 were purified to figure out the regulation mechanism of SNAC4/9 transcription factor involved in ethylene biosynthesis.The results of the study are as follows:1.The physicochemical property of proteins SNAC4-9 was analyzed,SNAC4/5/6/9 were basic protein and SNAC7/8 were acidic protein,the cis-acting elements of SNAC4-9 were also analyzed,the results showed that the promoter regions of SNAC4-9 included the basic transcription components such as TATAbox and CAATbox,there were also salt,drought and heat stress cis-acting elements,the presence of these elements revealed that SNAC4-9 may involved in the biotic and abiotic stress.Ethylene response element,ABA and GA response elements in promoter regions of SNAC4-9 indicated that these hormones can induce the expression of SNAC4-9.2.The prokaryptic expression vectors pET-SNAC4/5/7/8/9 were successful constructed,exogenous inducer IPTG was utilized to express the recombinant SNA C4/5/7/8/9,SNAC4/7/8/9 were inclusion body proteins and SNAC5 was soluble protein,after the optimizate of the expression program,we found that lowering temperature and the concentration of IPTG,prolonging induction time could incude the expression of solube protein only if the protein was colube.3.By using Ni column,we purified proteins SNAC4/9 with His-tag in the N-terminal based on affinity interaction between Ni and His-tag,and then we refolded it in order to improve the fineness and bioactivity of fusion protein.Also,gel was cutted to purified proteins SNAC4/9.4.EMSA was performed to indentify the combination of recombinant SNAC4/9 and probe LeACO1,LeACO4,LeACS2 and LeACS4 in vitro.The results indicated that SNAC4 specifically binding to the promoter region of probes LeACS2 and LeACS4,and it has more binding capacity with probe LeACS2.And SNAC9 has ability to combine probes LeACO1,LeACO4,LeACS2 and LeACS4.Together,these data indicated that the SNAC4/9 proteins bind to the promoter of ACS and ACO in vitro.Therefore,transcription factor SNAC4 affects maturity of tomato fruit through regulating the expression of LeACS2 and LeACS4 as key genes of ethylene synthesis,and SNAC9 can regulate the expression of LeACO1,LeACO4,LeACS2 and LeACS4 to modulate the biosynthesis of ethylene.