Construction Of Yeast Two-hybrid CDNA Library Of Pear Fruit And Identification Of Protein Interacting With Key Protein BZR1 In The BRs Signaling Pathway

The length of storage time of the fruit has a close relationship with the level of ethylene production. 'Nanguo' pear (Pyrus ussuriensis) is a climacteric fruit ripening by ethylene regulation. In recent years, more reports have confirmed that ethylene through interaction with BRs (Brassinolide) affects fruit maturation and storage, however the current studies unclear reveal the regulatory network between ethylene and BRs. We have found that ethylene production of'Nanguo'pear reduced during.storage in the treatment of 0.1mg·L-1 EBR (Brassinolide). In order to clarify the relationship between BRs and 'Nanguo' pear storage, in the transcriptome of'Nanguo'pear climacteric around (the maturity and after 10 days storage at room temperature), We have found 5 BR synthesis and signal transduction pathway related genes that are BRU1-like, BES1/BZR1-like1, BES1/BZR1-like4, CYP450-90A1, BZRl differentially expressed. It is found that BZR1 gene is induced by ethylene inhibitor (1-MCP) though semi-quantitative PCR analysis. For further study the role of BZR1 in fruit maturation, we have constructed a cDNA library of 'Nanguo' pear fruit and in BZRl performed yeast two-hybrid screening as bait. The test results are as follows:1. It is found that 5 BR synthesis andsignal transduction pathway-related genes (BRU1-like, BES1/BZR1-like1, BES1/BZR1-like4, CYP450-90A1, BZR1) differentially expressed by comparison transcriptome analysis of 'Nanguo' pear mature around. It is identified that BZRl gene is promoted by the ethylene inhibitor (1-MCP) through analysis fruits gene expression of'Nanguo'pear under the treatment of ethephon and 1-MCP.2. Construction cDNA library of the 'Nanguo' pear fruit is calculated storage capacity of 3×106 cfu and the conversion efficiency of 7.89×104 cfu?g-1, library titer was 5.28×108 cfu·ml-1 and the average length of the insertion fragment is 780 bp, so richness and integrity of library meet the requirement, this shows that the constructed yeast cDNA library of 'Nanguo' pear reaches standards of yeast screening libraries. The library screening test can be carried out.3. By the yeast two-hybrid screening system finally obtained 135 positive clones were sequenced to obtain 24 different interacting proteins, we found the ACC oxidase (ACO1) as the next study, co-transformation test to verify that the ACO1 and BZR1 bait have an interaction relationship.4. BRs signal transduction pathway transcription factor BZR1 with ethylene biosynthesis ACC oxidase (ACO1) has an interaction relationship, it inhibits the ACC oxidase (ACO1) protein activity, to regulate ethylene production of 'Nanguo' pear fruit, and then affect fruit ripening.