Screening And Validation Of Interacting Proteins Of Citrus NAC036

As an important origin of citrus,China has a number of local varieties with good quality characteristics.In 2000,a bud mutation of Ougan was found in Ouhai station of Wen Zhou,its peel still remained green while fruit ripening,which was named ?Qing'Ougan?.The appearance of the bud mutation provides valuable experimental materials for our study in citrus fruit ripening and senescence processes.The NAC(NAM;ATAF1/2;CUC1/2)transcription factors family contains numerous menbers and are widely distributed in terrestrial plants.It is reported that NAC transcription factors are involved in a variety of biological processes.In this study,NAC036 transcription factor was amplified from Qing'Ougan by PCR,and screened the interacting proteins via yeast two-hybrid system and bimolecular fluorescence complementation.This study provides insights into the possible functions of NAC036,which would contribute to exploring the relationship between NAC036 and the phenotype of Qing'Ougan,and revealing the regulatory mechanism of NAC036 in ripening and postharvest senescence process.The main results were as follows:1.The cDNA of Qing'Ougan flavedo as the template which was 210 days after flowering,NAC036 and it's short sequences were amplified by PCR,bait fusion vectors were constructed by cloning these sequences into pGBKT7 vector.After self-activation and toxicity tests,we found that all the bait vectors were inactive in AH109 and Y187 yeast strains but both have different levels of toxic effects for them,and the toxicity affected the growth.of yeast strains.2.After mating,twice nutritional stress screening and NCBI blasting,we found 10 transcription factors interacted with NAC036,they are AP2?ZFN?BIM2?GTE6?NAC72?NAC78?TCP13?NAC2?NAC1 and NAC29 respectively,most of them were NAC family members and related to signal transduction for stress and hormone.These results were in accordance with the previous research results that NAC transcription factor formed by homologous or heterologous protein complexes,regulating plant growth and biological or abiotic stress.3.Point to point to verify the accuracy of interactions,the transformation of NAC29 and NAC036 did not show bule yeast colonies,the others show blue clonies,we speculated the interaction between NAC29 and NAC036 might be a false positive,the remaining nine candidate proteins with NAC036 exist the possibility of interactions,but still needs further validation.4.In the experiment of Bimolecular Fluorescence Complementation,YFP vectors was constructed by the method of Gateway.NACa-PCL112 is the N-terminal of YFP,and TFs-PCL113 is the C-terminal of YFP,transforming them into GV3101 Agrobacterium respectively,blending bacteria liquid and injecting tobacco,observing the epidermal cells near injection hole by Laser confocal microscopy.The results showed that in YFP excitation wavelengths,the leaves of the combination of NAC036 with NAC2 and NAC1 were not detected fluorescent signal,speculating that its interactions with the bait of the gene as a false positive,while the AP2,ZFN,BIM2,GTE6,NAC72,NAC78 and TCP13 with the combination of NAC036 have strong fluorescence signal in the nucleus,indicating a higher possibility of the bait protein interactions.5.Based on the results of Yeast Two-hybrid and Bimolecular Fluorescence Complementation,we speculated that NAC2,NAC1 and NAC29 interacting with NAC036 are false positive,while AP2,ZFN,BIM2,GTE6,NAC72,NAC78 and TCP13 with NAC036 existed interaction.These results provided some ideas for further laboratory research.