Screening And Functional Analysis Of Up-Stream Regulatory Factors Of Pear DAM Gene

Pear is a kind of temperate deciduous fruit trees.Plants have to experience dormancy to survive in the extremely low temperature during winter.The release of dormancy of pear trees requires a certain period of chilling accumulation.If the chilling accumulation is insufficient during dormancy period,it will affect the growth and development of pear trees in the coming year,which will not only reduce the yield and quality of pear and even seriously endangers the tree life.Previous studies have found that the DAM genes,which belong to the SVP subfamily of the MADS-box transcription factor family,play a key regulatory role in the dormancy process of Rosaceae crops.Although studies focusing on DAM and its regulatory mechanisms have made some progress,there are still many unsolved problems.In order to clarify the specific regulation mode of DAM gene in dormancy,here we used ‘Dangshansu' pear floral buds as the plant material.Meanwhile,we used the promoter of PpyMADS71(PpyDAM3),which was previously identified as a key gene during bud endoodrmancy,to do yeast one-hybrid library screening and several upstream transcriptional factors were identified.The specific work and main results are as follows:1.Exploring the upstream transcription factors of PpyMADS71 in pearUsing yeast one-hybrid library screening method,three new transcription factors PpyERF060,PpyGBF,and PpyRAP2.4 that could interact with PpyMADS71 promoter were highlighted.Then,transcriptional abundance of these three genes during the dormancy cycle of ‘Dangshansu' pear was identified.We also conducted subcellular localization,yeast transcriptional activation assay and dual-luciferase assays to discover the possible functions of these proteins.It was primarily determined that the transcription factor PpyERF060,which was located in the nucleus of cells,was upregulated during the endo-dormancy stage,and can activate the expression of PpyMADS71 and may function in the upstream of PpyMADS71.2.Regulatory role of PpyERF060 on the promoter of PpyMADS71The yeast binding site mutation assays and the dual-luciferase assays were used to confirm that PpyERF060 could bind to the DRE1 element of the PpyMADS71 promoter.The result of transgenic pear calli further proved that PpyERF060 activates PpyMADS71's expression.Meanwhile,we explored the upstream genes of PpyERF060 and found that PpyEIL1 and PpyABF3 transcription factors could activate the expression of PpyERF060 and the binding site of PpyABF3 on the promoter of PpyERF060 was primarily determined.Furthermore,ethylene treatment on ‘Dangshansu' pear buds caused up-regulation of PpyERF60 and downregulation of PpyABF3.In addition,overexpression of PpyERF060 in pear calli caused inhibition of the transcription of PpyABF3.Based on the above results,we found that there is a regulatory network among PpyERF060,PpyABF3,and PpyMADS71,which could combine ethylene and ABA signaling pathway,and ultimately regulates the dormancy process.3.Research on the regulation of DAM gene during the chilling accumulationArtificial chilling accumulation was used to simulate the endo-dormancy process of ‘Dangshansu' pear buds,and the results confirmed that ‘Dangshansu' pear buds required more than 1,000 effective chilling accumulation hours to break the endodormancy.Among the 4 DAM genes found in previous studies,the expressions of PpyMADS28,PpyMADS29,and PpyMADS71 increased at the early stage of chilling accumulation and then gradually decreased during chilling accumulation,while PpyMADS31 was down-regulated continuously throughout the whole period of chilling accumulation,indicating two different expression patterns of DAM genes during dormancy cycle.We confirmed that the expression of PpyCYP707A3 continuously increased during the chilling accumulation.It might inhibit the transcription of PpyMADS71 by degrading ABA to participate in the dormant process.In addition,we found another transcription factor PpyMYB44 that responds to low temperature,could interact with the promoter of PpyMADS71 and inhibit transcription of PpyMADS71.