Gene Mining And Regulatory Network Analysis Of Peach Fruit Ripening-related Genes Based On RNA-seq

Peach(Prunus persica(L.)Batsch),a perennial deciduous tree belonging to the family Rosaceae,subgenus Prunus persica.It is one of the oldest fruit trees,which originated in northwest of China.Peach has been cultivated for a long time,and many excellent varieties have been bred.The most varieties of northern peach variety group,are harvested in summer and autumn and mainly used for fresh eating.Breeding peach varieties with different maturity plays an important role in prolonging the market supply period and improve the economic benefits of growers.Therefore,gene mining and regulatory network analysis of peach fruit ripening-related genes are important to enhance people’s understanding of fruit ripening.IAA and ARF,as key regulatory factors in auxin signal transduction pathway,play a very important role in fruit ripening.In this study,several IAA and ARF genes related to fruit development were compared the fruit transcriptome data of ’Kurakato Wase’ peach with its early-ripening mutant at different development stages.Besides,the regulatory network among these genes were analyzed.The main results are as follows:1.The transcriptome of ’Kurakato Wase’ and its early-maturing mutant fruits with different developmenal stagest were sequenced and analyzed.4395 differentially expressed genes were screened out in total.Through GO and KEGG metabolic pathway analysis,we found that most of the differential genes were concentrated in plant hormone signal transduction,secondary metabolite synthesis,carbon metabolism,photosynthesis and other pathways.According to the results,38 genes related to fruit ripening were screened,including 17 auxin,11 abscisic acid,and 10 ethylene biosynthesis and signal transduction genes.2.Based on transcriptome data,the weighted gene co-expression network was constructed.Based on pivotal modules turquoise and KEGG pathway analysis,ten ripening-related IAA genes were focused,and eight ARF transcription factors interacting with them were predicted.Comprehensive consideration of the gene expression level and trend,six genes,PpIAA6,PpIAA9,PpIAA28,PpARF1,PpARF4 and PpARF19,were further studied.Yeast two-hybrid analysis showed that PpARF4 and PpARF19 have autonomous activation activity,and PpARF1 does not interact with PpIAA6,PpIAA9 and PpIAA28.PpIAA6 and PpIAA9 can combine with themselves to form homodimer,and PpIAA9 can also form heterodimer with PpIAA6 and PpIAA28.The above six genes were cloned and the properties and structures of the proteins encoded,were analyzed by using bioinformatics methods.Instantaneous expression results of tobacco showed that PpARF1 and PpARF19 are transcription repressors;PpIAA6 and PpIAA9 could weaken the inhibition of PpARF1,while PpIAA28 could enhance the inhibition of PpARF1.PpIAA6 and PpIAA9 could enhance the inhibition of PpARF19,and PpIAA28 could weaken the inhibition of PpARF19.3.pRI-IAA6 and PHB-ARF4 overexpression vectors were successfully constructed.The cotyledons of tomato micro-Tom were transformed by Agrobacterium-mediated infection,and transgenic lines had been obtained.