Functional Analysis Of PpHSFA2D And PpLAZY1 Involved In Regulating Branch Angle In Peach(Prunus Persica)

Peach is native to China and is one of the most popular fruits in the world.The tree architecture of peach directly affects its cultivation mode,fruit yield and quality,etc.,which is very important to the development of the peach industry.Standard type which greatly increase the cost of manual pruning because of the larger branching angles,wider tree shapes,vigorous vegetative growth,and luxuriant branches and leaves,is the main cultivation of peach trees.’Pillar peach’ with smaller branching angles and crown could greatly reduced the cost of manual pruning for a certain number of fruiting branches could be produced without pruning,besides,pillar peach could realize the ’wide row and dense planting’ cultivation mode proposed by the current fruit tree production.Therefore,discovering the key genes that control the branch angle of peaches and analyzing the molecular mechanism of the formation of branch angle has become a research hotspot for researchers.In this study,the normal peach ’Okabo’ and the pillar peach ’Zhaoshouhong’ was used as main research object,PpHSFA2D and PpLAZY1 were screened as the key genes that regulate the formation of peach branch angle through functional verification,gene mutation and yeast twohybrid experiments.Which could help to deeply understand the mechanism of peach branch angle formation and provide theoretical support for the genetic improvement of the defined architecture of peach.1.Analysis of the transcriptome data of the one-year old normal peach ’Okubo’ and pillar peach’Zhaoshouhong’ showed that the expression level of PpHSFA2D gene in the pillar peach was significantly higher than that of the normal peach,and displayed a co-expression pattern with the PpLAZY1 gene.2.A pSAK277-35S:PpHSFA2D overexpression vector was successfully constructed and infected with Arabidopsis thaliana by Agrobacterium-mediated genetic transformation.Positive plants were obtained through screened and phenotypic observation,results showed that no significant difference was observed in plant height,number of nodes and rosette leaves of PpHSFA2D overexpression Arabidopsis T2 generation plants compared with wild type.However,the branch angle of transgenic lines was significantly smaller than that of wild type,showing a more compact phenotype overall.A L101-PpHSFA2D-YFP subcellular localization fusion expression vector was successfully constructed and injected into tobacco leaves using transient exprsssion technology.The results suggested that PpHSFA2D was located in the nucleus.3.The pGBKT7-PpHSFA2D vector was successfully constructed and transformed into AH 109 yeast strain with pGADT7-AD empty plasmid.The results showed that the PpHSFA2D had transcriptional self-activation activity.The PpHSAF2D was devided into 1-133aa,134-242aa and 243-359aa fragments,and the transcriptional self-activating activity was verified in 1-133aa.4.A pSAK277-35S::PpLAZY1 overexpression vector was constructed,agrobacterium-mediated stable genetic transformation method was used to transform tobacco leaf,and four transgenic lines were obtained.Compared with wild-type tobacco,the leaf angle of transgenic lines was smaller,and showed a more compact phenotype.5.The first two Leucines(L)in V conserved domain of PpLAZY1,gravity response element"EAR"domain(LxLxL),was mutated to Phenylalanine(F).And the mutant PpLAZY1-ΔEAR was constructed to overexpression vector and was used to transform Arabidopsis thaliana.6.The 134-359aa segment without self-activating domain were constructed(pGBKT7PpHSFA2D134～359),which was transferred with pGADT7-PpLAZY1 into AH 109 yeast strain.The results showed that PpHSFA2D could interact with PpLAZY1.