Identification,Expression Analysis And Functional Verification Of PpGA20ox Genes In Peach

Peach(Prunus persica)belongs to Prunus of Rosaceae and is one of the important economic fruit tree in China.The dwarf cultivation is gradually and widely used in modern production of peach,since it can reduces the length of the juvenile period,result in higher quality and yield,and allow for efficient mechanized management.However,the growth of peach trees is vigorous,and the cultivation requirements of dwarf dense planting can only be achieved through reasonable tree management.Gibberellin(GAs)has been identified as an plant hormone and plays a positive role in regulating plant height.The activity of the gibberellin 20-oxidases(GA20ox)is of particular critical role in determining GA concentration in many plant species.In order to study the possible regulatory role of PpGA20ox in the growth and development of peach trees,six PpGA20ox genes were identified from peach genome.Then,six PpGA20ox genes were cloned from ’QiuMiHong’ peach and its transcript levels in different tissue of ’QiuMiHong’ and subcellular localization was analyzed.The function of PpGA20ox genes were verified by ectopic expression of PpGA20ox genes in Arabidopsis.The main research results are as follows:1.Six PpGA20ox genes were identificated from peach genome,named as PpGA20oxl,PpGA20ox2,PpGA20ox3,PpGA20ox4,PpGA20ox5 and PpGA20ox6,respectively.The results showed that the CDS length of six PpGA20ox genes was 1134 bp,1164 bp,726 bp,1164 bp,1155 bp and 1155 bp.And the length of amino acid sequence is 378、388、242、388、385 and 385 amino acids,respectively.2.Chromosome distribution and gene structure analysis showed that PpGA20oxl and PpGA20ox2 were located on chromosome 2,and the others were located on chromosome 1.All PpGA20ox genes contain three exons and two introns.The sequence similarity among the six PpGA20ox proteins reach up to 43～73%.All GA20ox protein sequences from different species contain several conserved motifs,including"LPWKET",conservative H and D residues.These results indicated that the six PpGA20ox genes belong to the GA20ox gene family.3.Determination of the PpGA20ox transcript levels in young leave,stem tips,young stems,flower buds and fruit from ’Qiumihong’ was conducted by qRT-PCR.The results showed that the PpGA20oxl was mainly expressed in young leaves and shoot tips,whereas PpGA20ox2 was strongly expressed in flower bud;the transcript of PpGA20ox5 and PpGA20ox6 were only detected in flowers;PpGA20ox3 and PpGA20ox4 have very low transcript level in all detected tissues.Our results possibly indicated that the development of leaf and shoot tips was controlled by PpGA20oxl,and the regulation of flower development was affected by PpGA20ox2.4.In order to analyze the subcellular location of PpGA20ox proteins,the expression vector of 35S-PpGA20ox-GFP fusion proteins were successfully constructed by homologous recombination method and transiently expressed in tobacco leaf epidermis cells.The results showed that PpGA20ox1、PpGA20ox2 and PpGA20ox5 were all localized in the nucleus and cytoplasm.5.In order to verify the function of the PpGA20ox genes,the over-expression vectors of pSAK277-PpGA20ox1,pSAK277-PpGA20ox2 and pSAK277-PpGA20ox5 were constructed and transformed into Arabidopsis thaliana plants.The results showed that etopic expression of PpGA20ox1 and PpGA20ox2 genes in GA-deficient Arabidopsis mutants result in the vegetative growth of transgenic plants,such as larger leaves and earlier flowering time.The growth rate of transgenic plants slowed down obviously,and there was no significant difference in the final plant height between wild type and the mutant.Etopic expression PpGA20ox5 gene in wild Arabidopsis promote slightly final plant height compared to wild-type.These results revealed that PpGA20ox1,PpGA20ox2 and PpGA20ox5 genes can significantly promote the growth and flowering time of Arabidopsis thaliana.Taking all together,there are six GA20ox genes in peach genome,and each member contains three exons and two introns.PpGA20ox1 was mainly expressed in young leaves and shoot tips,whereas PpGA20ox2 was strongly expressed in flower bud.The results of functional verification of transgenic Arabidopsis showed that both PpGA20ox1 and PpGA20ox2 could significantly promote the vegetative and early reproductive growth and development of Arabidopsis thaliana.Based on the above results,it can be concluded that the content of GA in young leaves and shoot tips is mainly regulated by PpGA20ox1 and the content of GA in flower buds is controlled by PpGA20ox2.This study was focused on the relationship between PpGA20ox genes and peach height which provide fundamental molecular information to understand the mechanism of gibberellin regulating internode length of peach,and provide a theoretical basis for the breeding of new dwarf peach cultivars.