Cloning And Functional Analysis Of PpD2 And PpWRKY24 In Peach (Prunus Persica)

Peach is one of the most important fruits in northern China.The large growth of peach not only causes the tree closed,but also affects the distribution of photo synthetic products,resulting in the decline of fruit quality.Branch number,branch angle and internode length are important components of tree architecture.It is of great significance for peach cultivation,breeding and industrial development to regulate branch characteristics and cultivate peach trees with ideal plant type.Plant growth and development were affected by environmental factors(light,temperature,etc)and plant hormones due to generating a series of signal transmission and response.Brassionsteroids(BRs)play an important role in plant growth and development,while the study of BRs function on peach tree architecture,especially in branch development,was reported rarely.In this study,the effect of BR on peach tree architecture was analyzed using one-year-old ’Zhaoshouhong’ seedling.The candidate genes related to BR treatment were identified using transcriptome,and then cloned,the expression pattern in different tissues,subcellular localization,and functional analysis in transgenic Arabidopsis.The main results are as follows:1.The results showed that the number of internodes and branching rate in BR treated group were significantly increased than that in control.Transcriptome results showing that the expression levels of BR related genes PpD2 and PpWRKY24 in BR treatment group were significantly higher than that in control group,so these two genes were selected as candidate genes involved in BR regulation of peach tree architecture in further study.2.PpD2 and PpWRKY24 were successfully cloned from ’Zhaoshouhong’ using the cDNA of shoot tip by homologous cloning method.Sequence analysis showed that the full length of their CDS are 1458 bp and 1752 bp,respectively.The PpD2 and PpWRKY24 genes were located on chromosome 3 and 6,respectively.Structure analysis indicated that there were three exons and two introns in PpD2,four exons and two introns in Pp WRKY24 gene.3.The expression levels of PpD2 and Pp WRKY24 genes were detected in shoot tips,tender leaves,mature leaves,annual branches(upper,middle and lower parts)and phloem(upper and lower parts)at branch junction of ’zhaoshouhong’ using qRT-PCR.qRT-PCR analysis indicated the expression of PpD2 and PpWRKY24 exhibited tissue specific.The PpD2 gene expressed a highest level in middle and lower part of annual branches,while showed a lower level in mature leaves.The PpWRKY24 gene expressed a highest level in mature leaves and a lowest level in shoot tip tissue.4.The 35S-PpD2-GFP and 35S-PpWRKY24-GFP were constructed using homologous recombination method and transformed into tobacco leaf cells by Agrobacterium mediated transformation,respectively.The transient expression fluorescence results showed that PpD2 was located in plasma membrane and nucleus,while PpWRKY24 by laser confocal microscopy.5.It was found that the number and size of rosette leaves and branching angle were larger in PpD2 transgenic Arabidopsis lines than that in WT.Compared with WT,the number of lateral branches were significantly increased,and the length of second internode were significantly shorter in PpWRKY24 transgenic Arabidopsis.