Functional Study Of The Adventitious Rooting Related Oxygenase Gene ARRO-1 In Apple

The main breeding mode of apple is grafting.Due to its rapid forming,strong early fruiting,high yield per unit area,small canopy,convenient management,and suitable for mechanized operations,the dwarf dense planting has gradually become the mainstream development mode of the apple industry.Therefore,a series of changes have taken place in the selection of rootstocks.The first is rootstocks,which have developed roots,good solidity and adaptability,but no dwarfing effect,and followed by dwarf intermediate rootstocks,which has a certain dwarf effect,but the breeding of intermediate rootstock seedlings requires two grafts,and the procedure is cumbersome.In order to simplify the seedling process,the dwarf rootstock is directly used to regenerate adventitious roots as a dwarf rootstock.The dwarfing effect is better,but the formation of adventitious roots of dwarf rootstocks is difficult,and its root system is shallow,the root system is underdeveloped,and the soil fixation is poor.Therefore,they are less resistant to natural disasters such as the wind.If the problem of rooting difficulties of dwarf rootstocks is solved,it will bring huge economy benefit.It is reported that the adventitious rooting related oxygenase gene ARRO-1 is up-regulated in adventitious root induction stage,which is predicted to be related to the formation of adventitious roots of woody plants,but the biochemical function of ARRO-1 protein is unknown.Therefore,studying the function of the ARRO-1 gene is of great significance for revealing the molecular mechanism of adventitious root formation in woody plants.The main results of this study are as follows:(1)By the bioinformatics analyzing of ARRO-1 and its homologue genes DAO1 and DAO2 in Arabidopsis,it was found that the similarity of the c DNA sequences of ARRO-1,DAO1 and DAO2 was 78.72%.The amino acid sequence similarity of ARRO-1,DAO1 and DAO2 was 72.26%,and the similarity was higher.(2)The fusion expression vectors of ARRO-1,DAO1 and GFP driven by 35 S were constructed and expressed instantaneously in tobacco.Laser confocal microscopy showed that both ARRO-1 and DAO1 were localized in cytoplasm and nucleus.(3)We cloned 2.0 KB of the upstream CDS region of ARRO-1 gene and sequenced the promoter regulatory elements.Four GUS reporter gene expression vectors driven by ARRO-1 promoter fragments of different lengths were constructed by 5’deletion method.The expression specificity of ARRO-1 was studied by histochemical staining.It was found that ARRO-1 was mainly expressed in cotyledons.In addition,it was slightly expressed in flowers and pods.After IBA treatment,GUS staining was relatively deep.This indicates that the ARRO-1 promoter responds to IBA and its expression is regulated by IBA.In addition,we found that when the promoter is gradually deleted from the 5’-end deletion to877 bp,the promoter function is not affected,but when deleted to 328 bp,the GUS signal is relatively weak relative to 1602 bp,1202 bp,and 877 bp,the promoter function is affected.Therefore,we speculate that the DNA sequence between 877 bp and 328 bp contains important regulatory elements(sites)for gene expression.(4)The prokaryotic expression vector of ARRO-1 gene was constructed and transformed into E.coli BL-21 to express and purify ARRO-1 protein,and ARRO-1 antibody was prepared and purified by immunizing rabbit.Whether using pure protein expressed in vitro or using total protein of transgenic plants to test serum antibodies,and the results showed that we successfully prepared ARRO-1 antibody,which laid the foundation for the identification of protein levels of subsequent transgenic plants.(5)The 35S::ARRO-1 and 35S::DAO1 and p DAO1::ARRO-1,p DAO1::DAO1 plant binary expression vectors were constructed and transformed into wild-type Arabidopsis and T-DNA insertion mutants dao1-1 of DAO1 genes,respectively,thus obtained over-expression line and complementary line.Phenotypic analysis revealed that the phenotypes of underground parts of complementary line including the length of main roots and the regeneration ability of adventitious roots were restored to a great extent.(6)The 35S::ARRO-1-GUS and 35S::DAO1-GUS,pDAO1::ARRO-1-GUS,p DAO1::DAO1-GUS plant binary expression vectors were constructed and transformed into wild-type Arabidopsis thaliana.Thus transgenic plants of T1 generation were obtained,which prepared materials for subsequent study of tissue and organ specificity of gene expression.In summary,our research has a preliminary understanding of the function of ARRO-1,which is of great significance for revealing the molecular mechanism of the formation of adventitious roots in woody plants.