Gene Cloning And Construction Of Over-expression Vectors Of WOX Family Genes In Rosa Species

Rose (Rosa rugosa Thunb.) and Rosa multiflora, widely used in the garden, are important ornamental plants of rosaceous. Currently, For R. rugosa and R. multiflora most researches focus on the field of trait identification, tissue and organ culture, little is reported in fuctional gene isolation and gene engeeering.Previous reports shown that the WOX family genes regulated plant growth and key process of development, including to regulate plant embryos, meristems, leaves, flowers, roots and vascular tissue development, which also related to the low temperature resistance of plants. Whereas these researches mainly focuses on the model plant Arabidopsis.In this study, gene cloning sequence analyses were used to isolate WOX family genes, aiming to research their relationship in Rosa and Rosa multiflora. The main results were as follows:1. Based on transcriptome sequencing results, we obtained seven WOX(WUS homeobox) family genes, and amplified a full-length of genes from R. rugosa and R. multiflora by PCR and FPNI-PCR strategies. In R. rugosa, RrWOX1/A and RrWOXl/B homology with the AtWOXl in Arabidopsis; RrWOX4homology with the AtWOX4in Arabidopsis; RrWOX9homology with the AtWOX9in Arabidopsis; RrWOX10homology with the AtWOX10in Arabidopsis; RrWOX13homology with the AtWOX13in Arabidopsis; RrWUS homology with the AtWUS in Arabidopsis; Correspondingly, R. multiflora have RmWOXl/A, RmWOXl/B, RmWOX4, RmWOX9, RmWOX10, RmWOX13and RmWUS.2. DNA and amino acid analysis of the14Rosaceae WOX family genes,15Arabidopsis and10peach were undertaken. RrWOXl/B, RmWOX1/A and RmWOX9can not translated to complete amino acid sequence, while other11genes contain the homeodomain, which show the genes pattern of WOX family. For the genetical phytology of isolated WOX genes, RrWOXl/A, RmWOX1/A, RrWOX4, RmWOX4, RrWUS and RmWUS were subdivided in WUS clade, RrWOX9was subdivided in intermediate clade, and RrWOX10, RmWOX10, RrWOX13and RmWOX13was subdivided in ancient clade. WUS boxes were presented in all the six members of WUS clade. In addition, RrWUS and RmWUS cotain EAR-like motif. Through the above analysis, we preliminary confirmed the gene cloned in this study belonged to WOX family.3. In our experement, seven over-expression vectors were constructed. In which, The R. rugosa gDNA were used as template. pCAMBIA2300s-RrWOXl/A, pCAMBIA2300s-RrWOX1/B, pCAMBIA2300s-RrWOX4, pCAMBIA2300s-RrWOX9, pCAMBIA2300s-RrWOX10, pCAMBIA2300s-RrWOX13and pCAMBIA2300s RrWUS vectors were constructed respectively, and all these seven vectors were transformed into agrobacterium EHA105.