Cloning And Functional Study Of RhD14 Gene From Rosa Hybrida ’Dianhong’

Rose（Rosa hybrida）is the top ten cut flowers in the world.In addition to flower type and flower color,plant architechure is the main ornamental element of the rose,which determines the overall layout of the flowers and leaves,plant shape,resistance and pruning cost.The lateral branches are an important component of the aboveground plant architechure,directly determining the yield,quality,and production efficiency of roses.Therefore,it is of great theoretical and practical value to explore the mechanism and efficient regulation mode of rose lateral branching.The formation of rose lateral branches is affected by many factors,such as genetic factors,environmental factors and plant hormones,with plant hormones playing an important role in lateral branch regulation.Strigolactones（SLs）,as a new hormone regulating lateral branch development,will provide new directions and ideas for studying lateral branch regulation of rose.Dwarf 14（D14）is a substrate receptor that responds to SLs signals,and the gene encoding this protein is a key gene involved in the SLs signal transduction pathway.Rosa hybrida‘Dianhong’is a widely cultivated Chinese rose food variety in Yunnan at present.However,its strong apex dominance and limited lateral branching hinder the improvement of flower yield and economic benefits.Therefore,there is an urgent need for technology to regulate lateral branches and new varieties with excellent plant types for production.In order to explore the function of Rh D14 gene,this study cloned the CDS sequence Rh D14 gene from Rosa hybrida‘Dianhong’,and conducted bioinformatics analysis.At the same time,real-time quantitative PCR（q RT-PCR）was used to detect the gene expression in different tissues and under the treatment of toppings.The transient transformation system of tobacco was used to conduct subcellular localization of the protein encoded by the Rh D14 gene.The overexpressed vector was constructed and the genetic transformation of Arabidopsis thaliana was mediated by Agrobacterium tufaciens,in order to explore the function of Rh D14 gene in lateral branch regulation by observing phenotypic changes.The results are as follows:（1）The c DNA sequence of the cloned Rh D14 gene（Gen Bank accession number:OP009358）is 1094 bp in length,containing a complete open reading frame（ORF）of 1045 bp,encoding 347 amino acids residues.The molecular formula of the protein is C₁₇₃₈H₂₇₀₃N₄₄₁O₅₁₀S₁₅,with the relative molecular weight of 38.41 k Da,and the theoretical isoelectric point（p I）of 5.71.The main secondary structure of its amino acid sequence isα-helix and Cc（random coil）,and Rh D14 is a stable protein without transmembrane helical structure and signal peptide,which belongs to theα/βhydrolase family.Homologous sequence alignment and phylogenetic tree analysis showed that the amino acid sequence of Rh D14（OP009358）had the highest similarity of 98.05%with that of R.chinensis‘Old Blush’（XP＿024283944.1）,followed by Fragaria vesca subsp.vesca（XP＿004287076.1）of the same subfamily with the similarity for 89.75%.The amino acid sequence of D14 was similar to that of Prunus dulcus（BBH08393.1）,Prunus persica（XP＿007202641.2）,Prunus mume（XP＿008242593.1）,European Prunus avium（XP＿021807166.1）and Malus domestica（XP＿008386980）in the same family with the similarity ranging from 73.20%to73.97%.（2）The analysis of q RT-PCR showed that the Rh D14 gene was expressed in all of the test tissues（roots,stems,leaves,axillary buds,nodes,and apical buds）.Taking roots as the control,the expression levels of Rh D14 was the highest in stems,in which were significantly higher than that in roots（P<0.01）,The expression level of Rh D14 gene in stems and axillary buds was determined by q RT-PCR.It was found that the expression of Rh D14 gene was significantly up-regulated after decapitation and peaked at 12 h,indicating that decapitation could significantly（P<0.05）induce the expression of Rh D14 gene in stems and axillary buds,regulating the germination and elongation of axillary buds.（3）The overexpression vector p C1300s-35S-Rh D14-GFP containing kana mycin and hygromycin resistance was successfully constructed by connecting R h D14 with the overexpression vector p C1300s-35S-GFP.The vector was then tr ansferred to Agrobacterium tumefaciens GV3101,instantaneous infection of toba cco leaves by Agrobacterium-mediated method.The results showed that the pro tein encoded by Rh D14 was located in the cytoplasm and cytomembrane memb rane of the cell,as predicted by WOLF PSORT online.（4）Identification of positive plants:In this experiment,8 transformed and regenerated plants were obtained（5）from Columbia wild type Arabidopsis seeds,and 6 were identified as T₁positive plants.After sowing,T₂transgenic homozygous plants were produced.The selected pure and pure lines were further cultured,and the T₂generation transgenic homozygous plants were identified by DNA and RNA,and the positive conversion rate was about 88.89%.In order to further investigate the expression of Rh D14 gene in overexpressed Arabidopsis Thaliana plants,q RT-PCR was used to detect the relative expression of Rh D14 gene in some transgenic plants preliminarily identified.It was found that there were differences in the expression of Rh D14 gene among different plants.Compared with WT,the expression level of the three transgenic plants was 77.53,33.04and 24.64times.Statistical analysis of related phenotypic indicators showed that overexpression of Rh D14 gene was involved in regulating the Arabidopsis growth of roots,rosette leaves,stem diameter,plant height,pod size and pod maturity.The detection of related genes in Arabidopsis thaliana showed that the Rh D14 gene did not promote the expression of At D14 gene in Arabidopsis,but significantly promoted the expression of At SMXL7 gene.GID1A gene of GA signal transduction pathway in transgenic strains was also significantly higher than that in WT.