Cloning And Expression Analysis Of LAZY1 Genes Related To Creeping Growth In Rosa Rugosa

Rosa rugosa?Rosa rugosa?is an important ornamental plant of Rosa,which is not only of great economic value,but also widely used in urban and rural greening.In recent years,domestic and foreign scholars have mainly focused on flower color,flower fragrance,breeding,variety classification and flowering regulation and so on,but there is little research on rose plant type.Plant type is an important ornamental feature of garden plants.Good plant type not only contributes to the accumulation of plant biomass,but also enriches the ornamental elements of plants.The plant type is mainly controlled by the branching angle and branch direction of the aboveground part.At present,the genes related to branch regulation include LAZY1,TAC1,DRO and so on,but the molecular regulation mechanism of these genes is still in the initial stage.The materials for study are also concentrated in grasses and a few woody plants.Therefore,it is of great significance to study the cloning and expression of genes related to the plant type of rose,to elucidate the molecular regulation mechanism of the branch angle of rose,and then to improve the plant type traits and molecular breeding of roses.In this study,two transcriptional sequences of rose LAZY1 gene were isolated by using RT-PCR and RACE methods based on the data of related genes in NCBI database,and the structure of the two transcriptional sequences were analyzed.The physicochemical properties and phylogenetic relationships were analyzed by bioinformatics,and the expression differences of LAZY1 in different tissues and varieties of rose were analyzed by real-time fluorescence PCRX qRT-PCR.The main results were as follows:?1?Two transcripts of the rose LAZY1 gene were first cloned from the annual tender stem cDNA of Rosa rugosa,named RrLAZY1-a and RrLAZY1-b,GeneBank accession number are MG917092 and MG937048,respectively,and the open reading frame was 1194bp and1170bp,encoding 397,389 amino acids,respectively.?2?The molecular weight,hydrophobicity,phosphorylation and glycosylation sites,signal peptides,secondary structures,transmembrane domains and conserved domains of RrLAZY1-a and RrLAZY1-b sequences were analyzed by bioinformatics software.At the same time,the LAZY1 gene sequence of rose was analyzed by Blast,and phylogenetic tree was constructed.The results showed that the RrLAZY1-a and RrLAZY1-b proteins contained no conserved domains,and the molecular formulas were C₁₉₁₄H₃₀₄₃N₅₄₃O₆₂₈S₁₆6 and C₁₈₈₂H₂₉₈₈N₅₃₂O₆₁₇S₁₄,respectively.The secondary structures were mainly?-helix,irregular crimp and extended main chains,containing many phosphorylation sites and no glycosylation sites.The sequence analysis of non-secretory nucleophile protein.Blast showed that the similarity of RrLAZY1-a with RrLAZY1-b gene and peach tree was 85%,which was similar to that of other woody plants.The phylogenetic tree results showed that the LAZY1 gene of rose was closely related to LAZY1 of peach and sweet cherry,which was in accordance with the taxonomic classification of plants.?3?The expression of RrLAZY1-a gene in different tissues and varieties of rose was analyzed by using qRT-PCR.The results showed that rose RrLAZY1-a gene was expressed in different tissues.The highest expression was found in stem,the second in pistil and axillary bud,the lower in leaves,stamens and sepals,and the lowest in petals.The expression analysis of different varieties showed that rose RrLAZY1-a expression was the highest in the tender stem of'Zizhi',followed by'Zilongwuchi'and the lowest in'Pingzhi'.It is inferred that rose RrLAZY1-a gene negatively regulates the branching angle of rose and then regulates the plant type of rose by the role of suppressor.?4?The expression vectors of rose LAZY1 genepROK?-RrLAZY1-a and pROK?-RrLAZY1-b were successfully constructed,and Agrobacterium tumefaciens GV3101receptive states were transformed successfully.The subcellular localization analysis showed that both RrLAZY1-a and RrLAZY1-b proteins were located in the nucleus after infecting tobacco,and the function of RrLAZY1-a and RrLAZY1-b should be further verified after infecting tobacco.