Cloning And Expression Analysis Of Three WD40 Transcription Factors In Rosa Rugosa

Rosa rugosa,as a perennial famous flower plant of Rosaceae,it has gorgeous and rich flowers,unique fragrance,and is known as"queen of flowers".Rose planting conditions are wide,adaptability is very strong,so it is deeply loved by the majority of workers.The most important reason why roses have excellent planting conditions is that they are very drought-tolerant,so this is an important aspect of rose breeding.In addition,compared with the application of traditional Chinese famous flowers such as peony,rose and peony in landscape design,the prevalence of rose is lower.The reasons can be divided into two points.First,the thorny stems of roses cause many inconveniences in gardening,cultivation and management,and flower picking.Second,roses are mostly rosy,pink and white,with few roses of other colors,and their ornamental value is greatly reduced.Therefore,we use molecular techniques to study key regulatory genes,and found that WD40 protein is a family of proteins widely distributed in eukaryotes with a variety of biochemical and cellular biological functions.It has various functions such as signal transduction,RNA processing and vesicle transport,assembly of cytoskeleton,regulation of cell cycle and activation of MBW transcription complex.Therefore,it is important to study the function of WD40 protein in rose cultivars and how to participate in the life process of rose cultivars.In this experiment,the main materials were'Zizhi'?R.rugosa‘Zizhi'?,and there were'Baizizhi'?R.rugosa‘Baizizhi'?,?Fenzizhi??R.rugosa‘Fenzizhi'?,'Tianehuang'?R.rugosa‘Tianehuang'?,'Tangfen'?R.rugosa‘Tangfen'?,'Xizi'?R.rugosa‘Xizhi'?,'Zilongwochi'?R.rugosa‘Zilongwochi'?,'Hunchun'?R.rugosa‘Hunchun'?.Based on the sequence fragments in the transcriptome database,three WD40 transcription factors and their full-length DNA sequences were isolated by RT-PCR,and three transcription factors were identified.The genes were Gbeta?WD40-1?,RUP2?WD40-2?,TTG1?WD40-3?,which were comprehensively analyzed and identified by bioinformatics.The expression of three WD40genes in different rose varieties and tissues was analyzed by real-time fluorescence quantitative RT-PCR.The WD40-1 gene was stably transformed into tobacco.In order to verify the biological functions of Arabidopsis thaliana,the WD40-2 and WD40-3 genes were stably transformed into Arabidopsis thaliana.1.Three WD40 genes,namely WD40-1,GeneBank number MK034864,open reading frame 981 bp,encoding 326 amino acids,WD40-2,GeneBank number MK039443,open reading frame 1173 bp,encoding 390 amino acids,WD40-3,GeneBank MK039444,open reading frame 1041 bp,encoding 346 amino acids,which were cloned for the first time using the petal cDNA of`Zizhi'roses at half-blooming stage.An amino acid.2.WD40 proteins contain many highly conserved special motifs,starting from glycine-histidine dipeptide and ending at tryptophan-aspartate dipeptide.They contain about40 amino acid residues.Therefore,they are named WD40 motifs.Further studies show that WD motifs can contain 44-60 amino acid residues,and C-terminal has a variable region containing 11-24 amino acid residues.WD40 proteins generally contain 4-16 homeopathic repetitive WD40 motifs,which are conserved domains.3.The molecular formulas of WD40-1,WD40-2 and WD40-3 were C₁₆₀₁H₂₅₂₀N₄₅₀O₄₈₆S₁₁,C₃₄₁₅H₅₆₅₉N₁₁₇₃O₁₄₃₄S₃₁₃13 and C₁₇₂₃H₂₆₆₁N₄₆₅O₅₂₉S₁₂,respectively.The secondary structure prediction results showed that beta turn and Rancoil were the main secondary structures of the three proteins,and some of them contained phosphorylation sites and glycosylation sites without transmembrane domains.These three WD40 proteins were proved to be non-secretory proteins,and their basic physical and chemical properties were predicted to be unstable hydrophilic proteins.4.Three WD40 genes were expressed in different cultivars and tissues by real-time fluorescence quantitative PCR,but the expression patterns were significantly different.Generally speaking,all three genes are involved in plant development regulation and have certain specificity.The expression of WD40-1 gene was higher in petals and leaves,WD40-2gene was the highest in roots and sepals,and WD40-3 gene was the highest in leaves and stems.It is speculated that three genes are involved in different biosynthetic processes,showing three different WD40 protein functions.5.Three expression vectors of WD40 gene,pCAMBIA1304-WD40-1-WD40-2-WD40-3,were successfully constructed and transformed into Agrobacterium tumefaciens GV3101.Subsequently,WD40-1 transgenic tobacco plants,WD40-2 transgenic Arabidopsis and WD40-3 transgenic Arabidopsis plants were successfully obtained,and the transgenic plants were observed and analyzed.