Cloning And Expression Analysis Of Sex Related Genes RcACS5and RcHK1in Castor (Ricinus Communis L.)

With female inbred line YC2S and its sister male inbred line YC2F，the difference ofethylene release rate between different sex types was examined, the sex-related genesRcACS5and RcHK1were cloned, of which the bioinformatics and expression analysiswere conducted, the main results were as follows:1. It was proved by stem tip freehand section that YC2S and YC2F got into flowerprimordium differentiation stage during from five-leaf stage to six-leaf stage.2. Gas chromatography identification showed that there were no significantdifferences of ethylene release rate between pistillate line and monoecious line beforefive-leaf stage. The difference increased rapidly after six-leaf stage, and it was significantlyhigher in monoecious line than in pistillate line at bud and blossom period. It suggestedthat ethylene was one of the key factors determining sex in castor.3. The coding region of gene RcACS5was amplified with RT-PCR primers designedaccording to RcACS5（ACC synthase gene） sequence （XM002514115.1） published inGenbank. The sequence, with a length of1323bp, encoding a protein containing440aminoacids, of wahich the molecular weight of108.5kD and isoelectric point of5.82. Its primarystructure was mainly hydrophilic structure and no transmembrane domain, which has ahomology of80.04%with the product of Cs-ACS2in cucumber. Protein domain analysisshowed that the main function of RcACS5protein were mainly decarboxylation,deamination or transamination.The5’UTR sequence of the gene was obtained with hiTAIL PCR method, which thelength was422bp and was containing4TATA-boxes located at-10,-188,-296and-273respectively. In addition, it had other cis acting elements, such as carbon metabolism geneelement DOFCOREZM, cytokinin response element ARR1AT, promoter elementCAATBOX1and pollen specific expression element GTGANTG10.The3’UTR sequence of the gene was obtained with3’RACE method,312bp, includinga stop codon TAA and poly （A） tail of17bp. There was a homology of81.02%between the3’UTR without poly （A） tail and the predicted3’UTR.Fluorescent quantitative PCR analysis showed that the expression level of RcACS5genewas the highest in male flowers, followed by female flowers, the lowest was in leaves. Itwas suggested that the RcACS5gene was closely related to the development of maleflowers. 4. The coding region of RcHK1gene was amplified with RT-PCR primers designedaccording to RcHK1（histidine kinase gene） sequence （XM₀02514855.1） published inGenbank. The sequence, with a length of3018bp, encoding a protein containing975amino acids, with a molecular weight of109.1kD and isoelectric point of6.60. There wasa homology of68.13%between the RcHK1gene and the AHK3gene （related to thedevelopment of female gametes） in Arabidopsis thaliana. Bioinformatics analysis showedthat the protein was a transmembrane protein, related to signal transduction. So it wassuggested that RcHK1gene was a cytokinin receptor gene.The5’UTR sequence of439bp was obtained with hiTAIL PCR method, containing fiveTATA-boxes located at-172,-236,-305,-307and-340respectively, and twoAAGA-motifs located at-147and-395respectively. In addition, its cis acting elementscontained three cytokinin response elements ARR1AT.The3’UTR sequence of554bp was obtained with3’RACE method. There was ahomology of99%between the part before poly （A） tail and the predicted3’UTR.Fluorescent quantitative PCR analysis showed that the expression level of RcHK1genewas higher in YC2S than in YC2F generally. There was no significant difference fromthree-leaf stage to five-leaf stage. At six-leaf stage, the expression level in pistillate line wassignificantly higher than that in monoecious line. The difference continued to grow at budstage and came back to the similar level at blossom period finally. It suggested that RcHK1was closely related to the development of female flowers.