Cloning And Analysis Of Genes Encoding Ethylene Biosynthesis And Signal Transduction Elements From Herbaceous Peony (Paeonia Lactiflora Pall.)

Herbaceous peony is a traditional ornamental flower in China, which is suitablefor application of cut flowers. Paeonia lactiflora has a short flowering period whichaffects its application. Ethylene is a key regulator on the opening and senescenceprocess of herbaceous peony flowers. In this thesis, coding sequences of Actin andACO, and full-length cDNA of ACS, ethylene receptor ETR1and signal transductionkey components EIN3were isolated. and analyzed. And semi-quantitative RT-PCRsystem was established and the expression profile of Actin in different tiusses anddifferent developmental stages of herbaceous peony were semi-quantified withRT-PCR technique.Actin gene of herbaceous peony was successfully amplified with RT-PCR tech-nique. Sequencing result showed that Actin gene is1134bp length, encoding377amino acids, whose GenBank accession number was JX310002. and the homologoussimilarity in amino acid level with Actin from other plants was up to99%. It was pre-dicted that the molecular weight of Actin protein of herbaceous peony was41.7kD,and the isoelectric point (pI) was5.31. It was a hydrophilic and stable protein whichwas located in cytoplasm, without the transmembrane domain or signal peptide.Semi-quantitative RT-PCR analysis showed that the gene expression leves of Actingene in root, stem, leaf, flower and different developmental stages of herbaceous pe-ony were almost constant.The CDS of ACO gene from P. lactiflora, containing939bp, was successfullycloned, which encoded312amino acids with a molecular mass of35.2kDa, whoseaccession number in GenBank was JX445143, and its isoelectric point was5.24. Bio-informatics analysis showed that ACO is a non-secreted protein without the signalpeptide, mainly localized in the cytoplasm. It has more than90%homologous simi-larities with ACO ptoteins from other plants such as Paeonia suffruticosa, Actinidia deliciosa, Ziziphus jujuba., Hevea brasiliensis, respectively.The full-length cDNA sequence of ACS gene was cloned from P. lactiflora byRACE technique. It revealed that ACS gene contained1752bp, whose accessionnumber in GenBank was JX512359, which encoded492amino acids, with a molecu-lar mass of55.2kDa, and isoelectric point of7.13. Bioinformactic analysis showedthat ACS protein mainly was localized in the cytoplasm, without the signal peptide,and it was a non-secreted protein. It has more than70%homologous similarities withACS from other plants such as Paeonia suffruticosa, Eriobotrya japonica, Malus xdomestica. Pelargonium x hortorum. respectively.The full-length cDNA sequence of ETR1gene was cloned by RACE technique,whose accession number in GenBank was JX406435. Sequencing result indicated thatit contained2817bp, which encoded740amino acids, with a molecular mass of83.1kDa and isoelectric point of6.82. Bioinformatics analysis showed that ETR1wasmainly located in the vacuole, without signal peptide molecules. It has more than80%homologous similarities with ETR1from other plants such as Citrus sinensis,Eriobotrya japonica, Solanum lycopersicum. Lactuca sativa.The full-length cDNA sequence of EIN3gene from P. lactiflora, containing2739bp, was successfully cloned by RACE techniques, whose accession number in Gen-Bank was JX445144, which encoded548amino acids, with a molecular mass of61.5kDa and isoelectric point of5.55. Bioinformatics software predicted that EIN3protein,without the signal peptide, didn’t contain transmembrane protein, mainly localized inthe nucleus. It has more than50%homologous similarities with EIN3protein fromother plants such as Populus trichocarpa, Malus x domestica, Ricinus communis. re-spectively.