Cloning And Expression Analysis Of Flowering Genes In Carya Cathayensis

Flowering is qualitative change process in plant’s life, which from vegetative growth toreproductive growth and from sporophyte growth to gametophyte growth. Flowering is the importantprocess of plant development which has complex molecular regulation network. In recent decades,through the researching of the development of the flower by molecular biology technology especiallycloning skills, which greatly promote the research of flowering mechanism. Hickory(Carya cathayensisSarg)belongs to the Carya Nutt, which is the famous dried fruit in Zhejiang Province of China. It is amonoecism plant with pendulous catkin in staminate flowers and a short inflorescence in pistillateflowers. And the staminate and pistillate flowers of hickory is different in space and time. As the keyfactor in the yield and quality of hikory, flowering does help to explore the mechanism of floweringregulation. We had mastered the differentiation and development period of staminate and pistillateflowers of hickory through morphology.There is still a long way to explore the floral regulation ofhickory in molecular biology level. Our team had obtained the fragments of E3ubiquitin protein ligaseCOP1-like, serine/threonine-protein phosphatase, ubiquitin-activating enzyme E1, E3ubiquitin proteinligase RIN2-like and SPINDLY-like by using454sequence of Carya cathayensis. The the full-lengh ofthe five genes were obtained by RACE technology. The characteristics of the five proteins and therelationships among homologous genes from different species were analysed using bioinformaticsmethods. The real-time RT-PCR showed the expressions of the five genes in the process of staminateand pistillate floral development, which helped to explore the biological function of the five genes andelucidate the flowering molecular mechanisms in hickory. The main research conclusions are as follows:(1)The E3ubiquitin protein ligase COP1-like was obtained by PCR amplification from flower buds ofhickory with the full length of2331bp, which encodes a protein of707amino acid residues. It has twospecial structural domains named Ring zinc finger domain and WD-40repeat sequences domain, theamino acids hydrophobity analysis showed that its coding protein hydrophilicity is strong hydrophilic.And the hydrophilic aminos concentrated in the amino terminal and hydrophobic amino acids in thecarboxyl terminal; The serine/threonine-protein phosphatase has full length of1895bp, which encodes aprotein of626amino acid residues. It has two special structural domains named TPR domain and MPPdomain; ubiquitin-activating enzyme has full length of3346bp, which encodes a protein of1112aminoacid residues. It has two special repeat structural domains named E1-enzyme super family; E3 ubiquitin protein ligase RIN2-like has full length of1761bp, which encodes a protein of587amino acidresidues. It has special structural domain named Ring zinc finger domain domain; SPINDLY-like has fulllength of2274bp, which encodes a protein of754amino acid residues. It has two special structuraldomain snamed TPR super family domains.(2)Compared with the proteins reported in other plant species, the results showed that E3ubiquitinprotein ligase COP1-like has closest relationship with Ricinus communis and far relationship withTriticum urartu and Brachypodium distachyon; serine/threonine-protein phosphatase has closestrelationship with Vitis vinifera and far relationship with Zea mays; The ubiquitin-activating enzyme E1has closest relationship with Citrus sinensis and far relationship with Oryza brachyantha; The E3ubiquitin protein ligase RIN2-like has closest relationship with Fragaria vesca and far relationship withCricetulus griseus; And the SPINDLY-like has closest relationship with Rosa lucieae and far relationshiowith Cucumis sativus. Results show that the genetic evolution exist differences between species.(3)Real-time fluorescence quantitative PCR showed that E3ubiquitin protein ligase COP1-like,serine/threonine-protein phosphatase, ubiquitin-activating enzyme E1, E3ubiquitin protein ligaseRIN2-like and SPINDLY-like expressed in Stems, leaves, fruits and flower buds of hickory and thehighest expressions in flower buds. The expression of the five genes throughout the developmentprocess of the staminate and pistillate flowers of hickory with high expression in the middle of March inpistillate flowers and staminate flower in the middle of May.It can be speculated that E3ubiquitinprotein ligase COP1-like, serine/threonine-protein phosphatase, ubiquitin-activating enzyme E1, E3ubiquitin protein ligase RIN2-like and SPINDLY-like related to staminate and pistillate flowerdifferentiation of hickory.