The Cloning Of CsCOL4 Gene Of Tea Plant And Its Function With CsSVP Gene In Floral Transformation

Tea plant(Camellia sinensis(L.)0.Kuntze)is an important woody economic crop in China,Because of the rich natural and medicinal ingredients in tea leaf and tea flower,it has various health benefits and nutritional values for human.Therefore,at present,the development of economic benefits of tea plant mainly focuses on the extraction,purification and deep processing of the nutritional components of tea leaf and flower.Among them,tea flower buds will last for a year from differentiation to fruit ripening,This long-term reproductive growth is bound to compete with the vegetative growth of tea leaf buds for organic matter and nutrients,while flowering is the transition from vegetative growth stage to reproductive growth stage.Therefore,it is necessary to analyze the molecular mechanism of tea plant flower transition,and reasonably control of flowering time,so as to obtain high-yield tea leaf and high-quality tea flower,and provide theoretical support for the improvement and development of the tea industry.In the regulation pathways of floral induction,CO(CONSTANS)and SVP(SHORT VEGETATIVE PHASE)genes play a crucial role in photoperiod pathways and temperature pathways respectively.Among them,the CO gene acts as a bridge gene in the light signal pathway and controls the transition of light signal into flowering signal.The SVP gene,as a typical MADS-box transcription factor,participates in the temperature regulation pathway and regulates flowering by activating or inhibiting the expression of downstream flowering genes.In order to elucidate the function of the tea plant CsCOL4 and CsSVP genes in flowering transition.In this study,"Camellia sinesis cv.Ziyangzhong",a representative species of tea plants in Shaanxi province,was used as the material to clone cDNA full-length of CsCOL4 gene from young leaves of tea plant by homologous cloning and RACE technology,and further bioinformatics analysis and phylogenetic analysis were conducted on CsCOL4 and CsSVP genes.Then its expression characteristics were analyzed by real-time quantitative PCR.Finally,Arahidopsis thaliana was over-expressed to analyze its function.The purpose is to preliminary revealed the regulatory mechanism of these two genes during the tea plant flower transition.The main conclusions are:1.The 732 bp homologous conserved fragment,598 bp 3' end and 341 bp 5' end were successfully cloned by homologous cloning and RACE technology from the young leaves of "Camellia sinesis cv.Ziyangzhong",and the cDNA full length of 1434 bp was obtained by further sequence splicing.The start codon of this gene is ATG,the stop codon is TAA,and its CDS sequence length is 1080 bp,encoding 359 amino acids.This gene is highly similar to the COL4 homologous proteins of Nicotiana attenuata,Ipomoea nil,Vitis vinifera,Theobroma cacao and other species by BLASTN alignment and bioinformatics analysis,N-terminus has two complete B?box zinc finger domains,whose configurations are all expressed as C-X2-C-X8-C-X7-C-X2-C-X4-H-X8-H(X stands for any amino acid),the C-terminus has a highly conserved CCT domain.Therefore,it's named CsCOL4,the GeneBank accession number is MH479450.1.2.Bioinformatics and phylogenetic analysis of CsCOL4 and CsSVP encoded proteins showed that the CsCOL4 and CsSVP genes belong to the CO gene family and the MADS-box transcription factor,respectively.Both proteins are hydrophilic proteins and are mainly phosphorylated by serine.Phylogenetic analysis showed that the CsCOL4 and CsSVP encoded proteins are structurally conserved and related to the taxonomic classification of the species in the phylogenetic tree,which was the most similar to the Rhododendron plants,and had similarities with the grape plants.The homology between COL4 and SVP homologous proteins is positively correlated with the genetic relationship among different species.3.Real-time quantitative PCR results showed that CsCOL4 and CsSVP genes were expressed in stem,young leave,early flower buds,late flower buds,fruit of tea plant,but the expression levels were different.CsCOL4 gene is highly expressed in young leaves and stems,and gradually decreased in early flower buds,late flower buds and fruits.CsSVP gene expression is highest in early flower buds,followed by stems and young leaves,and is relatively low in late flower buds and fruits.It is speculated that only high expression of CsCOL4 gene in leaves could initiate transcription of downstream FT gene and thus regulate flowering time.CsSVP gene may regulate the flowering time mainly by participating in the regulation of formation of tloral meristem,and then participate in the regulation of plant vegetative growth process.4.Overexpression of Arabidopsis showed that twitch time and flowering time in 35s::CsCOL4 and 35s::CsSVP transgenic Arabidopsis were significantly advanced.When the 35s::CsSVP transgenic plants were twitched,the number of rosette leaves was significantly reduced,and the leaves were thin and grew slowly.These results indicated that both CsCOL4 and CsSVP genes could promote the early flowering of Arabidopsis.The CsSVP gene also reduced the number of rosette leaves and inhibited its vegetative growth.In addition,the number of flowers of 35s::CsCOL4 and 35s::CsSVP transgenic Arahidopsis increased significantly,indicating that both CsCOL4 and CsSVP genes have the function of regulating the formation of floral meristems,and the effect of CsSVP was more significant.5.Real-time quantitative PCR was used to further detect the expression of flowering time-related genes in 35s::CsCOL4 and 35s::CsSVP transgenic Arabidopsis.The results showed that the expression levels of FT and SOC1 in 35s::CsCOL4 and 35s::CsSVP transgenic Arabidopsis were significantly increased,and the expression level of FLC,a negative flowering regulation gene,were significantly decreased in 35s::CsSVP transgenic Arabidopsis.It is suggested that the early flowering phenomenon of 35s::CsCOL4 transgenic Arabidopsis may be caused by overexpression of CsCOL4 to promote the expression of FT and SOC1.The early flowering of 35s::CsSVP transgenic Arabidopsis may be caused by overexpression of CsSVP,which promotes the expression of FT and SOC1 and inhibits the expression of FLC.