Study On Molecular Cloning, Expression Pattern,Sequence Variation Of ECE Clade Genes And Over-expression Of PhTCP1in Petunia

TCP genes encode plant-specific transcription factors. The TCP gene family was first described in1999, as a small group of plant genes encoding proteins sharing the so-called TCP domain, a59-amino acid basic helix-loop-helix (bHLH) motif that allows DNA binding and protein-protein interactions. This domain was initially identified in four proteins encoded by apparently unrelated genes, from which the name ’TCP’ was derived:teosinte branched1(TB1) from maize (Zea mays), CYCLOIDEA (CYC) from snapdragon (Antirrhinum majus), and the PROLIFERATING CELL FACTORS1and2(PCF1and PCF2) from rice (Oryza sativa). Phylogenetic analyses indicate that two types of TCP protein can be distinguished:Class Ⅰ and Class Ⅱ. In angiosperms, class Ⅱ can be further subdivided into CIN clade and ECE clade (or CYC/TB1clade). The ECE clade experienced two duplications at the base of core eudicots, giving rise to three types of genes:CYC1, CYC2and CYC3. ECE-CYC1and ECE-CYC3clade genes are known as regulators in shoot branching. ECE-CYC2clade genes are known to play critical roles in patterning floral dorsoventral asymmetry (zygomorphy) in Antirrhinum majus and other plants with bilateral flowers. Petunia processes slightly zygomorphic flowers and rich shoot branching variations, and can be genetically modified easily. It should be a good material for studying the function of the ECE clade of TCP genes and creating novel flower. So we decided to clone the ECE clade TCP genes from petunia and study their functions.Degenerate primer PCR method was used to isolate the ECE clade TCP genes from petunia. RACE (rapid amplification of cDNA ends) method was used to clone the full-length cDNA sequences. Sequences analyses were carried out using the DNASTAR sequence analysis software. Real time quantitative PCR method was used to study the expression pattern of the ECE clade TCP genes in different petunia tissues. The main results are as follows:1. Cloning and analysis of the ECE clade TCP genes in petuniaGenomic DNA and cDNA from petunia inbred line GL8were used as templates. Primers for amplifying the TCP-R region sequence from gnomic DNA were designed from published ECE clade TCP genes, in the TCP domain (fowared primer) and the R domain (reverse primer). The obtained TCP-R region sequences which putatively belong to the ECE clade were used for designing RACE and TAIL PCR primers. Four full-length cDNA sequences and their corresponding gnomic sequences were obtained, and designated as PhTCP1PhTCP2, PhTCP3and PhTCP4respectivly. The GenBank accession numbers for cDNA are JQ400104～JQ400107, for genomic DNA are JF274249～JF274251and JQ400103. The four genes encode proteins of406,332,341and333amino acids respectively. The PhTCPl is a gene without intron. Phylogenetic analysis showed that PhTCPl belongs to the CYC3clade, PhTCP2belongs to CYC2clade, PhTCP3and PhTCP4belong to CYC1clade.2. Sequence variation of the petunia ECE clade TCP genesGenomic DNA sequences corresponding to protein-coding regions of the four TCP genes were amplified from P. axillaris, P. integrifolia and four P. hybrid varieties:V26, MD, GL8, DT and CD respectively. Analysis of the PhTCP1sequences showed no difference among the six materials. The PhTCP2sequences showed difference among them, with MD the highest and slight variation among the other materials. The PhTCP3sequences can be clearly divided into two clades, with P. integrifolia in one and the other5materials in another. The6materials also clustered to two groups according to the difference of PhTCP4sequences, with P. integrifolia and CD in one and the others in another.3. Expression analyses of the petunia ECE Clade TCP genesFluorescent quantitative PCR was employed to analyse the expression level of the4cloned genes in different organs of adult petunia plants. The results showed that all the four genes were expressed mainly in axillary buds, which supported a role during bud development. Besides expressing at high levels in axillary buds, PhTCP1also expressed at low levels in apical buds, flower buds, stems and leaves; PhTCP2expressed at low levels in roots and stems; PhTCP3expressed at high levels in stamens and low levels in roots and stems; and PhTCP4expressed at middle levels in leaves and low levels in big flower buds. The TCP gene expression levels in axillary buds at different nodes from10-leaf stage petunia seedlings were compared by qPCR to detect the change trends of expression levels of different genes along the main stem. PhTCPl and PhTCP3mRNA levels changed reversely along the main stem. PhTCPl levels were highest in nodes9-10that were near the apex, and lowest in cotyledonary nodes, far from the apex, while PhTCP3levels were highest in cotyledonary nodes, and lowest in nodes9-10. PhTCP2levels were higher in nodes1-7and cotyledonary nodes than in nodes7-10. PhTCP4mRNA expressed at relatively high levels in all detected nodes.4.Overexpression of PhTCP1gene in petuniaPhTCPl and PhTCP1::SRDX sequences were first cloned under the control of double35S promoter and35S terminator respectively in an auxiliary vector. Subsequently the expression box were excised from the auxiliary vector and inserted into the MCS of pCAMBIA2301binary transformation vector. After confirmed by restriction digestion and DNA sequence analysis, the destination vectors were designated as pCMF505and pCMF506, which were introduced into petunia V26via Agrobacterium tumefaciens-mediated leaf disc method.44and48independent transgenic lines transformed with pCMF505and pCMF506respectively were obtained and verified by GUS staining and PCR amplifying the target genes. The transgenic plants have been transferred to green house, and their phenotype changes are being investigated.