Cloning, Expression Analysis And Overexpression Of PhTCP2in Petunia

Transcription factor is a kind of DNA binding protein which specifically interacts with cis-acting elements in the promoter region of eukaryotic gene.The transcription factors of TCP gene family,which have a common TCP domain, only exist in plants. According to the characteristics of the structural domain, TCP genes can be divided into two subfamilies, class Ⅰ and class Ⅱ. With the differences of TCP domain, Class Ⅱ is futher subdivided into two clades, ECE clade and CIN clade, and the ECE clades contains three subclads, CYC1, CYC2and CYC3. The previous research showed that the TCP genes family participated in the development of bilateral symmetry type of flower, controlling the branching of plants and regulating the development of leaves.Until now, most researches on CYC2genes have been focused on floral organs, and the research on CYC2genes in other tissues is limited. mRNA in-situ hybridization and promoter analysis show that AtTCPl could regulate the development of plant tissues and it expresses in petioles, inflorence, stems, particular locations of leaves, the flower meristem in incipient stage and leaf-bud meristem. The PhTCP2gene, belonging to the branch of ECE-CYC2, may involve in the development of flowers and stems.In this paper, PhTCP2gene was cloned from Mitchell Diploid (MD), then its mRNA expression leves were analyzed by qRT-PCR. The functions of PhTCP2gene are analyzed using over-expression and silence technology.1.Cloning of PhTCP2On the basis of cDNA sequence of PhTCP2gene (GenBank accession number: JQ400105)(ShiHui. Zou, etc,2013) downloaded from Genbank database, specific primers PT02F/PT02R are designed and synthesized. The full-length CDS sequence of PhTCP2was obtained by PCR, using genomic DNA of petunia MD variety as template.2.The transcription analysis of PhTCP2geneThe expression quantity of PhTCP2in different development stages and tissues in MD are analyzed by means of fluorescence quantitative PCR. The results showed that the PhTCP2transcripts were detected in organs including stems, leaves, axillary buds, flower buds, and opening flowers, and accumulated highest in stems of20-leaf stage plants. PhTCP2was gound to be expressed at a low-level in flower buds, opening flowers and leaves. These evidences suggested that PhTCP2regulates growth and development of MD.3. Over-expression of PhTCP2Using gene engineering technique, the target gene PhTCP2is linked to cloning vector of MF500, and then constructs a plant expression vector by ligating the recombinant to pCAMBIA2301. PhTCP2gene was transformed into MD by the Agrobacterium-mediated transformation method. After that, through a series of resistance selection, GUS staining detection and PCR assay, transgenic MDs were identified. RT-PCR is applied to identity the expression of PhTCP2in transgenic MDs. Plants having a high PhTCP2expression were chosen for further study. The results of phenotype observation indicated that transgenic MDs were taller than wild types.4. Over-expression of PhTCP2-SRDX in MDGene-silencing vectors were constructed by fusing the sequence of PhTCP2with SRDX and then inserted into pGREEN binary vector. The recombination vector was transformed into MD using Agrobacterium-mediated method. After resistance screening, transgenic plants were obtained. Under the stress of cremart and polymerase chain reaction detection, Transgenic plants were identified by spraying BASTA(?) and PCR dectetion. The results of phenotype observation showed that the leaves of transgenic MD were rounder than wild types.