Isolation And Functional Verification Of CFCIN In Cymbidium Faberi

Cymbidium faberi, also known as Multi-flower orchid and Summer orchid etc, is a kind of terrestrial herbaceous plants, which belongs to Cymbidium, Orchidaceae. It is originated in China. It is the oldest cultivated and most popular orchid in China. The flowers are delicate, fragrance, together with high ornamental value and economic value. Therefore, people like them very much. Natural seed selection makes the wild source of Cymbidium damaged significantly. Moreover, the long cycles of crossbreeding and mutation breeding can not satisfy the market requirements. Genetic engineering techniques have direct purpose, through which can significantly shorter the breeding cycle. Furthermore, it can also overcome the distant hybridization incompatibility barriers. Therefore, the genetic engineering technique has become the most important method for cultivating new Cymbidium varieties. So far, there are few domestic or international reports on genetic engineering with Cymbidium.Here we chose Cymbidium as experimental material. CfCIN (gene of TCP family) in Cymbidium were isolated through genetic engineering technique. Gene sequences were analyzed, and then transgenic vector was created. Saintpaulia ionantha was transfected with the vector and transgenic plants were analyzed to described CfCIN function. Our research provide the theoretical bases for cultivating new Cymbidium varieties resources. Our main study results were listed as below.1. Gene cloning. Cymbidium was chosen as experiment material. Total RNA of flower bud was extracted used Trizol method. The full-length of CfCIN cDNA was amplified through RT-PCR, RACE technique. Amplified cDNA was confirmed by sequencing and homologous sequence analysis. The result showed that the cloned CfCIN was 1188bp, the Open Reading Frame (ORF) was 1161bp, encoding a protein with 386 amino acids. After compared CfCIN with TCP protein from other species, we found that CfCIN was highly homologous with TCP gene from Phalaenopsis, Dendrobium, etc. All of them contained typically conserved TCP-domain. Our systematic cluster analysis forecasted that CfCIN protein belonged to CIN subfamily.2. Construct transgenic vector. Firstly, CfCIN was cloned into pMD18 vector to produce pMD18-CfCIN. After analyzing the restriction enzyme cutting site on the plant vector pCAMBIA1302 and pMD18-CfCIN, restriction endonuclease cutting sites of Bgl Ⅱ and Spe I were chosen as cloning sites. The original plasmid pMD18-CfCIN and plant vector pCAMBIA1302 were performed double enzyme digestion and then ligation, transformation, generating transgenic vector. Results of double enzyme digestion and DNA sequencing showed that target expression vector of pCAMBIA1302-CfCIN was successfully created. The vector was used in the subsequent genetic transformation study.3. Genetic transformation. CfCIN gene was transformed into Saintpaulia ionantha by Agrobacterium-mediated method. Transgenic plants were identified by RT-PCR. CfCIN function was analyzed by comparing the phenotype of transgenic and wild-type plants.