Study On Cloning And Identification Of Genes Related To Flower Color And Genetic Transformation Of Orchid

Flower color is a significant ornamental character which influence the ornamental value oflandscape plant. Orchid is one of the most valuable flowers, and its flower color improvementhas always receive much concern in orchid breeding. However, the development of orchidbreeding by traditional methods is very slow because of its long growth periods and low settingpercentage. Orchid breeding by transgenic approach will compensate the defects of traditionalbreeding. This research aims to clone and analyse genes related to flower color which can beused in orchid genetic transformation, which will provide scientific basis to get new variety oforchid with novel color through flower color related gene transfer.Genes related in the process of flower color formation were cloned from Saintpaulia andPetunia by nested PCR and PCR, and plant express vectors were constructed with these genes.The function of these genes were analysed through model plant transformation, and genetictransformation of orchid were carried out. Some scientific results were obtained as follows:F3’5’H gene was cloned from Saintpaulia by nested PCR, The total length of the genesequence was1612bp, which encoded508amino acid. It contained two exons and one intronand there was a85bp intron between site894and site980. Analysis in the NCBI shown thatthe similarity between this gene and other Saintpaulia F3’5’H genes was94%-99%, andsimilarity between this gene and genes of other family was more than70%. The sequence wassubmitted to the GeneBank, and the accession number was JF261509.DFR gene was cloned from the Petunia. The sequence of this gene was1122bp，whichencoded373amino acids. The similarity degree of this gene and anther Petunia DFR gene onNCBI (GeneBank accession no. AF233639) was100%.Chalcone isomerase gene(CHI) was cloned from the Petunia. The total length of the genesequence was653bp,210amino acids were encoded, Contrast with the NCBI, the similaritydegree of this gene and anther Petunia CHI gene on NCBI (accession no. AB543054) was100%. Plant express vectors of pCAMBIA1304-senseCHS, pCAMBIA1304-antiCHS andpCAMBIA1304-F3’5’H were constructed to function verification and genetic transformationof orchids. Restriction sites NcoⅠ and SpeⅠ were introduced to the terminal of the CHS geneby PCR, and restriction sites BglⅡ and SpeⅠwere introduced to F3’5’H as well. These geneswere constructed to pCAMBIA1304by digesting and ligating. Recombinant plasmid weretransformed into Agrobacterium tumefaciens strain LBA4404and the engineering bacteriawhich would be used in plant transformation was obtained.In order to analysis the function of the CHS and F3’5’H gene,we transfered sense andantisense CHS gene and sense F3’5’H gene into tobacco, and the results shown that the contentof flavonoids changes in transgenic tobacco. The content of flavonoids increased by16.2%-31.6%compared to the wild type in sense CHS transgenic tobacoo, and reduced by44.5%-76.4%in antisense CHS gene transgenic tobacoo. In F3’5’H gene transgenic tobacoo,the flavonoid content increased by4.0%-16.3%, which was smaller then the sense CHStransgenic tobacoo.Studies on the minimum lethal concentration of hygromycin screening were carried out onF7and other types of orchids. The results shown that the minimum lethal concentration ofhygromycin of tropical Orchids was higher than Chinese orchids.5species of orchid weretransfered by sense and antisense CHS gene and sense F3’5’H gene, the protocorm-like Bodieswere regenerated after transformation screened by hygromycin.