Cloning Of Floral-specific Promoter, Transcription Factor And Transformation Into Lily

Lily is widely favored by people for the characters of the big flower, graceful posture, and strong fragrance. As one of the rare ornamental, it is suitable for use in the garden ,a shrub border, potted. Thus our study focused on the improvement of lily flower color and shape.In the first part, a polymerase chain reaction(PCR)amplification was used to isolate a flower-specific expression promoter PCHS from Arabidopsis genomic DNA. Sequence analysis revealed that PCHS was 531bp in total length and shared significant nucleotides identity 99% with Genbank databases, containing four flower-specific expression regulatory elements .Intermediate vector pPCHS was constructed by inserting the promoter to the upstream of GUS in pB1121,where the original CaMV35S promoter was replaced by the PCHS promoter. The flower,stem,leaf,and root of petunia hybrida were transformed via Agrobacterium tumefaciens. Histochemical localization of GUS activity indicated that,the PCHS promoter could confer a flower-specific expression pattern to GUS.In the second part, the research chooses carnation to be the materials, and extracts the total RNA of the petal. By Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technology, we obtained a floral-specific transcription factor CMB2 ,which is 637bp in lenth and had highly homologous 98%with that reported in NCBI. The result of Genscan indicated that it included an integrated ORF and encoded 204 amino acids.The analysis of conserved regions indicated that it had a very conserved MADS–box region and a conserved K-box region,which was identical with the typical structure of the MADS-box gene.Plant expression vector pPCHf,pPCCM were constructed through replaced the GUS in pPCHS by flower color gene Hf2 and floral-specific transcription factor CMB2 respectively. And construction of the PCM was replaced the GUS in pB1121 by CMB2. The construct was transformed to lily via Agrobacterium tumefaciens using the leaf disc method. Transgenetic plants were obtained by screening with the G418 resistance in the...