Study On MRNA Differential Display Of Flower Color Correlated Genes In Rhododendron Molle G.Don

Rhododendron molle(Blume)G.Don,which belongs to Rhododendron,is a kind of yellow flowering plant with important medicinal value.Recently a red variant of this species was discovered.The aim of our research is to find the differences in terms of gene expression between the yellow and red ones in order to provide a molecular basis for the color change.Total RNAs from yellow and red Rhododendron molle G.Don were isolated respectively and then subjected to DDRT-PCR using different palmer combinations of 5' random primers and 3' anchor primers.We effectively optimized the RNA extraction method as well as the overall reaction system,especially the annealing temperature of PCR reaction.From this work,we have developed a set of parameters suitable for DDRT-PCR analysis of Rhododendron molle.The PCR products were then analyzed by 6% denatured polyacrylamide gel electrophorisis.Ten differentially expressed bands could be seen after silver staining with the sizes ranging from 150bp to 1000bp.Some differentially expressed cDNA fragments derived from the petals of Rhododendron were recovered and purified from the silver-stained polyacrylamide gel and re-amplified by PCR.Only four of the purified.genes(E08,H06,A07 and E06) gave one specifically re-amplified band after agarose gel electrophoresis.These candidate genes were cloned to pMD18-T vector and sequenced.We searched the homologous sequences of these cDNA fragments in GenBank using basic local alignment search tool(blast).The results suggest that the sequence of cDNA fragment E08 has 83% similarity to gene encoding butyrate kinase; H06 has 87% similarity to gene encoding skikimate transporter; A07 has 83% similarity to gene encoding fusion protein.These fragments may be involved in the metabolic pathways of cyanin in Rhododendron molle G.Don.Interestingly,fragment E06 does not exhibit significant similarity to any known gene in the database,we therefore suspect that E06 maybe a fragment of a new gene encoding protein possibly related to color determination in Rhododendron molle G.Don.This paper could serve as a reference on the search of genes possibly involved in the determination of flower color.This knowledge could make it possible to change flower color by genetic engineering in the future.