DNA And CDNA Cloning And Sequence Analysis Of Phenylalanine Ammonialyase Gene (Pal) And Chalcone Synthase Gene (Chs) From Fagopyrum Tataricum

Flavonoids represent a class of secondary metabolites which is widespread in the plant kingdom and may serve specific functions in flower pigmentation,plant defence against pathogens,and they also have generous medical activity to human.Their composition and distribution has important function to plant physiology.Flavanoids are products of phenylalanine metabolism branch path,and its key enzyme genes have been cloned from many plant materials.Becouase Fagopyrum tataricum is the only alimentary crop containing abundant flavanoid,the studies on flavanoids content,component and distribution in F.tataricum are developed recently.However,the research about flavanoid biosynthetic pathway is weak on F.tataricum.In this study,the technics of Genome-Walking and RT-PCR are used to clone Phenylalanine Ammonialyase Gene(Pal) and Chalcone Synthase Gene(Chs) from F.tataricum,which is helpful to clear the gene function and molecular evolution of Pal and Chs,to develop flavanoids biosynthesis control and transgenic breeding study.Our main findings are as follows:1.According to the conservative fragment of Pal gene from F.symosum and Chs genes from other plants,the primers were designed to amplify the two genes' fragmentsh from genomic DNA extracted from F.tataricums(cultivated in Xichang,Sichuan Province) by PCR.The results showed the products were amplificated with 493bp and 554bp,and the blasting of nucleotide and amino acide proved they were the conservative fragments of Pal gene and Chs gene,respectively.2.By using Genome-Walking technic,each 9 primers for Pal gene and Chs gene were designed on the basis of known sequences respectivly,and used to amplificate their unknown DNA sequence of the 3'-Terminal and 5'- Terminal from F.tataricums DNA. The sequence analysis indicated that we obtained the full length of Pal gene and Chs gene DNA sequences with 2617bp and 1632bp,respectively,and their amino acid sequences translated from the DNA sequences removal of putative intron were coincident with characterization of Phenylalanine Ammonialyase(PAL) and Chalcone Synthase(PAL) from plant.Then,two-pair special primes were designed and amplified the full length DNA of Pal gene and Chs gene of F.tataricums. 3.Two-step RT-PCR was performed with total RNA extracted from flower F. tataricum.Pal gene cDNA of F.tataricums was amplified,cloned and sequenced.The results comfirm the ORF of Pal gene cDNA is 2166bp with full-length coding 721 aa,and its DNA sequence has an intron locating between 420bp and 870bp.Nnucleotide and amino acid sequence blasting indicates Pal gene of F.tataricums has the low homology with other plants with 43%-74%and 66%-75%,respectively.Although there are several conservative fragments in its amino acid sequence,N-Terminal sequence takes on difference enormously.4.Total RNA extracted from flower F.tataricum was uesd into Two-step RT-PCR. Chs gene cDNA of F.tataricums was amplified,cloned and sequenced.The results demonstrate that the ORF of Chs gene cDNA is 1188bp with full-length coding 395aa,and its DNA sequence has an intron locating between 182bp and 625bp.Nnucleotide and amino acid sequence blasting indicates Chs gene of F.tataricums has the high homology with other plants with 45%-85%and 81%-94%,respectively.Furthermore,four conservative domains locate in neighborhood of its active center composed of four amino acid residues:MMYQQGC,VGQ(A/S)LF(G/S),AHPGG(P/Q)AILD and YGNMSSACVLFI.