Cloning And Sequence Analysis For The Key Genes In Anthocyanin Biosynthesis Pathway Of Acer Palmatum

Acer palmatum,one of perennial herbs of the genus Aceraceae,is one kind of important and famous foliage plant with multicolor leaves,which has an extensive application and attention. However,the molecular mechanism of color emerging involved-leaf plant are poorly understood. Furthermore,the cloning and expression of genes involved in coloration of leaf in Acer palmatum are not reported up to now. The limited understanding about the mechanism of color emerging in colored-leaf delayed the development of molecular breeding and leaf color engeneering of foliage plant with Acer palmatum representative. Because anthocyanins play a key role informing colorful leaf,in this dissertation, four key enzyme genes in this pathway has been cloned by using bioinformatic method and RT-PCR.We got the main results listed below:1. Clumn plant RNAout, TRIzol reagent method and improved CTAB methods were chosen for comparison analysis of total RNA extraction from Acer palmatum. These results demonstrate that the improved CTAB method is suitable for the total RNA isolation from the leaf of Acer palmatum.2. Total RNA was isolated from the leaf of Acer palmatum and acted as template for reverse transcription polymerase chain reaction ( RT-PCR). The full-length cDNA encoding a transcriptional factor was cloned via RACE-PCR method and the cDNA contained an ORF of 1104bp encoding 337 amino acids, and four conserved WD repeat motifs were presented in the C-terminal of amino acid sequences,. Through BLAST software in NCBI,the nucleotide sequence and the amino acid sequence of Acer palmatum were analyzed. The results showed that the nucleotide and amino acid sequences of the cDNA in Acer palmatum were highly homologous with those of the WD40 transcription factor in other species, which suggested the sequence is WD40 transcription factor gene . The gene was accepted by Genbank (Accession numberJQ796698).3. The full-length CHS Structural gene of Acer palmatum was cloned via RT-PCR method and the cDNA contained an ORF of 1314bp encoding 438 amino acids.Through BLAST software in NCBI,the nucleotide sequence and the amino acid sequence of the leaf of Acer palmatum were analyzed. The results showed that the nucleotide and amino acid sequences of the cDNA in Acer palmatum were highly homologous with those of the CHS structure gene in other species, and have a same all active site with Acer nikoense Maxim, which suggested the sequence is CHS gene.The sequence was accepted by Genbank (Accession number JQ796699).4. The genome DNA was isolated from the leaf of Acer palmatum and acted as template for polymerase chain reaction. The F3H gene was cloned via PCR method and the sequence is 2237bp encoding 285 amino acids, containing two introns and five 2-ODD conserved motifs. Through BLAST software in NCBI,the nucleotide sequence and the amino acid sequence of the leaf of Acer palmatum were analyzed. The results showed that the nucleotide and amino acid sequences of the DNA in Acer palmatum were highly homologous with those of the F3H genes in other species, which suggested the sequence is F3H gene.The sequence was accepted by Genbank (Accession number JX070083).5. Genome DNA of Acer palmatum was acted as template, a ANS structural gene was cloned via PCR method and the sequence is 448bp.