Molecular Cloning And Expression Of Chalcone Synthase Gene And Phenylalanine Ammonia-lyase Gene From Ginkgo Biloba

The flavonoids of Ginkgo biloba had many pharmaceutical properties for human health. The studies on increasing flavonoids in Ginkgo biloba had been more and more popular and important. Until now, many methods had been carried out on increasing flavonoids in Ginkgo biloba, and it was a available method via physiological and biochemical to regulate flavonoids content. At the same time, it will be a a available method by gene engineering to increase flavonoids content in the future. There were many enzymes in flavonoids metabolic synthesis pathway, but chalcone synthase (CHS) and phenylalanine ammonia-lyase(PAL) was the key enzyme. So, carrying out the studies on these two enzymes was very important valuable for theory and application.DNA and RNA were isolated respectively from Ginkgo biloba leaves by CTAB method, and a CHS full-length cDNA and a PAL gene fragment was cloned from Ginkgo biloba. Temporal expression of CHS and PAL gene was analysed. In order to study the interrelation of expression level of CHS and PAL gene fluctuation of CHS and PAL activity and flavonoids accumulation, a detailed investigation and analysis was carried out. The results as following:1. The DNA and RNA were isolated respectively from Ginkgo biloba leaves by CTAB method. The result showed that DNA was high quality, and could be digested completely with EcoR I . RNA obtained from Ginkgo leaves, which Gel electrophoretogram revealed that there were 28s rRNA and 18s rRNA, and A260/A280 approximated to 2.0, cDNA could be used to RAPD amplifying. These results indicated that RNA was high purity and good quality. The CTAB method was especially useful for the DNA and RNA extraction of plant materials that contained plenty of polysaccharide and polyphenol.2. The genomic DNA sequence of CHS gene fragment was cloned from Ginkgo biloba by using a pair degenerate primers. Which was 863bp long, and GenBank accession number was AY563038. CHS gene 31 end genomic DNA sequence was cloned by thermal asymmetric interlaced PCR (TAIL-PCR) method, its length was 1238bp, and GenBank accession number was AY945936.3. A CHS gen; was cloned from Ginkgo biloba by rapid amplification cDNA ends (RACE) method, and it was the first gene of key enzymes involved in flavonoids metabolic pathway in G biloba. The full-length (named as Gbchs) was 1608bp with poly(A) tailing and it contained a 1173bp open reading frame (ORF) encoding a 391 amino acid protein, and GenBank accession number was DQ054841. Gbchs was found to have extensive homology with those of other plant CHS genes via multiplealignments. The active sites in CHS protein of Medicago sativa were also found in GbCHS. Molecular modeling of GbCHS indicated that the three-dimensional structure of GbCHS strongly resembled that of M. sativa (MsCHS), implying GbCHS may have similar functions with MsCHS. Phylogenetic tree analysis revealed that GbCHS had closest relationship with CHSs from gymnosperm plants. The genomic Southern blot analysis indicated that Gbchs belonged to a multigene family.4. A PAL gene fragment (named as Gbpal) was cloned form Ginkgo biloba by using a pair degenerate primers, its length was 862bp and encoded a 287 amino acid protein, and GenBank accession number was AY578145. Gbpal was found to have extensive homology with those of other plant PAL genes via multiple alignments. The active sites in PAL protein of Oryza sativa and Zea mays were also found in GbPAL. Phylogenetic tree analysis revealed that Gbpal had closest relationship with PALs from gymnosperm plants. The genomic Southern blot analysis indicated that PAL of Gingo biloba belonged to a multigene family.5. It was analysised for the change of CHS mRNA relative expression and PAL mRNA during the growth and development of Ginkgo biloba leaves via semi-quantitative RT-PCR method, and linearity regression analysis was took on it with curve of CHS activity PAL activity and flavonoids accumulation. The results showed that fluctuation of CHS mRNA relative expression and CHS activity was identical, CHS activity may be replaced by CHS mRNA relative expression. During the whole process of growth and development of Ginkgo biloba leaves, the fluctuation of CHS mRNA relative expression and flavonoids accumulation was almost same, the expression level of CHS mRNA directly determined flavonoids accumulation; The fluctuation of PAL mRNA relative expression and PAL activity was identical, PAL activity may be replaced by PAL mRNA relative expression. During the evening of growth and development of Ginkgo biloba leaves, the fluctuation of PAL mRNA relative expression and flavonoids accumulation was identical, the expression level of PAL mRNA played a key role in flavonoids biosynthetic pathway.