Cloning, Identification Of Poplar Phenylalanine Ammonia-Lyase Gene And The Construction Of Its Expressional Vectors

Poplar is one of the most important tree species in China, due to its fast growing and easy pulping. How to improve its timer utilization efficiency remains a problem for tree breeders. This study focuses on reducing its lignin content by manipulation of lignin biosynthesis enzyme gene.PAL (Phenylalanine Ammonia-Lyase, EC 4.3.1.5) is the first enzyme ahead the multibranched phenylpropanoid metabolism including the lignin synthesis in higher plants. In the present study, we have amplified the PAL gene fragment using mRNA extracted from the developing xylem, cloned it in a T-vector, identified by sequencing and further constructed in the plant expression vectors. The major results of this dissertation are as follows:We have designed the forward and reverse primer and used them to amplify the PAL gene from poplar (Populus euramericana 74/76) developing xylem mRNA. The amplified fragment is about 600 base pairs as we expected. This indicated our designation of the primers is adequate.The fragment was cloned into pGEM-T vector. The insert was identified by restriction enzyme, PCR amplification and sequencing. The sequence of the amplified DNA fragment was 565 base pairs. Alignment with the Populus kitakamiensis cDNA sequence of PAL retrieved from EMBL nucleotide acid database (accession number D30656) showed that the first 400 base pairs in both sequences were almost identical. Therefore we believed that the fragment was amplified from the PAL gene. To introduce proper restriction sites for subsequent cloning into the plant expression vector, both the pGEM-7Zf (+) vector and the pGEM-T-PAL were cut by EcoR I to release the PAL fragment and the pGEM-7Zf (+) fragment. The two fragment were joined together to produce a new plasmid pGEM-7Zf (+)-PAL.The pGEM-7Zf (+)-PAL, the binary vector pBI121 and pBin438 were cut by Xba I and BamH I to release the PAL fragment and the large fragments of pBI121 and pBin438. After ligation of the PAL fragment with pBI121 or pBin438 large fragment, we obtained two expressional vectors, named pBI121-PAL and pBin438-PAL respectively. The vectors were further transformed into Agrobacterium tumefeciens LBA4404. The two constructions with sense and anti-sense direction of the PAL gene will be further introduced into poplar using Agrobacterium-mediated transformation to obtain the transgenic poplar plants. Both the PAL activity and the lignin content of the transgenic trees will be measured to study the effect on expression both sense and anti-sense PAL RNAs.