Study On The Construction Of TCS Gene Expressional Vectors And The Transformation Of Poplar

Poplar is one of the most important tree species in China. Due to its wide distribution, abundance of species, easy propagation, simple cultivation, strong adaptability, fast growing and excellent timber, poplar is mainly used for commercial forest, breakwind and afforestation. But poplar strains suffer from many kinds of disease and insect pest seriously, in addition to use of chemical pesticides over a long period of time, forestry production and construction of forestry ecology sustained great economic losses. Therefore the control of poplar disease and insect pest is an urgent problem to be solved and also is a most important task to us.Trichosanthin is obtained from root tuber of Trichosanthes kirilowii. TCS is ribosome inactivating protein (RIP) of single chain. TCS can resist 7 kinds of virus (for example tobacco mosaic virus), 9 kinds of pathogen fungi (for example rice blast) and 4 kinds of insect pest (for example aphides). TCS gene was transformed into poplar for boosting up anti-adversity and avoiding hurt by disease and pest. It can make no pollution, not to kill natural enemy, protecting biodiversity and keeping ecological balance.The plant expression vectors pC-tPro-TCS-GUS, pC-tPro-TCS, pC-pPro-2-TCS, pC-pPro-1-TCS-GUS and pC-pPro-1-TCS were designed and constructed with TCS gene. There were TCS gene and TCS promoter fragment in the pC-tPro-TCS-GUS and pC-tPro-TCS expression vectors. TCS gene was regulated by PAL inducible promoter in the pC-pPro-2-TCS, pC-pPro-1-TCS-GUS and pC-pPro-1-TCS expression vectors. The plant expression vector pC-tPro-TCS-GUS was constructed with TCS promoter and TCS structure gene integrated into upstream of instant expression of GUS gene carried by pCambia1381 expression vector. The plant expression vector pC-tPro-TCS was constructed with TCS promoter and TCS structure gene inserted into the multicolone sites of plasmid pCambial301. TCS gene has no instant expression of GUS gene in plasmid pC-tPro-TCS. The plant expression vector pC-pPro-2-TCS was constructed with TCS structure gene integrated into downstream of PAL inducible promoter carried by pPPG expression vector. TCS gene was controled by linked promoter-PAL. The ATG translation start codon of plasmid pPPG was found 12bp in front of the ATG translation start codon of TCS gene. It could translate four amino acids of fusion at N-terminal of recombinant trichosanthin. A transcription termination signal was found downstream of TCS gene. TCS gene was inserted between non-translated region of PAL promoter and upstream of instant expression of GUS gene carried by pPPG expression vector to construct the recombinant expression vectors pC-pPro-1-TCS and pC-pPro-1-TCS-GUS. TCS gene was controled by linked promoter- PAL. It couldn't translate fusion amino acids at N-terminal of recombinant trichosanthin. An instant expression of GUS gene was found downstream of TCS gene in the pC-pPro-1-TCS-GUS expression vector. A transcription termination signal was found downstream of TCS gene in the pC-pPro-1-TCS expression vector.The plant expression vectors pC-tPro-TCS-GUS, pC-tPro-TCS, pC-pPro-2-TCS, pC-pPro-1-TCS-GUS and pC-pPro-1-TCS were transformed into Agrobacterium tumefeciens LBA4404. TCS structure gene was amplified from the plasmids in Agrobacterium tumefeciens LBA4404. The result of the PCR analysis showed the five plant expression vectors had separately transformed into Agrobacterium tumefeciens LBA4404 .The five expression vectors with TCS gene were introduced for the first time into poplar (Populus simonii'X P. nigra) using Agrobacterium-mediated transformation. It provided essential data for further study of TCS gene in wide-ranging resistance to plant disease and insect pest, and also laid a theoretical foundation for obtaining more transgenic trees.