Molecular Cloning Study Of A Cystatin Gene From Populus Tomentosa

The technology of gene control is one of the technological systems of sustainable pests management in forest (SPMF). Finding self-determination property right functional gene can do better for our country's forestry protection workers to use this technology. The cysteine proteinase inhibitor (cystatin) (CPI) is a protein super family and exists comprehensively in animal and plant. The plant CPI has been regarded as a new family of the CPI super family (Phytocystatin). Because it can kill insects by inhibiting the digestive function of the cysteine proteinase in gut, it has been used in introducing gene engineering comprehensively as an important insect-resistant factor. This study focuses on the molecular cloning, sequencing and identification of the CPI gene of Populus tomentosa and the analysis of this gene. The major results of this dissertation were as follows:According the conserved amino acid motifs of the Phytocystatin, we had designed the special primers and used them to amplify the conserved sequence fragment of the CPI gene from the cambium of Populus tomentosa. The amplified fragment was about 165 base pairs as we expected. This indicated that our designation of the premiers was adequate.A cDNA fragment was obtained from the RNA of the cambium of Populus tomentos through RT-PCR. This fragment was cloned into pGEM-Teasy vector. The insert was identified by PCR amplification and sequencing. The sequence of the amplified cDNA fragment was 696 base pairs. Alignment with all phytocystatins in Genbank showed that they were identical greatly. Therefore it is believed that the fragment was amplified fromthe CPI gene.Using the same premiers a DNA fragment from the genome DNA of Populus tomentosa was obtained with the PCR amplification. It was 1950bp in length. After it was cloned in pGEM-Teasy vector, the insert was identified by PCR amplification and sequencing. The alignment with CPI cDNA could identify the concrete location of the extron and intron.