| As a principal species of woody oil plants in the South of China, Camellia oleifera is one of the four most famous edible oil woody plants including Elaeis guineensis, Olea europaea and Cocos nucifera. Camellia oil is mostly composed of unsaturated fatty acids (UFA), such as oleic acid and linoleic acid, and the average content of UFA is above 90%. So Camellia oil is a high quality edible oil. However, because of the hight frequency of pest and diseases happening, there are many serious impact on the development and economic value of Camellia oleifera. At present, phenylalanine ammonia-lyase(PAL) is been more researched as well as plant disease resistance, which is also the key enzyme that catalyzes the first reaction in phenylpropanoid pathway, Phenylpropanoid has an important part in plant growth, disease resistance, resilience and plant supporting system. Therefore, the genetic manipulation to PAL from Camellia oleifera has an important practical significance. This thesis carried out a number of studies in gene clone,identification and expression vector construction of PAL Gene from C. oleifera.We have reached the flowing conclusion:1. Molecular clone and bioinformatics analysis of PAL Gene from C. oleifera. In the constructed cDNA library of Camellia oleifera by our laboratory,we identifyed the length cDNA of PAL gene, which has 2118bp. After we compared with the nucnic acid database in other species,we believed that we have get the full length of PAL encoding 705 amino acids. Using a series of bioinformatics analysis,such as isoelectric point, molecular weight, membrane structure, hydrophobicity, functional secondary structure prediction analysis. We found that the hydrophobic index of PAL was from-2.656 to 3.244,and in the protein of PAL the 110,340 and 480 amino acids showed hydrophilic.PAL protein has two obvious transmembrane domain,in whose secondary structure a-helix was 52.55%,βfold was 6.23%, random coil and other structures reached 41.22%. Looking at the overall structure of PAL protein, we found that a helix was the main structural elements of PAL from Camellia oleifera, at the same time,random coil were scattered throughout the protein.2.Full-length genome sequence of PAL Gene from C. oleifera Use the total DNA as a template,cloning Camellia PAL genomic sequence by PCR reaction,the result showed that the gene has an intron,which length is 768bp, situated downstream of start codon about 372bp, and this intron had the conserved sequence:GT/AG. 3.Prokaryotic expression of PAL Gene from C. oleifera.The fragment encoding PAL protein from Camellia was inserted into a prokaryotic expression vector pET-28a and was overexpressed in E coli BL21.We lowered the incubation temperature to 20℃for 20h. extracted bacterial crude enzyme,detected enzyme activity of the recombinant protein,the conclusion was that optimum temperature of protein is 70℃, the optimum pH is 8.7. And under optimum conditions, PAL enzyme activity was 2.583×10-2U, at last,we calculate the PAL specific activity was 36.38μmol/min·g pro. Compared with other literature reports, the recombinant protein specific activity was lower than soybean, parsley,but higher than the red yeast,as a whole, the recombinant protein's activity is not very high, the reseason for this phenomenon was there are some differences between different species.4.Eukaryotic expression. of PAL Gene from C. oleifera.pBI121 vector and The fragment encoding PAL were both digested by Xba I and BamH 1, and purificated, connected, at last constructed the plant expression vector pBI-PAL.We transformated the plant expression vector into wild-type Arabidopsis, obtained To generation seeds, screened by the resistance medium,received a number of positive plants with full length PAL gene.We extracted DNA from seven positive plants. After using PCR techniques,we can conclue that there are five transgenic Arabidopsis with PAL. |
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