Study On The Structure And Function Of The4CL Gene Promoters And APX In Populus Tomentosa

Chinese white poplar(Populus tomentosa Carr.) is a kind of native tree widely planted in northern China. With the characteristics of wide distribution, fast-growing speed, strong environmental stress resistance, high wood yield and excellent material quality, they are widely used in paper and other industries. The knowledge of the regulation of lignin and flavonoid synthesis and the precise control of antioxidant system in Populus tomentosa are limited.4-coumarate:Coenzyme A ligase (4CL) is located on the branch point of phenylpropanoid biosynthesis pathway.4CLs are encoded by an array of gene family members in plants. In this thesis, the promoters of four4CL gene family members were cloned from Populus tomentosa genome and the promoter-GUS fusion constructs were generated and transformed into tobacco. The tissue-specific expression patterns directed by the promoters were analyzed.5’deletion analysis were carried based on the bioinformatic prediction of cis-regulatory elements, and EMSA of synthetic probe were used to identify the key regulatory domains and cis-acting elements of Pto4CL2promoter.Ascorbate peroxidase (APX) is a key enzyme of the ascorbic acid-glutathione (AsA-GSH) cycle, which is an important H2O2scavenging system. Three recombinant PtoAPX isoenzymes were expressed and purified. The enzyme activity and kinetic parameters were measured. Antigen peptides representing APX enzyme epitopes were synthesized and used to raise multi-clonal rabbit antibodies. The subcellular localization of PtoAPX isoenzymes was detected by immunogold electron microscopy. The PtoAPX-GFP fusion proteins were expressed in transgenic tobacco and used for confirming the subcellular localization by laser-scanning confocal microscopy. The PtoAPX gene over-expression vector was constructed and transformed into tobacco. Salt tolerance of the transgenic plants was measured.Based on the above mentioned experiments, the following results were obtained:1. The promoter regions of four4CL gene family members were cloned. A series of tissue-specific expression and stress response regulatory cis-elements were predicted in these promoters by bioinformatics analysis.2. The plant expression vectors of the four promoter-GUS reporter gene were constructed and transformed into tobacco, respectively. Activity analysis showed that the GUS expression sites and expression patterns driven by four promoters are different. The expression patterns regulated by Pto4CL4p and Pto4CL5p were similar. The tissue-specific expression patterns directed by Pto4CL2p and Pto4CL3p were also developmental stage specific.3. The expression patterns and activity of GUS directed by full-length and5’-delete Pto4CL2promoters were compared in transgenic tobacco to identify the regulatory regions of the promoter. The results showed that the-317to-292region associated with the expression in the epidermis and petals, and the deletion of the-266to-252nt region resulted in the loss of tissue specificity and a dramatic reduction in GUS activity.4. Electrophoretic mobility shift assays (EMSA) validated that an adenine and cytosine-rich (AC) element (-264to-255nt) and an abscisic acid-responsive element (ABRE)(-242to-235nt) in the Pto4CL2promoter would have functions for the complex expression profile and efficient basal expression, respectively. These results characterized the key elements regulating the expression of class II4CL promoters in higher plants for the first time.5. The recombinant PtoAPX2, PtoAPX6and PtoAPX7proteins were expressed and purified. Enzymatic kinetic analysis found that their Km values to H2O2are close but the Vmax values of PtoAPX6and PtoAPX7to H2O2are10times higher than that of PtoAPX2, indicating that the clearance of H2O2by PtoAPX6and PtoAPX7was more efficient than that by PtoAPX2. The Km and Vmax values of PtoAPX7to ascorbate (AsA) are higher than others, which suggests that PtoAPX7could be responsible for the removal of AsA in high concentration. PtoAPX6and PtoAPX7have the highest activity at20℃and weak alkaline environment, while PtoAPX6is able to maintain a high activity in a wide pH range.6. Polypeptides were designed and synthesized to obtain the PtoAPX isozyme targeting polyclonal antibodies. With the help of these antibodies, PtoAPX6was located to chloroplast and PtoAPX7was located to mitochondria in poplar cells by immunogold electron microscopy. The subcellular location was consistent with that of PtoAPX6and PtoAPX7-GFP fusion protein detected in transgenic tobacco cells. PtoAPX7was the first mitochondria targeting APX found in woody plant.7. The loss of water content, H2O2and MDA level of transgenic tobacco harboring sense PtoAPX7were significantly higher than those of the wild-type plants, which indicates that the salt tolerance of sense PtoAPX7transgenic tobacco was much higher. It suggests that PtoAPX7over-expression can effectively improve the salt resistance of plants.Structural characterization of the promoter belonging to class II4CL would lay a foundation for revealing the machinery of the regulational spatial and temporal expression. And it will provide a theoretical basis for the regulation of the secondary metabolites biosynthesis in Populus tomentosa.The study of PtoAPX enzymatic properties, subcellular localization and salt resistence of transgenic plants laid an important foundation for the machinery of antioxidant system precise regulation in woody plants.