Toxic Effect Of Heavy Metal On Sparganium Stoloniferum And Its Gene Cloning Of Phenylalanine Ammonia Lyase

Heavy metals pollution becomes worse and worse. It not only reduces the quality of traditional Chinese medicine and drugs’safety, but also affects the international competitiveness of Chinese medicine directly. Heavy metal pollution on the effectiveness of medicinal plants and its security has become one of the key issues affected the quality of research in medicine. Its impact on the quality of medicinal plants may inhibit the formation and induce the expression of related genes interfere with the metabolic processes in medicine which lead to the quality changed. Through simulating heavy metal pollution in the water environment, using modern analytical techniques to analyze its structure, physiological and biochemical changes under the corresponding of environment changes. That will be an example of the impact on the traditional Chinese medicines’quality. The results of theoretical and practical significance will be guiding the production of medicinal plants and assessing of the safety of aquatic environmental risk in Chinese herbal medicines, adapting to understand the mechanism of aquatic ecological environment and then improving the quality of traditional Chinese medicine. This research’s contents and results include the following aspects:1. Studies on the effects of heavy metals on physiological ecology of Sparganium stoloniferum Buch.-Ham.A pot experiment conducted to investigate the influence of mercury and cadmium on the physiological index and the ultrastructure alterations in S. stoloniferum. Six concentrations of Hg2+、Cd2+ ranging from 0 to 100 mg-Kg-1 processe the medicinal plant of S. stoloniferum. Under the treatments of Hg2+、Cd2+, the photosynthetic rate, transpiration rate, chlorophyll content of leaves decrease with Hg2+Cd2+ concentration increased, photosynthetic rate and intercellular carbon dioxide concentration is significantly negatively correlated. The antioxidant enzymes that peroxidase (POD),catalase (CAT) and superoxide dismutase (SOD) showe some resistance under low concentrations, while showe inhibition under high concentrations. The activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) are induced in high level to eliminate and resolve ROS in order to stand up for stability of membrane systems. With more serious damages, the activities of protective enzymes decrease, the defense systems of cells could not protect cells, malondialdehyde (MDA) contents of plant cellular increase, and the integrality of cell membrane is destroyed.2. Studies on the effects of heavy metals on ultrastructure of Sparganium stoloniferum Buch.-Ham.With the increasing of Hg2+、Cd2+ concentrations, the number of chloroplast in diachyma cells is decreasing. The Shapes of chloroplast changed from oval to ball.The thylakoid system is also injured, and grana lamella is getting dim. The number of mitochondria is determined by Hg2+、Cd2+ concentrations, and the shape changes from rod-like to round or ellipse. The number of mitochondria changes with different concentrations of heavy metals Hg2+, Cd2+,the mitochondrial membrane structure and crest blurred, even the formation of vacuoles. Nuclei are also subject to varying concentrations of damage, nucleolus disappears, the disintegration of nuclei at high concentrations; while the membrane is also hurt, mainly for continuously forming intracellular membrane to deform the cell protrusions. The result demonstrates that in the same concentrations, the duress of Cd2+ on the photosynthetic characteristics, physiological indexes and ultrastructure in S. stoloniferum are higher than Hg2+. stoloniferum grow better in 10mg·Kg-1 of Hg2+、Cd2+, which show high resistance.3. Gene cloning, sequence analysis and effect of heavy mentals on enzyme activity of phenylalanine ammonia lyase in Sparganium stoloniferum Buch.-Ham.To clone the full-length cDNA encoding phenylalanine ammonia lyase (PAL),which is the key enzyme that links primary metabolism to secondary metabolism in S. stoloniferum and to perform bioinformatic analysis. With the total RNA as template, the full length cDNA of PAL in S. stoloniferum was cloned through homology-based cloning approach and rapid amplification of cDNA ends (RACE) technique. The bioinformatics of the cloning PAL gene was analyzed by DNAMAN and ExPASy. The full-length cDNA (2413 bp) of PAL gene was obtained (GenBank accession number KF633470), with an open reading frame (ORF) of 2151 bp and encoding 716 amino acid polypeptides. The relative molecular mass of PAL calculated was 1.98 x 105, the isoelectric point was 4.84, and there was no signal peptide in PAL. The protein sequence contained the active center sequence GTITASGDLVPLSYIAG.The cDNA encoding PAL from S. stoloniferum is cloned and reported for the first time. This work provides a scientific basis for exploring the biosynthetic pathway of the medicinal ingredient and improving its quality in S. stoloniferum.