| Alisma orientalis(Sam.)Juzep.is a perennial aquatic herbal belong to Alisma,Alismataceae.The rhizomes are used as common Chinese medicine that have the functions of clearing damp and promoting diuresis,expel the heat-evil,hypolipidemic,and so on.Heavy metal pollution has become one of the important ecological problem increasly,with a serious threat to the quality and yield of Chinese traditional medicine.It may be that the expression of genes related are inhibited and induction,and then intervent to metabolism.This research was conducted according to the characteristics of Chinese medicine research and combined with the evaluation of traditional Chinese medicine quality standards.We studied the key enzyme squalene synthase prokaryotic expression,for offering the foundation for understanding the molecular mechanism of heavy metal pollution.In addition,we did the research of Hg2+,Cd2+ single stress on A.orientalis physiological and biochemical,ultrastructural,heavy metal accumulation regularity,active component content and other aspects.The results would guide the actual production better,offer a theoretical basis for guiding the development of environmental quality assessment.The contents and results of the thesis studying mainly include the following aspects:1 Expression,purification,and polyclonal antibody preparation of squalene synthase from A.orientalisThe squalene synthase cDNA(JX866770)from A.orientalis was subcloned into a bacterial expression vector pCzn1 in frame with N-terminal HIS6-tag,results showed a prokaryotic expression vector pCzn1-SS was successfully constructed.Then recombinant vector pCzn1-SS was introduced into Escherichia coli strain BL21(ROSETA).Recombinant sesquiterpene cyclase was induced by adding IPTG(Isopropyl-beta-D-thiogalactoside).SDS-PAGE analysis showed that the specific bands size was 46 kD as the same to expected,but the protein was inclusion body.Then we purified it with denaturation and renaturation method.Fusion proteins 6xHis-SS was used to immunize rabbits to develop a polyclonal antiserum.The antiserum was purified by antigen affinity purified chromatography column coupling of protein and agarose.The purified antibody was detected by ELISA and purified antibody titer is larger than 512 k.The concentration of purified antibody was 0.22 mg/mL determined by BCA protein concentration kit.The purity of antibody purification was more than 95%analyzed by coomassie brilliant blue staining.Finally,the rabbit antiserum was evaluated by western blot and showed its specility.Antiserum showed specific binding with the obtained protein from tuber,petioles,and leaf of A.orientalis.2 Relationship between photosynthetic characteristics and environmental factors in leaves of A.orientalisThe light-response,diurnal photosynthesis course and environmental factors in leaves of A.orientalis were measured by LI-6400 Portable Photosynthesis System.The results showed that the net photosynthetic rate(Pn),stomatal conductance(Gs)and water use efficiency(WUE)showed a diurnal variation of bimodal curve.The transpiration rate(Tr)and stomatal limitation(Ls)all had a single peak diurnal variation.The intercellular CO2 concentration(Ci)were a single valley type of diurnal variation.Correlation analysis indicated that there were positive correlations between Pn of A.orientalis and photosynthetic active radiation(PAR),Gs,Tr or WUE,but negative correlations between Pn and relative air humidity(RH),Ci or Ls.We got the conclusionsthat A.orientalis is the type of stomatal limitation and sun plant.The net photosynthetic rate of A.orientalis leaves has an obvious "midday depression"phenomenon.The results suggest that stomatal conductance,water use efficiency,photosynthetic active radiation and transpiration rate are the main environmental factors influencing the A.orientalis photosynthetic rate.This study could offer certain theoretical reference for A.orientalis rational cultivation,as well as provide a scientific basis for improving the quality and production of medicinal materials.3 Effects of mercury and cadmium on the growth,physiological indexes and ultrastructure in A.orientalisChina’s "Surface Water Quality Standard(GB3838-2002)" and "Soil Environmental Quality Standard(GB15618-1995)" as the main guiding principles,we conducted the research of Hg2+,Cd2+ single stress on A.orientalis physiological and biochemical,ultrastructural.The results showed that black spots appeared on leaves of A.orientalis under Cd2+ pollution,while under Hg2+ pollution,they had no spots and lost the green color evenly.And each organ tolerance from weak to strong was in order of old leaves,leaves,buds.The plants height and biomass reduced with the rise of concentration of Hg2+and Cd2+.In both cases,the chLorophyll content of leaves decreased.Lower concentration of Hg2+ and Cd2+ increased chlorophyll content,photosynthesis rate,activity of SOD,POD,CAT and protein content of leaves,while higher concentration and longer time of pollution decreased their physiological index.Membrane lipid peroxidation degree deepened,malondialdehyde(MDA)content showed tendency of rising.Ultrastructural observation showed chloroplast swelling to collapse,starch grains and lipid ball appear,mitochondria deformate and its cristae swelled and decreased,membrane was damaged,and nuclear membrane was broken.The nuclear membrane was broken,nucleoli disappeared.4 Accumulation and translocation of Hg and Cd in A.orientalisMercury and cadmium content in different parts of A.orientalis,water and soil were detected by liquid chromatography with inductively coupled plasma mass spectrometry(HPLC-ICP-MS),which indicated the ’regularity of harmful substances in A.orientalis.The results showed that mercury and cadmium content in different parts of the plant were tuber>root>leaf.When the concentration of Hg2+and Cd2+was 5 mg/L,the heavy metal content in tuber was 0.3781 mg/kg and 2.5020 mg/kg,which were more than the prescribed standards in "green trade standards of foreign trade and economic cooperation medicinal plants&preprations".A.orientalis showed a greater absorptive capacity of Cd concentration in rhizosphere soil.The accumulation coefficient of Cd was greater than Hg in the same concentration and the same parts of A.orientalis,and mainly concentrated in the tuber.Transfer coefficient of mercury and cadmium in A.orientalis is less than 1,and arising along with the concentrations.5 Effects of mercury and cadmium on the active component in the A.orientalisThe content of 23-acetyl alisol B in the rhizomes of A.orientalis was detected with HPLC method.Its content decreased obviously with the increase of hg2+ and Cd2+ concentration.When the pollution concentration was 10.0 mg/L,the component content decreased 55.32%/41.05%compared to the control respectively.Hg2+,Cd2+pollution inhibited the synthesis and accumulation of the active components... |
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