Establishment Of Analytical Method For Amino Acids In Rice And Its Response To Mercury Stress

Plant amino acids are a class of polar small molecules in plant metabolites.Their metabolism can indicate plant physiological state.Their synthesis pathways are complex and strictly regulated,especially when plants are facing biological or abiotic stresses.Because the physiological functions of specific amino acids are different,the response to stress will be different.Accumulation of some amino acids is one of the adaptation ways.In addition,amino acids molecules will be associated with metal ions in plants and participate in the detoxification process of metals.Mercury?Hg?is a typical heavy metal contaminated element.Its organic form,methylmercury?MeHg?,can be produced by biological process and enter the food chain,which seriously threatens human health.It has been found that rice grown in abandoned Hg mining areas can accumulate MeHg,but the migration pathway of Hg in rice growth and the mechanism of Hg bioaccumulation in rice are rarely reported.To better understand the key factors controlling Hg in rice,amino acids,the essential composite of protein were selected for investigation,providing a prospective for alleviating the problem of Hg contamination in rice.In the thesis,a method for the determination of amino acids in rice was established.The distribution of amino acids in rice at organ level and subcellular level was determined by potted rice experiment in soil culture and by collecting rice samples in Hg mining area,respectively.The changes of amino acids in rice under different Hg concentrations were compared,and the coupling effect of amino acids and Hg in rice was further discussed.The main work and data results are as follows:A method for the determination of 18 kinds of amino acids in rice was established by pre-column derivatization of AQC and UPLC,using amino acid column and alpha-aminobutyric acid?Aaba?as internal standard substance.Within the concentration range of 0.025-0.5?mol·mL^-1,the correlation coefficients of each amino acid are between 0.9923-0.9999,which indicates that the peak area and concentration of amino acid show a very good linear relationship.The selectivity of each peak is good,the intra-day and inter-day accuracy of peak area and retention time is good,the detection limit and quantitative limit are low,and the recovery rate of amino acid is better than 80%,mostly in the range of 90-110%.The results of all methods showed that the method was sensitive,accurate and accurate for the determination of amino acids in rice.Responses of amino acids at organ level to mercury in rice showed that the total amino acids?TAA?contents in rice organs were 1.4-2.3 mg 100mg^-1,0.79-1.4 mg 100mg^-1,2.9-4.9 mg 100mg^-1and 2.4-5.0 mg 100mg^-1,respectively.Amino acids were divided into five groups according to side chain structure:aliphatic?Ali?,aromatic?Aro?,heterocyclic?Het?,sulfur-containing?Sur?and hydroxyl amino acids?Hyd?.Ali content was the highest in all organs of rice,while Sur content was the lowest.The correlation between TAA and total mercury?THg?in rice organs was root>stalk>leaf>grain from strong to weak,which was consistent with the accumulation trend of THg in rice organs.The significant correlation between TAA and MeHg was root>leaf>stalk>grain,and the correlation of TAA-THg in different organs was root>stalk>leaf>grain.This may be due to the different binding ability of amino acids in stalk and leaf to THg and MeHg.Amino acids were classified according to side chain structure,and the correlation of various amino acids?AA?with THg and MeHg in rice organs was analyzed.It was found that the correlation of fatty amino acids in root,stalk and leaf with THg and MeHg was the most significant,while the correlation of Sur with THg and MeHg in rice was the most significant.Studies on the subcellular levels of THg and TAA in rice organs showed that the subcellular distribution of TAA in rice organs was organelle?Or?>cell wall?Cw?>cell fluid?Cf?.The highest and lowest amino acids in Cw were Ali and Sur,respectively.There was a negative correlation between THg-TAA in organelles of rice,but there was no obvious regularity in Cw and Cf of organs.The correlation between various amino acids and THg in rice was analyzed.There was a weak correlation between THg and amino acids in root,leaf and panicle subcellular components.However,significantly positive correlations between THg and amino acids in stalk cell wall,as well as between sulfur-based amino acids Sur and Ali?Hyd in panicle Cf were observed.