Explore The Absorption And Transporter Of Cordyceps Militaris For Selenite And The Related Gene

To preliminary explore the cordyceps militaris transport pathway of selenite,determine the protein in selenite transportation and regulatory mechanism,this study through the following experiments respectively,different pH,selenite concentration on the effects of the cordyceps militaris selenium absorption and the middle elemental selenium selenium absorption process of electron microscope,cordyceps militaris absorption kinetics experiment and comparison of selenite of cordyceps militaris under different inhibitors on the change of the selenium absorption,to explore the different cases of selenium may transport way,and through bioinformatics method,from the protein database than the possible transshipment protein-coding genes,Through experiment under the condition of transporters corresponding m RNA expression quantity change,based on the analysis of the above experimental results from many aspects validation,explore the cordyceps militaris absorption selenite possible regulating mechanism in the process.The main results are as follows:1.Effects of different selenite characteristics（pH value）and concentration on the selenium transport pathway of cordyceps militaris.By biomass,selenium absorption amount,unit mass selenium content point of view,selenium-rich selenium-rich indexes such as efficiency,through factor analysis foundation,the simplified and stable selenium-rich optimal conditions,pH = 8,selenite concentration for 30 mg/L,the optimal average biomass is 2.067 mg/L,selenium absorption amount to an average of 2.715 mg,selenium-rich quantity units of 10.756 mg/g,selenium-rich efficiency is about 90%.By single factor and double factors experiment to prove the existence of cordyceps militaris again according to different forms of a variety of transport pathway of selenite,and the different forms of selenite(SeO₃^2-,HSeO₃^-,H₂SeO₃)on transport protein affinity,under the condition of pH = 8 SeO₃^2-> pH = 5 conditions HSeO₃^-> pH = 3 under the condition of H₂SeO₃.In addition,different ionic form of hydrochloride in aqueous solution,the molecular weight,molecular polarity,take charge of different,there are differences in such aspects as space structure,to a certain extent,can be considered to different substances,found in the results of different selenite form of activation and the regulation effects of cordyceps militaris transporters,and cordyceps militaris for different absorption and assimilation of the compatibility of selenite is different.2.Effects of different selenite characteristics（pH value）and concentrations on the production of elemental selenium in cordyceps militaris.In the process of enrichment of selenium,the "red selenium phenomenon" appeared in some microorganisms,and there were nano-level single selenium,and the principle was used for electron microscopy.Under the same conditions of pH = 5,different selenite concentration conditions,with selenium concentrations from the gradual adjustment 20 mg/L to 200 mg/L,and found experiment elemental selenium produced mainly in the intracellular,and show the cordyceps militaris for the absorption and assimilation of selenite saturation phenomenon.Conditions and in the same concentration of selenite（25 mg/L）under the condition of different pH value（pH = 3,5,8）,the elemental selenium in selenium-rich cultured cordyceps militaris hyphae had obvious different particle number,to a certain extent,also illustrates the different pH value under the condition of different forms of transport pathway of selenite differences,including pH = 3 when transfer efficiency and low selenium content inside the cell.3.Kinetic characteristics of selenium transport pathway in cordyceps militaris.The linear equation for the absorption of selenite in pH=3 is y=0.002917x+0.02509,and the determination coefficient R²=0.9492;The concentration fitting curve is a nonlinear curve.Km=76.92 mg L^-1 h-1mg^-1,Vm=0.8986,and the determination coefficient R²=0.8986.Linear equation is pH = 8,y = 0.007752 + 0.02506 x,determination coefficient R² = 0.8454 and pH = 8 non linear equation fitting,the determination coefficient R² is 0.8422,indicating there H₂SeO₃ transportation is given priority to with passive transport,HSeO₃^-transportation is given priority to with active transportation,but SeO₃^2-transport may be to active transportation and passive role at the same time.Through the experimental results of selenite and the results of electron microscopy,it is concluded that there are many different ways to transport the cordyceps sinensis(SeO₃^2-,HSeO₃^-,H₂SeO₃)in different forms.4.The amount of inhibitor was used to investigate the selenite transporter protein in cordyceps militaris.Using water channel protein inhibitors and anion channel protein inhibitors in different pH conditions,in view of cordyceps militaris on different absorbed form selenite inhibition experiment,the results showed that water channel protein in selenite transportation is an important transporter,and SeO₃^2-,HSeO₃^-,sensitive anion channel mainly by volume.By using phosphate and sulfate in different pH conditions,the experiment was carried out to inhibit the morphological absorption of different selenite.pH = 3,high phosphorus and low phosphorus condition,low phosphorus treatment after inhibition rate reached 40%,high phosphorus treatment after inhibition rate reached 51%,pH = 8,high phosphorus treatment cordyceps militaris absorb the content of selenite there is no significant difference,low phosphorus treatment after inhibition rate can reach 14%;At pH=8,there was a significant difference in the absorption of selenium in the low-sulfur treatment group compared with the control group.The inhibition rate of low sulfur treatment was 42%,and the inhibition rate of high sulfur treatment was 19.5%.PH = 5 with low phosphorus and low sulphur can absorb significant differences with the control,low sulphur and low phosphorus treatment after inhibition rate can reach 27%,and a single adding phosphate or sulfate experimental results under the condition of pH = 5 no inhibition phenomenon.The results can be used to determine the phosphate transport pathway and sulphate transport pathway of cordyceps sinensis,but the transport efficiency is related to the morphology of selenite.5.The biological information of the selenite transporter protein in cordyceps militaris and its regulatory mechanism were explored.Choose Os NIP2 bioinformatics method,according to the literature: 1 transporter（sulfuric acid）,pho84,pho89 transporter（phosphoric acid）,SUL2 transporter（sulfuric acid）and cordyceps militaris protein database for protein sequence alignment select various assumptions of sulfuric acid in cordyceps militaris turn proteins,transporters,sulfuric acid,phosphate transporters,and combined with crop cultivation science experimental results design filter condition,by RT-PCR technique to detect the expression of gene,reliable screening selenium transport protein coding genes,Os NIP2:1-analogue CCM₀0972,Pho84-analogue CCM₀9308,sul2-analogue CCM₀0881.Finally,through the design of different cultivation conditions on the assumption that transporters m RNA level of control experiment,the experiment of genes expression in accord with in this paper,the experimental results of cultivated experiment conclusion,cordyceps militaris water channel protein genes is regulated by the selenite form and concentration,and phosphate transport protein gene is affected by pH,selenite and regulation of concentration sulfate transporters may have the same sulfate and saccharomyces cerevisiae SUL2 depend on the deactivation mechanism.