Study On Regulation Mechanism And Effects Of Different Phosphorus Levels On The Arsenic Absorptionof Panax Notoginseng

Objective:Radix notoginseng（Chinese medicine）is the dry root and rhizome of Panax notoginseng（Burk.）F.H.Chen.Yunnan and Guangxi are its growing area.It is one of traditional and precious herbs in China and one of authentic medicinal materials in Yunnan.It has important medicinal and high economic value.Wenshan Prefecture is regarded as local area of Panax notoginseng,but it is seriously affected by the soil and excessive use of pestic.This study intends to use the principle of"phosphorus and arsenic,a pair of similar elements,have similar chemical properties"to change the phosphorus level.In order as to clarify the influence on the absorption and accumulation of notoginseng arsenic,and further discover to the relevant regulation mechanism.The results provide data support and theoretical references to the arsenic pollution of Panax notoginseng.Methods:1.Low phosphorus（3.7 mg/L）,medium phosphorus（136 mg/L）and high phosphorus（272mg/L）,3 phosphorus levels and low arsenic（4 mg/kg）,high arsenic（100 mg/kg）were used to treat the Panax notoginseng seedlings.All the Panax notoginseng seedlings were divided into8 groups:low phosphorus and no arsenic（lPnAs）,low phosphorus and low arsenic（lPlAs）,low phosphorus and high arsenic（lPhAs）,medium phosphorus and low arsenic（mPlAs）,medium phosphorus and high arsenic（mPhAs）,high phosphorus and non-arsenic（hPnAs）,high phosphorus and low arsenic（hPlAs）,high phosphorus and high arsenic（hPhAs）,8treatment groups and medium phosphorus arsenic-free（mPnAs）control group.By measuring the Panax notoginseng biomass,the content of saponin,phosphorus and arsenic in different plant parts and the concentration of available phosphorus and arsenic in the matrix,and to make sure of effects of phosphorus and arsenic on the absorption,growth and saponin accumulation of Panax notoginseng.The test of phosphorus was carried out by ultraviolet spectrophotometry;the determination of arsenic was carried out by atomic fluorescence spectrometry,and saponin content used HPLC.Simultaneous was calculated phosphorus and arsenic absorption transfer factors.2.Through interaction treatment of phosphorus and arsenic,the changes of antioxidant system in roots of Panax notoginseng were clarified.Determination of antioxidant system includes（superoxide dismutase,SOD）,（peroxidase,POD）,（catalase,CAT）activity and antioxidant substances（malondialdehyde,MDA）,（reduced glutathione,GSH）,（proline,Pro）,β-carotenoid content.SOD,CAT,MDA,GSH,Pro,β-Carotenoids were determined by ultraviolet spectrophotometry and POD was determined by guaiacol method.3.Selected lPnAs,lPhAs,mPhAs,hPnAs,hPhAs as 5 treatment groups and mPnAs as 1control group fresh plant roots,and sent them to the biotechnology company for transcriptome sequencing;analyzed transcriptome results and drew phosphorus arsenic based on the transcriptome results and the differential heat map of key enzymes in saponin synthesis was processed,and the phosphate transporter（Pht）gene in response to phosphorus and arsenic induction and stress was screened.Bioinformatics analysis was performed by various online software.4.Through qPCR,yeast heterologous expression,clear the target Pht gene expression characteristics,transport efficiency,mechanism of action and other functions.Results:1.Compared with group of arsenic-free low arsenic and high arsenic,the content of phosphorus and arsenic in stems and leaves of Panax notoginseng increased.Under the same arsenic level,the content of phosphorus and arsenic in roots and leaves decreased under different phosphorus treatments.Compared with arsenic-free arsenic and high arsenic,the biomass of Panax notoginseng decreased.In general,high phosphorus levels can promote the accumulation of phosphorus and arsenic in the roots of Panax notoginseng;the medium level of phosphorus can promote the increase of biomass in Panax notoginseng.2.Medium phosphorus level is beneficial to resist arsenic stress and promote the accumulation of notoginsenoside R₁,ginsenoside R_g1,R_b1 and panax notoginseng saponins,that is,medium phosphorus concentration resists low concentration arsenic stress and promotes the accumulation of notoginsenoside R₁ and ginsenoside R_b1.3.Under the same phosphorus level,high arsenic stress increases the content of SOD,POD,CAT and antioxidant substances MDA,GSH and Pro in Panax notoginseng.Under the same arsenic level,the medium phosphorus level decreases lowers the antioxidant activity and enzyme activity（SOD,POD,CAT）and antioxidant substances（MDA,GSH,Pro）conten in Panax notoginseng.4.RNA-seq technology the transcriptome of Panax notoginseng tissue is analyzed:by quality of RNA extracted from Panax notoginseng and the repeatability of transcriptome sequencing samples are found to be good;low phosphorus and high arsenic stress inducing more genes up-regulated.The results of Nr library annotation analysis showes that the matching degree of Panax notoginseng Unigenes and vitis vinifera is 4.5%;GO function clustering,KOG functional classification and KEGG metabolic pathway and functional analysis find that differential genes are mainly enriched in metabolic pathways in biological processes and cellular component;differentially expressed genes in metabolic pathways are mainly enriched in four pathways:ribosome,carbon metabolism,biosynthesis of endoplasmic reticulum protein processing amino acids,PI3K-Akt signaling pathway;and differential genes of cellular components are mainly enriched in Nucleic acid cells;differential genes of molecular function are mainly catalytic activities of various enzymes.Among the 25 classifications of KOG,translation,ribosome structure and biotransformation account for the largest proportion.The differential heat map of saponin synthesis key enzyme gene show that the genes of CYP450,FPS and CAS are different under different arsenic and phosphorus stresses.5.According to the results of the Panax notoginseng transcriptome sequencing and BLAST alignment analysis,32 strip phosphate transporter genes are screened from the transcript,including 11 intact ORFs;then,11 genes are subjected to NJ phylogenetic tree analysis.The results show that 11 genes belonged to 4 subfamilies:Pht1,Pht3,Pht4 and PhO.PnPht1;2,PnPht1;4,PnPht1;5 belongs to Pht1,PnPht2;3,PnPht2;7,PnPht2;8 belongs to Pht2,PnPht4;1,PnPht4;10,PnPht4;12,PnPht4;13 belongs to Pht4,and PnPhO;17 belongs to PHO.Through qPCR experiments,the mRNA expression of 10 genes under the interaction of phosphorus and arsenic was investigated.The results show that PnPht4;1、PnPht1;2、PnPht1;4、PnPht1;5、PnPht4;12、PnPht2;7 and PnPhO;17 the up-regulation is larger,compaired to gene cloning status,105.59,74.83,85.04,3.3,4.37,84.54,2.18,1.5 times（compared with mPnAs）;6.Bioinformatics prediction analysis show that the PnPht1;2,PnPht1;4,PnPht1;5 and PnPhO;17 ORFs amino acid lengths are 527 aa,541 aa,545 aa and 273 aa,respectively;the isoelectric point and protein relative molecular weight are 9.08,9.43,8.87,5.55 and 57.53kDa,59.43 kDa,59.04 kDa and 30.77 kDa,and 4 genes have more hydrophobic amino acids than hydrophilic amino acids;PnPht1;2,PnPht1;4,PnPht1;5 all contain 11 transmembrane domains;PnPhO;17 does not contain a transmembrane domain.7.Yeast heterologous expression experiments show that PnPht1;2,PnPht1;4,PnPht1;5 and PnPhO;17 protein can complement the yeast mutant MB192 of phosphorus deletion transporter and enhance yeast phosphate transport function.The yeast growth of Pht1;2,Pht1;4,Pht1;5 genes is better than that of PnPhO;17 gene,indicating that PnPht1;2,PnPht1;4,PnPht1;5 and PnPhO;17 can be in the form of phosphorus deficiency.Phosphorus transport function in yeast,and the phosphate transport capacity encoded by PnPht1;2,PnPht1;4,PnPht1;5 is significantly better than that of PnPhO;17 encoded phosphate transport capacity.In addition,subcellular localization results showe that both PnPht1;2,PnPht1;4genes are localized on the cell membrane.Conclusion:In this paper,the effects of different phosphorus levels on the absorption of Panax notoginseng arsenic and related regulatory mechanisms are discussed from the physiological,enzymatic and molecular aspects.It is found that under the conditions of sufficient phosphorus supply（medium phosphorus and high phosphorus）,arsenic stress can be alleviated to some extent,and reduce the absorption of arsenic by Panax notoginseng,especially under high arsenic stress,whose effect is more obvious.The biosorption efficiency of arsenic is directly reduced sharply;the lower phosphorus treatment,the medium phosphorus level can also promote the growth,saponin synthesis and accumulation of Panax notoginseng.Antioxidant system analysis show that high arsenic stress caused the strengthening of antioxidant mechanism of Panax notoginseng,and sufficient phosphorus source（medium phosphorus,high phosphorus）would decrease this mechanism,indicating that sufficient phosphorus can reduce arsenic stress.By transcriptome analysis and qPCR experiments under the interaction of phosphorus and arsenic,lPhAs cause significant changes in the metabolism pathway and cellular components of Panax notoginseng compared with mPnAs and hPnAs;some high-affinity phosphate transporter genes（Pht1）in response to arsenic stress,the expression is significantly up-regulated,but the appropriate and high-phosphorus treatment decreased the level of Pht1 gene up-regulation.This indicate that sufficient phosphorus levels could change the absorption of phosphorus and arsenic by Panax notoginseng by regulating Pht1 expression.The heterologous expression of yeast further confirmed that PnPht1;2,PnPht1;4,PnPht1;5 had strong phosphate transporting effect under low phosphorus stress.