Study On The Mechanism Of BnaALMT7.2 In The Root System Of Brassica Napus In Response To Aluminum Toxicity

Acid soil accounts for about 40%-50%of the world’s potential cultivated land,which is the main limiting factor of food production.With the aggravation of global soil acidification,aluminum toxicity and phosphorus deficiency have become the most common problems in acid soils.In the soil with acid p H,the solubility of aluminum ion increases,on the one hand,it will be toxic to plants,on the other hand,it can complex with phosphate,leading to phosphorus deficiency.Many studies have shown that aluminum can stimulate the secretion of organic acids in plants.This process is mainly realized by the aluminum activated malate transporter(ALMT)and multidrug and toxic compound extrusion(MATE).Phosphorus is an essential nutrient for plant growth and development.When plants suffer from aluminum toxicity and phosphorus deficiency,their growth will be greatly inhibited.Therefore,how to reduce aluminum toxicity and increase available phosphorus content in acid soil has become an important research direction.In this study,a candidate gene,Bna ALMT7.2(gene ID:Bna A04g15700D),was identified for aluminum tolerance in Brassica napus by measuring the aluminum content in stems of 131 Brassica napus germplasms and genome-wide association analysis(GWAS)based on genome re sequencing data.Subsequently,a pair of rapeseed varieties with significant difference in aluminum accumulation ability were selected as the core research materials.Then,the physiological differences in root growth rate,fluorescence staining,H~+kinetics and comparative transcriptomics were analyzed under different aluminum stress and low phosphorus stress.Subsequently,several key genes related to aluminum toxicity in plants including Bna ALMT7.2 were analyzed by real-time fluorescence quantitative PCR(QRT PCR).Finally,through subcellular localization of Bna ALMT7.2,phenotype and function analysis of heterologous transgenic plants,the molecular mechanism of Bna ALMT7.2 tolerance to aluminum toxicity in Brassica napus L.and the regulatory function of phosphorus were elucidated.The main results are as follows:(1)Screening,identification and physiological experiment determination of rape varieties.In this study,we measured the aluminum content in the stems of 131 rapeseed core germplasm resources.Combined with the completed genome-wide re sequencing data,we found a significant association interval through genome-wide association analysis(GWAS).In this interval,we identified a functional protein Bna ALMT7.2 that can participate in the tolerance of aluminum stress in rapeseed.Subsequently,we plan to carry out in-depth research on the molecular mechanism of aluminum tolerance in Brassica napus based on Bna ALMT7.2 function.(2)A pair of aluminum-tolerant and aluminum-sensitive rapeseed varieties were selected from the existing 131 rapeseed core germplasm resources and named aluminum-sensitive(Al-S)and aluminum-tolerant(Al-T)respectively.In order to study the differences in the tolerance of rape to aluminum toxicity under different phosphorus concentrations,this study designed four sets of experimental treatment conditions(labeled:+P-Al,+P+Al,-P-Al,-P+Al).Subsequently,the root growth rate,root aluminum accumulation and root tip H~+dynamics characteristics of the selected varieties were measured at the seedling stage.The results showed that the root growth rate of Al-T varieties was faster than that of Al-S,the root length was longer,the root accumulation of aluminum concentration was higher,and the Al-T rape varieties had stronger ability to tolerate low phosphorus stress.(3)Molecular analysis of different aluminum-tolerant rape varieties.In this study,after the above four treatments were carried out on the roots of Al-T and Al-S,the roots of different aluminum tolerance cultivars,the rape root seedlings under the four treatments were sampled in sections,and they were divided into 0-2cm and 2-4cm and>4cm.Subsequently,transcriptomics analysis and fluorescence quantitative PCR(q RT-PCR)detection and analysis were performed on samples of different segments.By analyzing the differentially expressed genes(DEGs),it was found that some enzymes related to cell wall changed after aluminum treatment,such as glucokinase and pectinase in xyloglucosan,and the enzymes related to antioxidant enzyme system also changed significantly.Through the GO enrichment analysis of DEGs,it is found that Al-S is mainly concentrated in some antioxidant enzyme activities in plants,while Al-T is concentrated in cell wall enzymes,such as xylosyltransferase and glucosyltransferase.Through the KEGG enrichment of DEGs,it is found that the Al-S enrichment pathway is mainly primary metabolites,while Al-T is secondary metabolites.After that,through the quantitative analysis of the key genes related to aluminum in the above three segments of roots,it was found that the three genes Bna ALMT7.2,Bna ALMT1.1 and Bna ALS3.1 were all expressed at the highest level at 0-2cm from the root tip of rape.As the root system goes up,the expression level gradually decreases.Under aluminum stress conditions,the expression levels of these three genes in rape-sensitive Al-S were significantly higher than those in Al-T varieties.(4)Sequence analysis of Bna ALMT7.2 and its molecular mechanism in the process of rape tolerance to aluminum toxicity.Phylogenetic tree analysis showed that the rape ALMT family had 34 members,among which Bna ALMT7 had three members.In order to clarify the function of Bna ALMT7.2,this study transformed it into Arabidopsis wild-type Col-0 and malate transport deletion mutant atalmt1,respectively,and obtained two overexpression lines(Bna ALMT7.2/Col-0,OX-1,OX-2)and two functional reverting lines(Bna ALMT7.2/atalmt1,CO).Through different phosphorus and aluminum treatments,it was found that the root growth of Arabidopsis thaliana was reduced overall under the conditions of phosphorus deficiency.Under the conditions of aluminum stress,the root lengths of the two overexpression lines were longer than those of the wild type,while the root lengths of the reverting lines were longer than those of the wild type.The wild type is similar,indicating that Bna ALMT7.2 has anti-aluminum function in Arabidopsis.