Functional Mechanism Of SbNRT1.1B In Sweet Sorghum Responding To Cd Stress

Due to mining,smelting,industrial waste discharge and excessive use of pesticides and fertilisers containing heavy metals,the problem of heavy metal pollution in China’s soils is becoming increasingly serious,with cadmium（Cd）pollution being the most serious.Not only does soil Cd pollution inhibit plant growth,but Cd can also be transferred and enriched through the food chain,endangering human health.Therefore,it is imperative to tackle the problem of soil Cd contamination.Traditional physical and chemical remediation techniques are costly,technically demanding,destroy soil structure and are prone to secondary contamination,whereas phytoextraction remediation techniques are of great interest because of their green,economic and sustainable advantages.Sweet sorghum（Sorghum bicolor L.Moench）is an annual C4 energy crop of the grass family,with a wide range of adaptations,high biomass and Cd tolerance,and has great potential for remediation of Cd-contaminated soils.Plant biomass and Cd accumulation capacity are directly related to the efficiency of phytoremediation of Cd-contaminated soils.Nitrogen not only affects plant biomass,but is also closely related to plant uptake and accumulation of Cd.Previous studies found that nitrate nitrogen significantly promoted Cd accumulation in sweet sorghum compared to ammonium nitrogen,and that the sweet sorghum nitrate transporter gene SbNRT1.1B is a key gene in the promotion of Cd accumulation in sweet sorghum by nitrate nitrogen,but the mechanisms involved are not yet clear.Therefore,this thesis aims to elucidate the mechanism by which sweet sorghum SbNRT1.1B responds to Cd stress,with the following main results:1.The SbNRT1.1B gene was cloned by PCR and was 2264 bp in length with an open reading frame（ORF）of 1788 bp.The gene was characterized using bioinformatics and encoded 595 amino acids with a molecular weight of 63.58 k Da,a theoretical isoelectric point of 8.44 and 11 transmembrane structural domains.The promoter region of SbNRT1.1B contains response elements such as gibberellin,abscisic acid,methyl jasmonate and a cis-acting element involved in the low temperature response.Subcellular localization predicts that the gene is localized to the plasma membrane,chloroplast-like vesicle membrane,Golgi apparatus and endoplasmic reticulum（membrane）.We further analysed the subcellular localisation of sweet sorghum SbNRT1.1B by constructing a green fluorescent（GFP）subcellular localisation vector using a tobacco leaf transient expression system,which showed that SbNRT1.1B was localised only to the plastid and no GFP signal was detected at the plasma membrane.Phylogenetic evolutionary tree analysis revealed the highest amino acid sequence similarity between sweet sorghum SbNRT1.1B and maize Zm NRT1.1B and very low similarity to Arabidopsis At NRT1.1.At the sweet sorghum seedling stage,tissue-specific expression analysis of SbNRT1.1B revealed the highest expression in roots,followed by stems and the lowest in leaves,while short-term Cd treatments at 6 h,24 h and 3 d significantly induced SbNRT1.1B expression in roots,stems and leaves,and SbNRT1.1B expression in roots,stems and leaves after 5 d Cd treatment was not significantly different from that of CK.At the nodulation stage of sweet sorghum,SbNRT1.1B expression was lowest in roots and similarly significantly higher in leaves,leaf sheaths and internodes than in roots,while long-term Cd stress had no significant effect on SbNRT1.1B expression in roots and internodes,but Cd significantly up-regulated SbNRT1.1B expression in leaves,especially older leaves,and down-regulated SbNRT1.1B expression in leaf sheaths However,Cd significantly up-regulated SbNRT1.1B expression in leaves,especially older leaves,and down-regulated SbNRT1.1B expression in leaf sheaths.In conclusion,SbNRT1.1B showed a strong response to Cd stress at different fertility stages and different times of Cd stress in sweet sorghum;2.The cloned sweet sorghum SbNRT1.1B gene was constructed as an overexpression vector and heterologously transformed with SbNRT1.1B into Arabidopsis thaliana on a Col-0 background by the flower dipping method,and similarly transformed with SbNRT1.1B back into Arabidopsis thaliana NRT1.1 loss-of-function mutant chl1-5（Col-0 background）.Finally,seven heterozygous overexpression SbNRT1.1B independent strains and eight complementation SbNRT1.1B independent strains were obtained,of which seven overexpression strains expressed 1.5-3.5 times more SbNRT1.1B than wild type（WT）and eight complementation strains expressed 1.6-4times more SbNRT1.1B than WT.Two overexpression（OE-29-7 and OE-26-9）and backfill SbNRT1.1B strains（C-76-1 and C-46-1）were selected to investigate the response mechanism of SbNRT1.1B to Cd stress.It was found that Col-0,overexpressed SbNRT1.1B（OE-29-7 and OE-26-9）,chl1-5 and back-supplemented SbNRT1.1B（C-76-1and C-46-1）strains with nitrate-nitrogen as the sole nitrogen source had the best main root length,above-ground fresh weight and root fresh weight compared to NH₄NO₃,CO（NH₂）₂and（NH₄）₂SO₄as nitrogen sources.Optimal.The overexpression SbNRT1.1B strains（OE-29-7 and OE-26-9）had significantly higher primary root length,fresh weight,nitrate-nitrogen content and chlorophyll content than Col-0.Similarly,the back-complemented SbNRT1.1B strains（C-76-1 and C-46-1）had significantly higher primary root length,fresh weight,nitrate-nitrogen content and chlorophyll content than the mutant chl1-5.However,Cd stress significantly reduced the primary root length,fresh weight,nitrate-nitrogen content and chlorophyll content of overexpressed SbNRT1.1B strains（OE-29-7 and OE-26-9）and Col-0,but Cd stress significantly increased the primary root length,fresh weight and nitrate-nitrogen content of back-complemented SbNRT1.1B strains（C-76-1 and C-46-1）.Both the plate and hydroponic methods showed that aboveground and root Cd concentrations were significantly higher in the overexpressed SbNRT1.1B strains（OE-29-7 and OE-26-9）than in the back-supplemented SbNRT1.1B strains（C-76-1 and C-46-1）,followed by Col-0,with the mutant chl1-5 having significantly lower aboveground and root Cd concentrations than the other strains;3.Transcriptome sequencing data analysed for changes in differentially expressed genes（DEGs）in SbNRT1.1B strain C-76-1 and mutant chl1-5 root lines under Cd stress and found a total of 82 DEGs,of which 46 were up-regulated and 36 were down-regulated.The GO enrichment analysis showed the highest enrichment of sulphur-containing compounds metabolic processes.Whether the response of SbNRT1.1B to Cd stress was dependent on the GSH synthesis pathway was investigated by using the glutathione（GSH）synthesis inhibitor BSO（buthionine-sulfinamide）.Col-0,overexpressing SbNRT1.1B（OE-29-7 and OE-26-9）,chl1-5 and back-complemented SbNRT1.1B（C-76-1 and C-46-1）strains were used as experimental materials,and four treatments,CK,BSO,Cd and Cd+BSO,were set up to determine primary root length,fresh weight,GSH,oxidized glutathione（GSSG）,non protein sulfhydryl（NPT）and phytochelating peptides（PCs）,as well as the expression levels of important genes in the GSH pathway.The results showed that Cd stress significantly induced above-ground and root levels of NPT,GSH,GSSG and PCs in all lines,and also up-regulated the expression levels of GSH1,GSH2,PCS1 and PCS2.BSO significantly suppressed the expression levels of Col-0,overexpressed SbNRT1.1B（OE-29-7 and OE-26-9）and back-complemented SbNRT1.1B（C-76-1 and C-46-1）strains in Cd stress-induced levels of NPT,GSH,GSSG,and PCs and expression levels of the corresponding genes,but BSO had no significant effect on the levels of NPT,GSH,and PCs and expression levels of GSH1,GSH2,PCS1,and PCS2 in chl1-5 under Cd stress,suggesting that SbNRT1.1B responds to GSH synthesis through GSH-dependent synthetic pathway in response to Cd stress.In summary,the overexpression or back-complementation of SbNRT1.1B strai ns enhance plant biomass through the uptake of more nitrate ions by promoting c hlorophyll synthesis and photosynthesis on the one hand,and assist in the synthes is of more PCs dependent on the GSH synthesis pathway on the other hand,whi ch are chelated with Cd and stored in leaf or root vesicles to detoxify Cd stress,thereby enhancing Cd tolerance,increasing Cd accumulation and promoting plant efficiency in remediating Cd-contaminated soil.This thesis provides a theoretical basis and technical support for the use of genetic engineering to enhance sweet s orghum remediation of heavy metal contaminated soils.