Analysis Of The Mechanism Of Rice Transcription Factor OsNAC3 Tolerant To Cadmium

Cadmium pollution is a negative product of today’s heavy industrial development.Cd is a non-essential element,which enters plants through the transporter of essential elements and interferes with photosynthesis,respiration,water and nutrient uptake,thus affecting plant growth and development.In this study,we obtained the rice cadmium tolerance gene Os NAC3 based on a previous screening using a yeast library,and conducted a preliminary identification and mechanistic exploration of its functions involved in response to cadmium stress in rice,and the main results are as follows:The transgenic rice material overexpressing FLAG-35S:NAC3-GFP was constructed,and the target genes corresponding to the Os NAC3 transcription factor binding DNA fragments were resolved using Ch IP-seq,and GO and KEGG analyses revealed that three Motifs,DOF2.5,SWI14,and ABI4,were enriched at the Os NAC3 binding site;meanwhile,the genes containing the three Motifs were screened using the Jaspar database,and the target genes were verified by luciferase reporter assay,and Os NAC3 was found to enhance the expression of LUC reporter genes driven by the promoters of the heat shock protein Os HSPRO1,heavy metal associated isoprenylated plant protein Os HIPP42,wound induced protein Os WI12,and prephenate dehydratase domain containing protein Os PDD,indicating that these genes are the target genes of Os NAC3.To further investigate the cadmium tolerance of Os NAC3 target genes,transient expression of Os HSPRO1,Os HIPP42,Os WI12,and Os PDD in tobacco leaves was followed by injection of 800μM Cd（NO₃）₂ after 3 days,and it was found that the diameter of blight spots transformed with these four genes was reduced;DAB（Diaminobezidin）histochemical staining showed the accumulation of H₂O₂ was significantly reduced in the region with transient expression of Os NAC3 target genes;meanwhile,functional complementation of yeast muntant assay revealed that Os HSPRO1,Os HIPP42,Os WI12,and Os PDD conferred cadmium tolerance to the cadmium-sensitive yeast V2Tycf1,allowing it to grow normally on medium containing 50μM Cd Cl₂.The above results indicate that Os HSPRO1,Os HIPP42,Os WI12,and Os PDD as the target of Os NAC3 genes are cadmium-tolerant.To investigate the function of Os NAC3,the seed germination status of Os NAC3overexpression and osnac3 knockout transgenic material was analyzed under 100μM Cd（NO₃）₂ treatment conditions,and it was found that overexpression of Os NAC3 could alleviate the effect of cadmium stress on seed radicle and embryo development.The seedling growth of Os NAC3 overexpression and osnac3 knockout transgenic materials were subsequently observed under the treatment conditions of 20%PEG6000,100 m M Na Cl,4°C and 100μM Cd（NO₃）₂,and it was found that under the treatment conditions of 20%PEG6000,100 m M Na Cl and 100μM Cd（NO₃）₂ The plant height,root length and fresh weight of Os NAC3 overexpression material were significantly higher than those of osnac3 knockout transgenic material.Under natural growth conditions,Os NAC3 overexpression and osnac3knockout transgenic materials were treated with 100 m M Cd（NO₃）₂ for 3 weeks at the gestation stage,and the contents of Cd,Zn,Fe,Mn,and Cu elements in roots,stems,leave blades and grains were determined by ICP-MS.Os NAC3 overexpression material was inhibited for the uptake and accumulation of Cd,Fe and Mn elements,but promoted the uptake and accumulation of Zn elements.The above results indicate that Os NAC3 enhances cadmium tolerance in rice by regulating the expression of target genes Os HSPRO1,Os HIPP42,Os WI12 and Os PDD,providing a genetic resource and theoretical basis for cadmium tolerance breeding in rice.