Screening Of Cadmium Sensitive Mutants And Cloning And Functional Analysis Of Causal Genes In Arabidopsis

Cadmium(Cd)is a common heavy metal with environmental toxicity,Plants grown in Cd-contaminated soil can suffer from Cd toxicity.The toxic effects of Cd t includes inhibiting the germination of seeds,hindering plant growth and promoting the programmed cell death.but the specific mechanism involved is still to be studied.Complexation of Cd by phytochelatins(PCs)is the most important way to detoxify Cd in plants.The absence of PCs in plants can lead to a significant increase in Cd toxicity.Little is known about the other mechanisms of Cd detoxification that are independent of the PCs pathway.The aim of this study was to investigate Cd detoxification mechanism beyond PCs-dependent pathway in the model plant species Arabidopsis thaliana.In this study,we used the mutagen ethylmethylsulfone(EMS)to construct a library of Arabidopsis thaliana mutants in the background of the PC synthase function defective mutant cad1-3.A Cd hyper sensitive mutant,named 7-19,was isolated.We generated a 7-19 × cad1-3 F2 population,We seek out the target gene By generated a 719 x cad1-3 F2 population,whole genome sequencing,derived cleavage ampfied polymorphic sequence site validation and Phenotype validation of transgenic complementation line of candidate genes.The gene is named CDT-R,which is a TNL(TIR-NLR)disease-resistant gene that belongs to the NLR(Nucleotide-binding domain and leucine-rich repeat)category.Also rename the mutant 7-19 to cdt-r cad1-3.the root and shoot parts of cdt-r cad1-3 mutants are more susceptible to Cd toxicity than cad1-3 can be seen from the? MS(Mulashige and Skog)agar plates or soil medium culture tests.In ? MS plates with 30 μM Cd,the primary root elongation of cdt-r cad1-3 was 30%-50%of only 30%-50%of cad1-3 and the fresh weight of plant was reduced by half.CDT-R mutation site is located at the last base of the last intron,changing from G to A.CDT-R post-transcriptional variable shear changes,over-cutting seven bases,resulting in early termination of protein translation.Mutation CDT-R protein length is 95 amino acids shorter than the wild type.CDT-R T-DNA insertion mutants and constructed CRISPR/Cas9 editing lines also showed that CDT-R functional loss caused Cd sensitivity in Arabidopsis thaliana,suggesting that CDT-R plays a role in Cd tolerance both in the presence or absence of the PCs detoxification mechanism.The phenotypic recovery lines were obtained using CDT-R coding frame fused GFP coding sequence driven by the CDT-R the native promoter.The GFP fluorescent signal was observed in the endodermal cells of Arabidopsis thaliana roots,with a subcellular localization in both the cytoplasm and the nucleus.Cd can affect auxin homeostasis,which in turn may affect plant growth.We introduced auxin fluorescent probe DII-VENUS into cad1-3 and 7-19 by hybridization.In the presence of Cd treatment,the DII-VENUS fluorescence intensity in the root tips of 7-19 stronger thancad1-3.This suggests that CDT-R mutation resulted in decreased auxin level in the root tips in the presence of Cd.In summary,CDT-R encodes a nuclear and cytoplasmic co-localized protein that plays a role in Cd tolerance by regulating the steady state of auxin in Arabidopsis thaliana.