Functional Validation Of Cadmium Candidate Genes In Brassica Napus L.

Environmental pollution is increasingly serious,especially the heavy metal cadmium pollution in soil.Therefore,it is imperative to seek an effective way to reduce cadmium pollution.In recent years,it is confirmed that phytoremediation technology can effectively and safely handle soil pollution.Rape is one of the main oil crops widely cultivated in China.It is also one of the potential plant resources for Cd contaminated land remediation.Based on the candidate genes related to cadmium stress in Brassica napus obtained by GWAS,11 genes were verified in this study using Arabidopsis and Brassica napus as materials.The main research contents and results are as follows:1.Comparing the studied Brassica napus gene with the homologous gene in the model crop Arabidopsis,and the functional annotation analysis was performed.It was found that the homologous genes At NRT1.8、At ALDH2B4 and At ARF1 in Arabidopsis were involved in cadmium ion reaction,and other genes belonged to different enzymes and participated in cell antioxidant reaction.Gene Bna A09g32780 D is an unknown gene with unknown function.2.Ten cadmium related candidate genes of Brassica napus were constructed and overexpressed into Columbia Arabidopsis.Transgenic positive seeds were obtained for phenotypic identification.This paper mainly studies the characters of plants under cadmium stress.We found that the growth of Bna C03g65260D(Bn GSTF14)were significantly different from those of wild-type Arabidopsis and blank control group.3.In order to further verify the response of overexpressed plants to cadmium stress,we measured the content of cadmium ion in the positive lines.The results showed that cadmium ion content of Bna C03g65260D(Bn GSTF14),Bna A09g32780 D and Bna C05g01540D(Bn GSTF7)positive seedlings differed 20-30μg per unit weight before and after cadmium treatment.The difference was significant.4.We also constructed a CRISPR/Cas9 knockout vector for genetic transformation in Brassica napus to verify its function.In conclusion,this study found that Bn GSTF14 and Bna A09g32780 D could significantly reduce the cadmium ion content in plants,and Bn GSTF7 could improve the cadmium tolerance of plants.Bn GSTF14 and Bn GSTF7 are both glutathione s-transferases,which are related to the chelation detoxification of cadmium ions.Bna A09g32780 D is a gene with unknown function,it is involved in transmembrane transport.