Editing GmHIPP26 Gene By CRISPR/Cas9 And Its Function Under Cadmium Stress In Soybean(Glycine Max L.)

Food security is closely related to human health and heavy metal pollution in soil has posed a great threat to crop yield and quality.HIPP and HPP are heavy metal transport-related plant protein and act as balancing and detoxifying the heavy metal in plant.Here,the soybean GmHIPP26 mutants were generated by CRISPR/cas9 gene editing technology in order to study the function of GmHIPP26 gene under cadmium stress treatment.The main results were as follows:1.Bioinformatics analysis of GmHIPP26 and spatio-temporal expression of GmHIPP26 in response to heavy metal stress showed that GmHIPP26 was a family genes with a conserved domain of HMA in soybean genome,mainly distributed on soybean chromosome 12,and their protein sequence was highly similar.The relative expression of GmHIPP26 in leaves increased with the increase of Cd Cl₂concentration,and reached the highest point after 48 h treatment,which was 70 times of the normal expression.At the same time,the expression of GmHIPP26 in root was much lower than that of leaves.2.Soybean GmHIPP26 mutants were generated by Agrobacterium tumefaciens mediated transformation methos using CRISPR/cas9-GmHIPP26 as the vector and cotyledonary nodes as explants.Three soybean mutants were positively confirmed by sequencing.At the same time,the agronomic traits were no significant difference between mutants and wild type in the field experiment.3.Study on function of GmHIPP26 mutants under cadmium stress.Under cadmium stress treatment,the plant height,dry weight of aboveground,dry weight of underground,and the root shoot ratio in GmHIPP26 mutants were lower than those of wild type.The root length,number of root tips,and branches of the mutants decreased significantly besides the average root diameter increased significantly.Root microstructure showed that the root epidermal cells were irregular and the diameter of pericycle narrowed in the mutant roots.While primary xylem and epigenetic xylem cells of mutants was no significant difference under cadmium stress.However,the root cells in wild-type plants were normal.The cadmium stress treatment damaged not only the roots but also the leaves in mutant plants.The results showed the electrical conductivity,O^2-and H₂O₂ accumulation were increased in the leaves of mutants.But the antioxidant enzyme activity were decreased.At the same time,the accumulation of cadmium in roots of mutant was 4 times that of the wild type.But the accumulation of cadmium in leaves was no obviously different.From these results,GmHIPP26 gene might play an important role in alleviating cadmium stress and cadmium transport in plants.The mechanism of GmHIPP26 gene involved in cadmium transport needs to be further studied in plants.