Functional Study Of Maize Phytochelatin Synthase Gene ZmPCS1 Under Cadmium Stress

Maize（Zea Mays L.）is an important crop for food,feed and industrial raw materials,and is one of the main crops in China and all over the world.However,maize is often affected by abiotic stress such as heavy metals during its growth process,which leads to the decline of its yield and quality.Among them,the heavy metal cadmium（Cd）is a non-essential element for plant growth,which can inhibit the growth and development of plants.Therefore,it is of great theoretical and practical significance to excavate Cd tolerance genes and analyze their functions for maize production and breeding of superior maize varieties.In response to the toxic effects of heavy metals,plants have developed a series of metal detoxification mechanisms,such as rejection,segregation,chelation and organic ligands.PCS catalyzed by phytochelate protein synthase（PCs）plays an important role in detoxification of metal ions in plants and other organisms.Studies have reported that the transcriptional expression of PCS genes in Arabidopsis thaliana,rice and wheat can be significantly increased under the induction of cadmium,and the overexpression of PCS transgenic plants can significantly improve the tolerance of plants to the heavy metal cadmium.However,little is known about whether the PCS gene in maize has a similar function and how it acts.In this study,chelatopeptin synthase gene ZmPCS1 was cloned from maize B73 inbred line by homologous comparison,and its function was studied in different systems of Arabidopsis thaliana,yeast and tobacco by means of molecular biology.The specific results are as follows:1.Sequence analysis showed that the full length of ZmPCS1 gene was 1524 bp,encoding 507 amino acids.Amino acid sequence alignment shows that ZmPCS1 has the same amino acid sequence as PCS of other species,and has two typical protein domains:Phytochelatin and phytochelatin-C.2.Tissue expression pattern analysis showed that ZmPCS1 gene was expressed in all tissues of maize,and the highest expression was found in filaments and leaves.3.Analysis of induced expression pattern showed that the expression level of ZmPCS1in maize roots and leaves under 100 m M Cd Cl₂treatment increased firstly and then decreased,and the expression level reached the highest at 6 h and 1 h after treatment,respectively.4.Transient expression analysis of maize protoplasts and tobacco indicated that ZmPCS1 was localized in the cytoplasm.5.The complementary experiment of Cadmium-sensitive yeast mutant（ycf1）showed that ZmPCS1 could significantly restore the growth phenotype and growth rate of ycf1,indicating that ZmPCS1 could improve the tolerance of cadmium in yeast.6.After ZmPCS1 gene was introduced into Arabidopsis homologous mutants（atpcs）,the plants were supplemented.The cadmium tolerance test showed that the plants could significantly restore the growth defect phenotype of the mutants,such as root length and fresh weight.7.ZmPCS1 overexpression vector was constructed and introduced into wild-type Arabidopsis thaliana to obtain overexpressed transgenic plants.Cadmium treatment.Compared with the wild type,ZmPCS1 overexpression increased root length,fresh weight,and MDA content,suggesting that ZmPCS1 can improve cadmium tolerance in Arabidopsis thaliana.In addition,the growth of overexpressed plants under cadmium stress was inhibited after treatment with the inhibitor BSO,suggesting that ZmPCS1 indeed regulates plant cadmium tolerance by regulating the synthesis of PCS.8.Compared with the wild type,under cadmium stress,the NBT staining results showed that the accumulation of ROS in the overexpressed plants decreased,while the activities of SOD and POD increased,suggesting that ZmPCS1 may also regulate the tolerance of plants to cadmium by regulating the balance of ROS.9.After transient expression of ZmPCS1 in tobacco leaves,the tobacco plants with transient expression of ZmPCS1 under cadmium stress can alleviate the toxic effect of cadmium compared with the wild type,suggesting that ZmPCS1 can improve the tolerance of tobacco to cadmium.