Analysis Of WRKY Family Gene In Potentilla Sericea And Identification Of Cadmium-resistance Function Of PsWRKY40

Cadmium（Cd）is a heavy metal with strong migration ability.The accumulation of Cd will affect the growth and physiological process of plants,causing different degrees of damage to plants;Cd enriched in the human body through the food chain will cause a variety of diseases.Potentilla sericea is a plant of the Rosaceae genus Potentilla,with extensive management,strong stress resistance,high ornamental value,and strong tolerance to heavy metals such as lead and cadmium.It is excellently beneficial for soil fixation and ecological protection.At present,most of the researches on Potentilla sericea are focused on the physiology of adversity,and there is a lack of research at the molecular level.As one of the largest transcription factor families in higher plants,the WRKY transcription factor family is widely involved in abiotic stress responses in plants.In this study,the WRKY family genes were mined and the effect of Ps WRKY40 gene on plant Cd tolerance was analyzed based on the Cd stress transcriptome data obtained by the research group.This lays a theoretical foundation for remediation and sustainable utilization of soil.The main results are as follows:（1）A total of 50 members of the WRKY family were screened out from the transcriptome data obtained in the previous study,and Group I,II,and III subgroups contained 10,32,and 8 members,respectively.Phylogenetic analysis showed that the WRKY family of Arabidopsis thaliana had similar evolutionary relationship with the Arabidopsis thaliana,and Group IIa and IIb and Group IId and IIe were all closely related.The 50 members are all hydrophilic proteins,48 of which are only located in the nucleus,and 2 are located in both cell membrane and nucleus.Most of the conserved domains of each member correspond to some conserved motifs,and a conserved domain may contain 1 to 4 conserved motifs;the conserved motifs of each subfamily are similar to the type,quantity and distribution of domains.The expression data of Cd stress transcriptome were analyzed,and it was found that most of the genes in the WRKY family of Potentilla sericea had higher expression levels under CK and Cd treatments,but the expression changes of each gene were not consistent.The c400859.graph＿c0 gene,which was clustered with At WRKY40 and was significantly up-regulated after Cd stress,was selected and named Ps WRKY40.（2）The Ps WRKY40 gene with an ORF length of 978 bp and encoding 325 amino acids was successfully cloned using the c DNA obtained by reverse transcription of the RNA of Potentilla sericea.Ps WRKY40 protein contains a WRKY conserved domain in the 169-227 aa region,and the zinc finger structure is C₂H₂ type,which is the closest relative to Fragaria vesca subsp.vesca and Rosa chinensis.Under the treatments of Na Cl,mannitol,Zn SO₄ and Cd Cl₂,the expression levels of Ps WRKY40 in root,stem and leaf of Potentilla sericea were all increased,and showed a rapid response to stress in the roots;Ps WRKY40 was more sensitive to Na Cl and Cd Cl₂ treatment,with highest expression in roots under Na Cl treatment,and in stems under Cd Cl₂ treatment.The overexpression vector PBI121-Ps WRKY40-GFP was successfully constructed using Xba I and Sma I restriction endonucleases and T4 ligase,and Agrobacterium EHA105 was used to infect Arabidopsis through the flower dip method,and six stably inherited T3 generation Ps WRKY40 transgenic Arabidopsis overexpressing line were screened out;the fluorescent signal of protein Ps WRKY40-GFP appeared in the nucleus of overexpressing Arabidopsis leaf protoplasts.（4）Seeds and plants of Arabidopsis wild-type and overexpression lines were treated with Cd Cl₂.With the increase of stress concentration,the seed germination rate generally showed a downward trend,but the seed germination rate of Arabidopsis thaliana overexpressing Ps WRKY40 was significantly higher than that of the wild type.With the prolongation of treatment time,the plants appeared chlorosis and wilting,but the growth state of overexpressed Arabidopsis were better.The measurement results of physiological indicators showed that under Cd Cl₂ treatment,the contents of O^-_2·,H₂O₂,MDA and relative conductivity of transgenic Arabidopsis increased but were significantly lower than those of wild type,and the activities of SOD,POD,CAT and proline increased significantly but higher than those of the wild type,the chlorophyll content was also significantly higher than that of the wild type after the decrease.The expression levels of At SOD,At POD and At CAT genes were significantly up-regulated,and the expression level of transgenic Arabidopsis was higher,which was significantly different from the wild type.The above results show that overexpression of Ps WRKY40 gene can significantly enhance the antioxidant enzyme activity of plants under Cd stress,maintain the self-regulation ability of plants,and alleviate peroxidative damage.This preliminarily proved that Ps WRKY40 gene has the function of regulating plant tolerance to Cd.