Functional Analysis Of WRKY33 Gene In Response To Heavy Metal Cadmium Stress In Arabidopsis Thaliana

With the expansion of human activities and the acceleration of industrialization process,the problem of heavy metal pollution to crops in the soil is becoming more serious.Therefore,it is of great theoretical value and practical significance to study the function of plant heavy metal resistance-related genes and their response mechanisms to heavy metal stress.In this subject,WRKY33 gene of Arabidopsis thaliana WRKY transcription factor family is taken as the research object to explore its function and mechanism in regulating the response of heavy metal cadmium stress.The results are as follows:1.Studies on the expression patterns of WRKY33 gene showed that: The expression of WRKY33 gene in Arabidopsis was tissue-specific,with the highest expression in the root;subcellular localization showed that WRKY33 protein was localized in the nucleus;WRKY33 gene was significantly induced by cadmium stress.2.The T-DNA insertion mutants wrky33-1 and wrky33-3 showed more sensitive to cadmium stress than wild type.35S:WRKY33 recombinant vector was constructed,and WRKY33-overexpressing transgenic lines were obtained through transgenic technology.Analysis of the phenotype suggested that WRKY33-overexpressing transgenic lines showed significant enhanced tolerance compared to wild type under cadmium stress.These results indicated that WRKY33 gene plays a positive role in the regulation of cadmium tolerance in Arabidopsis.3.Through pharmacological experiment and expression analysis of genes involved in phytochelatin(PC)synthesis,it has been proved that WRKY33-mediated cadmium tolerance is independent of glutathione(GSH)-dependent PC synthesis pathway.4.The expression levels of downstream genes related to cadmium stress were analyzed by q RT-PCR.It was found that expression of PAD3 gene and ATL31 gene was induced by cadmium stress,and WRKY33 gene affected the expression levels of PAD3 and ATL31 genes under cadmium stress,suggesting that PAD3 and ATL31 genes may be targets of WRKY33,and WRKY33 may regulate cadmium tolerance in Arabidopsis through transcriptional regulation of them under cadmium stress.5.Cadmium content in WRKY33-overexpressing transgenic lines was significantly lower than wild type,indicating that WRKY33 gene may positively regulate cadmium stress tolerance by inhibiting the uptake of cadmium or promoting the extrusion of cadmium in Arabidopsis.This study proved that WRKY33 gene is a functional gene which can positively regulate Arabidopsis tolerance to cadmium,and it can provide new gene resources for crop genetic improvement.The specific regulatory mechanism of WRKY33 gene to downstream target gene under cadmium stress needs further study.