Cloning And Functional Analysis Of FhWRKY33 From Fagopyrum Hailuogouense

Fagopyrum hailuogouense is a new buckwheat species belonging to Fagopyrum Mill,Polygonaceae family,which has been discovered in recent years.This species mainly grows near Hailuogou glacier,which is an ideal material for excavating stress-resistant genes.WRKY transcription factors are one of the most important families of plant transcription factors,which are widely involved in plant growth and development,abiotic stress and biological stress.In this study,Fagopyrum hailuogouense was used as experimental material to screen the Fh WRKY33 gene with significant differential expression by analyzing the transcriptome data at different temperatures.Through gene cloning,biological analysis,subcellular localization,transcriptional activation analysis,expression pattern analysis and Arabidopsis genetic transformation,the biological function of Fh WKRY33 was preliminarily explored.The main results are as follows:1.According to the transcriptome data of Fagopyrum hailuogouense,the CDS sequence of Fh WKRY33 gene was successfully cloned.According to bioinformatics analysis:the coding region of the sequence is 1566bp,encoding 521 amino acids,with a molecular weight of 56.85 k Da and an isoelectric point of 6.26.It is an unstable protein and hydrophilic protein.Fh WKRY33 contains two WRKY domains and two C₂H₂zinc finger domain,belonging to the WRKY family Group I.2.The subcellular localization analysis of Arabidopsis protoplasts showed that Fh WKRY33 was located in the nucleus and was a nuclear localization protein.Transactivation analysis assay showed:Fh WKRY33 had transcriptional activation activity in yeast.3.Semi-quantitative PCR analysis showed that Fh WKRY33 had different expression patterns under different stresses.Under the stress of low temperature,Na Cl,PEG6000 and ABA,the peak time of Fh WKRY33 expression in 24 h was 3 h,24 h,6 h and 3 h,respectively.4.Fh WRKY33 overexpression vector was constructed and transferred into Arabidopsis thaliana by Agrobacterium-mediated method.Finally,Fh WRKY33 transgenic Arabidopsis was obtained.The physiological indexes of transgenic Arabidopsis leaves were detected,the results showed that the activities of antioxidant enzymes(POD,SOD,CAT)and Proline(Pro)content of Fh WKRY33 transgenic Arabidopsis leaves were significantly higher than those of wild type(P<0.01),while the content of malondialdehyde(MDA)was significantly lower than that of wild type(P<0.05).The results showed that Fh WKRY33 could enhance the resistance to cold,salt,PEG6000 and ABA.