Functional Analysis Of SaWRKY7 From Sedum Alfredii Involved In Cadmium Tolerance

Sedum alfredii Hance is genus Sedum in Crassulaceae.It is an important model plant for studying the mechanism of heavy metal accumulation,tolerance and adaptive evolution in plants.WRKY transcription factor family members were induced when subjecting to various environmental factors in stress and played an important regulatory role in plants'resistance to biotic and abiotic stress.Two genes encoding a member of plant WRKY family were isolated from hyperaccumulating?HE?and non-hyperaccumulating ecotype?NHE?plants according to the cDNA library of S.alfredii and they were named as SaWRKY7h and SaWRKY7n,respectively.Meanwhile,the function of these transcription factor WRKY7 were investigated through sequence alignment,expression pattern analysis under the cadmium analysis,subcellular localization,transcriptional activation test,and heterologous expression in Arabidopsis thaliana.The main results are as follows:1.SaWRKY7h gene was isolated from S.alfredii.The full length of the open reading frame was 1011 bp,encoding 336 amino acids.The molecular formula was C₃₀₀₆H₅₀₀₃N₁₀₁₁O₁₂₃₉S₂₇₄,the molecular weight was 83.91 KD,and the isoelectric point was 5.03.SaWRKY7n gene was isolated from non-hyperaccumulative Sedum alfredii.The open reading frame length was 1011bp,encoding 336 amino acids.The molecular formula was C₃₀₁₆H₅₀₂₃N₁₀₁₁O₁₂₄₄S₂₆₉,the molecular weight was 83.97 KD,and the isoelectric point was 5.03.The results of phylogenetic tree showed that they are closest to AtWRKY7 in A.thaliana,so they are named SaWRKY7h/n and belonged to class IId of WRKY gene family.2.Quantitative Real-time PCR?qRT-PCR?results showed that SaWRKY7h was constitutively expressed in all tissues of S.alfredii with highest expression in roots,followed by stems and leaves.Under cadmium stress,SaWRKY7h's expression was induced in the roots,stems and leaves peaking at 0.5 h.The SaWRKY7h's transcript accumulation was highly induced in roots,reaching a maximum of 13 fold of the initial value,4.5 folds in stems and 3.7 folds in leaves,respectively.3.The transcriptional activation activity test of SaWRKY7h/n showed that neither gene had transcriptional self-activation activity.Subcellular localization showed that SaWRKY7h/n are located in the nucleus of A.thaliana protoplasts.These suggests that they may play novel roles in the nucleus.4.The function of SaWRKY7h was validated by constructing over-expressing of SaWRKY7h/n in A.thaliana.Phenotypic analysis showed that SaWRKY7h transgenic lines were less tolerant to cadmium than wild-type.The chlorophyll fluorescence parameters showed that SaWRKY7 transgenic lines were more seriously damaged by cadmium stress.DAB and NBT staining results showed that transgenic A.thaliana was more seriously poisoned by reactive oxygen species.The results of cadmium content and translocation factors analysis showed that the transgenic lines had higher ability of transporting cadmium to the shoots and could accumulate more cadmium,thus causing greater cadmium toxicity.These results indicate that over-expression of SaWRKY7 may transports cadmium to the ground,leading to more serious cadmium toxicity.5.And certain softwares were used to analyze the cis-acting elements within the SaWRKY7h promoter.And a 1971 bp SaWRKY7h promoter sequence was amplified based on the S.alfredii genome data and named as pro-SaWRKY7h.The pro-SaWRKY7h positive transgenic A.thaliana were selected for GUS staining,showing that all SaWRKY7h were constitutively expressed,indicated that the gene was a constitutively expressed gene.