Preliminary Study On The Role Of AtSCL30? In Iron-deficiency Tolerance Of Arabidopsis

Iron is one of the most abundant elements on earth,but it often exits in neutral and alkaline soil in the form of insoluble ferric hydroxides,leading to the greatly reducing bioavailability of iron,thus development of plants and human beings are influenced by iron deficiency.Many transcription factors are involved in the process of iron uptake and regulation in plants.In recent years,great progress has been made on the research of the regulation mechanism of iron signal,but of which remain very few on the study of post-transcriptional splicing regulation.Splicing factors play a very important role in the process of precursor mRNA to the mature RNA,which is associated with many biological and abiotic stresses.SR proteins are a kind of splicing factors.We idetified 8 homozygous mutants of SR protein genes,which were then under different kinds of stresses.And finally At3g13570 was determined as the gene we need to study the function in the iron deficiency in deepth,which encodes AtSCL30? protein,by means of genetic and molecular biology techniques.We obtained 15 different mutants of SR protein genes,of which T-DNA insert in intron or exon,and identified 8 different kinds of homozygous mutants,which were then under different stress treatments:NaCl stress,ABA stress,Cu stress and iron deficiency stress.The results showed that shoot growth of mutant and sr34b were significantly inhibited compared with wild type by 90 mm NaCl stress;root growth of mutant scl30a was less sensitive than wild type to 0.8?M ABA stress;root growth of mutant scl30a and scl33 were more resistant than wild type to 50?M Cu2+ stress;shoot iron content of mutant scl30a was significantly higher than the wild type when treated with iron deficiency stress.Then we choose SCL subfamily to make a amino acid alignment and genetic structure analysis,the results showed:amino acid sequence of SCL33 was highly homologous with SCL30a while which of SCL28 and SCL30 were highly homologous.In the end,we determine to study the role of AtSCL30? in iron deficiency stress.So we analyzed the expression,splicing pattern and localization of AtSCL30?.We found that AtSCL30? can produce 5 different transcripts.The expression of AtSCL30? was induced by iron deficiency while the treatment time was 1 day,3 days,5 days or 7 days,and achieved the lowest on the third day,the shortest transcript was induced most significantly.The result also showed that AtSCL30? expressed in different tissues and had the lowest expression in the 50 days' old rosette compared with other tissues by fluorescence quantitative PCR analysis.The plant complementation vector of pGWB504-AtSCL30? was constructed and transformed into the mutant atscl30a,which carry the GFP(green fluorescent protein)label.We analyzed the tissue and subcellular localization by observing the expression of GFP in the native genomic context.Research results showed that AtSCL30? was expressed in the whole root,and mainly located in the nuclear speckles by observing the expression of GFP in the cell of the native genomic context.The nuclear speckles was distinct and bigger under iron deficiency compared with that under iron sufficiency,which also confirmed that AtSCL30? is influenced by iron deficiency.However,we could not identify localization of other tissues in this way.In order to further study the biological function of AtSCL30?,overexpression vector of AtSCL30? was constructed and transformed into Arbidopsis wild type.Fe content,chlorophyll content,FCR activity and the expression of genes related with iron deficiency in wild-type and transgenic Arabidopsis under iron deficiency were tested.The results showed that after growth on 1/2 MS medium discontaining Fe for 12 days,root length of Arabidopsis over-expressing AtSCL30? were significantly longer than that of the wild type,but there were no significant difference between the mutant and wild type.Under hydroponic conditions,after iron deficiency treatment for 8 days,compared with the wild-type,Fe content and chlorophyll content in the shoot of mutant and over-expressing lines significantly decreased,Fe content in the root of over-expressing lines also decresaed;FCR activity in the over-expressing AtSCL30? is significantly lower than the wild type;the expression of FR02 was increased under iron deficiency in the wild type,but the level was lower significantly in the mutant.These results indicated that AtSCL30? can participate in the response to iron deficiency in arabidopsis and knock out and overexpression of the gene can alleviate iron deficiency to some degree,but the specific regulating machanism need to study in deepth.