Prokaryotic Expression Of A WRKY Transcriptional Factor Gene SUSIRI From Rice And Isolation Of Its Target Gene Sequence By Whole Genome PCR Method

Transcription factors are protein that play an important role in the regulation of plant growth and development,as well as plant response to environment.They ususlly contains a DNA-binding domain,a transcription regulation domain,an oligomerization site and a ncleaar localization signal.Transcription factors regulate the expression of target genens by binding to the cis-elements in the regulatory sequence of the gene with special protein functional domain.WRKY proteins are one of the important transcription factors in plants,whicn involved in growth,development,metabolism and biotic or abiotic stress responses of the plant.In this paper, the SUSIRI gene---- a WRKY familiy gene that was cloned from the Oryza sativa sub. Japonica var. Nipponbare,has been studied in aim to clarifiy its biological function from the following three aspects:1,Based on nucleotide sequence of the cloned SUSIRI gene, the structure and functions of its predicted protein were analyzed by bioinformatics tools,including its conserved domain,Subcellular localization,putative functions and so on. Furthermore,the flurescence expression vector of pNSUGFP driven by actin promoter was constructed by fusing the SUSIRI gene and GFP gene and was used to transform into epidermis cell of onion by Gene gun,the result indicated that the SUSIRI protein could be localized in nucleolus.2,Both PET-SU and PQE-SU procaryotic expression vector were constructed by inserting the SUSIRI gene into the PET-28a and PQE30 vector respectively.After addition with IPTG,a 63kD targeted protein was observed to express in PET-SU/BL21(DE3)plysS combination and the expression of SUSIRI protein maximized in 2-3h after IPTG induction. The result indicated that the WRKY protein SUSIRI can be obtained by procaryotic expression,but the expressed amount was so little that it was only detected by western bloting instead of by Coomassie brilliant blue staining.3,By use of the pull-down method for isolating the target genes binding to SUSIRI protein from the total DNA of rice,the lysate of E.coli which containing expressed SUSIRI protein was applied to catch the DNA target and the obtained 21 fragments were sequenced. Based on blast in NCBI,Gramene database and PLACE database,these sequences were identified to be Photosystem I P700 chlorophyII a apoprotein A1 gene,Chloroplast envelope membrane protein gene,Transposon protein gene and some unknown protein genes.There were 16 nucleotide sequences containing the core element of W-box,with 13 of them contain W-box element and 5 of them containing sugar responsive element.All these elements are the known targeting sites of most reported WRKY family transcriptional factors.