| Rice is one of the world’s major cereal crops and the staple food of more than half of the world’s population(50%-80%).Because of the single cropping pattern,it is very easy to be invaded by foreign pathogens,which seriously affects the rice yield and quality.Thus,it is necessary to solve these problems by finding and using resistance genes.Numerous studies have shown that WRKY transcription factors play a key role in plant response to biotic and abiotic stresses.These studies not only help us to discover the important role of the WRKY gene in disease resistance,but also help us to develop new disease resistance materials for rice breeding.The early work of the lab cloned the gene sequences of the OsWRKY7 and OmWRKY7,and did a preliminary study on its molecular function and disease resistance response.It was indicated that OsWRKY7 and OmWRKY7 proteins were mainly localised in cell nucleus of rice protoplast,and have transcriptional activation activity in yeast cell.Both OsWRKY7 and OmWRKY7 genes were expressed in roots,stems,leaves and flowers and the highest expression were found in the leaves.OsWRKY7 and OmWRKY7 genes may regulate the response of rice to biotic stress(bacterial leaf blight),but the underline mechanisms of regulation were not clear.In order to find the components involved in the same regulatory pathway controlled by OsWRKY7 and OmWRKY7 genes,we constructed the corresponding prokaryotic expression vector OsWRKY7-pGEX-4T-1 and OmWRKY7-pGEX-4T-1 and optimized the expression conditions to obtain highly purified OsWRKY7-GST and OmWRKY7-GST fusion proteins.Then using GST pull-down technique and mass spectrometry to identify the proteins that have possible interactions with target proteins,and using yeast two-hybridization to validate the interactions.We also tested the self-interaction of the target proteins by BiFC technology;constructed the OsWRKY7 CRISPR/Cas9 transgenic plants and conducted preliminary study on its disease resistance.The main results are shown below:1.Prokaryotic expression vector construction and protein expressionWe constructed prokaryotic expression vector OsWRKY7-ipGEX-4T-1 and OmWRKY7-pGEX-4T-1,then transformed them into BL21(DE3)competent cells for fusion protein expression and condition optimization.The results showed that the expressed OsWRKY7-GST and OmWRKY7-GST fusion proteins were soluble and the best expression condition was induced by 0.5mM IPTG for 6h at 20℃.2.Fusion protein purification and GST pull-down screening for interacting proteinUnder the optimal induction condition,the OsWRKY7-GST and OmWRKY7-GST fusion proteins were purified and pull down with the total protein of the plant tissue.By the mass spectrometry identification and analysis,85 proteins were pulled down by both the OsWRKY7-GST and OmWRKY7-GST fusion proteins.According to NCBI,RAP-DB,Uniprot and Chinese rice database,these proteins were known as starch synthesis related proteins,receptor proteins in signal transduction pathways,oxidation-reduction proteins,protein synthesis and metabolism related proteins,defense related proteins,respiration related proteins,photosynthetic related proteins,and other unknown functional proteins.3.Verification the protein interaction using the yeast two-hybridization testThrough the analysis of the structure characteristics of Os/OmWRKY7 protein,truncated proteins were designed at the C-terminal,WRKY domain and N-terminal,and the corresponding self-activating vector was constructed to test activation activity in yeast.The transcription activation domain was identified between the 51-75 amino acids in the N-terminal of both Os/OmWRKY7 protein.The truncated protein Os/OmWRKY7-NT3 without self-activating activity was used as bait protein for yeast two-hybridization to verify the interactions identified from pull down assay.The results showed that the selected four proteins(OsAlbal,OsCSP2,OsGF14e,OsRACKIA)were not interacted with the target protein in Y2H,possibly because the target protein was truncated and changed its conformation,which affected the protein function.It was also possible that the interaction between the candidate interaction protein and the target protein was indirect.4.The self-interaction analysis of OsWRKY7 and OmWRKY7 proteinsWe constructed the BiFC vector by fusing OsWRKY7 and OmWRKY7 proteins with nYFP and cYFP and transient expressed in tobacco leaves.The results showed that OsWRKY7 and OmWRKY7 had strong self-interaction in nuclear area outside nucleolus,demonstrated that they can form homologous dimers or multimers in the nucleus,that was the characteristics shared by the WRKY transcription factor family.5.Construction and preliminary analysis of OsWRKY7 CRISPR/Cas9 transgenic plantsA CRISPR/Cas9 knockout vector of OsWRKY7 gene was constructed and transgenic plants were obtained.The target region of the positive T0 transgenic plants was amplified for sequence analysis.The results showed that among the 33 T0 transgenic plants,19 plants were mutated and no mutation occurred in the rest of 14 plants,the mutation rate was 57.6%.Among the plants with mutation,13 plants have homozygous mutations,the homozygous mutation rate was 39.4%,and 6 plants have multiple hybrid mutations.Three type of homozygous mutation patterns were found:type 1 had an "A" deletion before the 3rd bp upstream of PAM site;type 2 had a "T"deletion before the 2nd bp upstream of PAM site;type 3 had two s"CA" deletion before the 3rd bp upstream of PAM site.All of the three mutations led to the missing of the initiator codon ATG.The multiple mutation had a hybrid sequencing peak at the target site,it suggested that different forms of mutation were produced in these lines.The disease resistance of OsWRKY7 CRISPR/Cas9 plants was preliminarily tested.T,plants from lines with homozygous mutation were used for bacterial blight infection.The results of the inoculation experiment showed that lesion lengths in OsWRKY7 knockout plants were obviously shorter than that in the susceptible wild type,indicating that the OsWRKY7 gene may act as a negative regulator of rice in response to bacterial blight. |
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