Screening And Separation Of Brassica SI Key Components Of MLPK Interaction Protein

Self-incompatibility(SI)is commonly found in flowering plants,and is a protective mechanism for the protection of genetic diversity in the long-term evolution of plants.The in-depth study of the SI signal transduction mechanism can provide new content for clarifying the signal transduction of plant intercellular signals,and can also provide a theoretical basis for the production of hybrids by using self-incompatibility lines.In the past,it was generally believed that the signal transduction of the spores self-incompatibility of Brassicaceae was transferred through the SRK-ARC1-Exo70A1 pathway.However,recent studies have shown that the ARC1-Exo70A1 signaling pathway may be only a branch of the signal transduction pathway of the downstream SRK,the suppression expression of ARC1 or over expression of Exo70A1 in transgenic Brassica napus can only partially break the self-incompatibility of materials.There may be other SI signal components and signal transfer branches,which transmit SI signals together with ARC1-Exo70A1 pathway.In this study,the coding sequences of SRK,SCR,MLPK,ARC1 and Exo70A1 genes were amplified by self-compatible Brassica rapa SCM1 and SCM2 and self-incompatible Brassica oleracea A4,and the expression of SI related genes was analyzed.The MLPK short cut bait protein based on yeast two hybrid system was successfully constructed and verified by interaction with ARC1 short cut.After screening the PUB family gene of Brassica oleracea,we screened the PUB family gene by constructing the MLPK short cut bait protein.The following results are obtained:(1)cloning and expression analysis of SI genes from Brassica oleracea and Brassica rapaUsing the stigma and pollen cDNA of Brassica oleracea A4,Brassica rapa SCM1 and SCM2 as template,the genes of SRK,SCR,MLPKf1,MLPKf2,MLPKn1,ARC1 and Exo70A1 of the three materials were amplified by high fidelity enzyme,and the expression of these related genes in the three materials was analyzed by fluorescence quantitative PCR.The sequence analysis shows that SCR,ARC1 and Exo70A1 amino acid sequences have high conservatism in Brassica oleracea and Brassica rapa.The SRK gene of Brassica oleracea is S28 haplotype.Both the full-length SRK gene and the deletion mutant can be amplified.The SRK gene of the two kind of Brassica rapa is f2 haplotype.Both the full-length SRK gene and the deletion mutant can be amplified.There was a critical amino acid mutation in MLPK of Brassica rapa material.The 194 position of glycine G mutated to arginine R,which lost the ability of auto-phosphorylation.After the analysis of the expression of SI gene,it was found that all the key components of SI could be expressed normally in Brassica oleracea and Brassica rapa materials,and the expression of different SI components in different materials has different level.(2)Construction of MLPK bait protein and its interaction with ARC1According to protein domain analysis,MLPK and ARC1 short cut bodies were constructed respectively.After bioinformatics analysis of the MLPK and ARC1 domains,it was found that MLPK contains a protein kinase domain;ARC1 contains an arm repeat region,a U-box,a leucine zipper,and a coiled coil.MLPK is a receptor kinase localized on the membrane.We constructed a short-cut of the MLPK removed membrane localization sequence,subcloned the constructed MLPK short-cut into pGADT7,and subcloned the constructed ARC1 short-cut into pGBKT7.The PEG/LiAc method was used to transform into AH109 yeast competent state.The constructed MLPK and ARC1 short segments showed no auto-activation and toxicity.Interaction analysis showed that the interaction between MLPK-M3M4 and ARC1 arms was the strongest,and MLPK-M2M4 could interact with the ARC1 arm repeats,and other combinations could not interact with each other.(3)Subcellular localization of MLPK short-cutsTo determine whether MLPK cannot be introduced into the nucleus have an influence on the interaction between full-length MLPK and ARC1 cannot be detected by yeast two-hybridization.The subcellular localization experiment was performed to observe the MLPK short-truncated which removed region of the membrane localization signal.After subcloning of the MLPK short truncated subunit into the 1391 vector,subcellular localization was performed by infecting onion epidermal cells with Agrobacterium EHA105,and the fluorescence was observed under a confocal microscope.The results showed that the full length of the fusion protein GFP-MLPK was only expressed on the onion cell membrane,and the fusion protein GFP-MLPK-core was expressed in the nucleus,cytoplasm,and cell membrane.(4)Excavation and analysis of PUB family genesUsing HMMER software to search the full-genome of PUB conserved domain PF04564,using domain prediction SMART software to analyze whether the PUB gene obtained by HMMER software contains PUB domain,and using the MEGA evolution analysis software to carry out evolution analysis of PUB amino acid.This study identified 96 Brassica oleracea PUB members,101 Brassica rapa PUB members,70 Arabidopsis lyrata PUB members,and 62 Arabidopsis thaliana PUB members.According to the composition of the PUB gene domain,Brassica oleracea PUB genes was divided into 6 categories,Brassica rapa PUB genes was divided into 12 categories,Arabidopsis lyrata PUB genes was divided into 6 categories,and Arabidopsis thaliana PUB genes was divided into 8 categories.Although the PUB family genes of Brassica oleracea are composed of different domains,the U-box domain sequences are conserved.In the PUB protein evolution relationship of Brassica oleracea,BoPUB95,BoPUB25,and ARC1 are on one large branch,and BoPUB25 and ARC1 have the closest evolution relationship.BoPUB30,BoPUB3,BoPUB56,BoPUB43,BoPUB75,and BoPUB69 are in the same evolutionary branch.(5)MLPK bait protein screens the Brassica oleracea PUB family members that interact with itAll the PUB sequences in the same branch as ARC1 were screened,named BoPUB43,BoPUB75,BoPUB30,BoPUB3,BoPUB56,BoPUB69,BoPUB95,and BoPUB25.The constructed MLPK short-cut was subcloned into pGADT7,and the constructed BoPUB short-cut was subcloned into pGBKT7,and transformed into AH109 yeast competent state by PEG/LiAc method.The constructed MLPK and BoPUB short-cutlets had no auto-activation and Toxicity,using the MLPK-core region and the arm repeat region of the PUB gene for interaction detection,it was found that the arm repeat regions of BoPUB43,BoPUB75,and BoPUB25 interact with MLPK-M3M4,and the interaction intensity with BoPUB25 is the highest.