Analysis On The Interaction Between M-locus Protein Kinase And Plant U-box Protein In Brassica Oleracea

Self-incompatibility(SI) is a complex and common genetic mechanism formed by plants in the long-term evolution process in order to limit self-destruction and promote hybridization,which is also a hybrid seed production of cruciferous crops.It is widely used in the important trait,and its stigma papilloma cells specifically reject the response from self-pollen pollen to cross-pollination,which provides an ideal model system for studying the signal transmission mechanism of plants.Brassica plants such as Brassica Oleracea are expressed as sporophytic self-incompatibility.It is generally believed that after self-flowering or the same haplotype pollen falls on the stigma,the SCR(S-locus cysteine-rich protein)in the pollen and the SRK(S-locus receptor kinase)in the stigma interact in a haplotype-specific manner.This interaction will cause the SRK conformational change to release Thioredoxin-h-like protein 1 and 2(THL1 / THL2),which was bound to the original kinase domain,and stimulate SRK kinase activity to interact with MLPK(M-locus protein kinase,MLPK),recruit and phosphorylate ARC1(Arm repeat containing 1,ARC1).Then,ARC1 binds and ubiquitinates to mediate the degradation of Exo70A1 by the proteasome.Then,the self-incompatibility reaction is achieved through a cascade reaction.For the above signal transmission process of self-incompatibility,there are still two aspects to be further studied.Firstly,the molecular mechanism by which MLPK participates in signal transduction is still not clear.In this respect,SRK can phosphorylate MLPK,at the same time,both SRK and MLPK can phosphorylate ARC1,and MLPK has a stronger phosphorylation effect on ARC1 than SRK on ARC1.Therefore,it is unclear that the phosphorylation of ARC1 during self-incompatibility signaling is due to MLPK recruiting and phosphorylation of ARC1 alone or whether SRK performs the function with MLPK.Secondly,there is also question about the signal transduction pathway of SRK-ARC-Exo70A1,which is currently widely believed.Antisense inhibition of ARC1 expression in Brassica napus and Arabidopsis lyrata and overexpression of Exo70A1 in Brassica napus can only partially break the self-incompatibility signal,thus suggesting that there may be other unknowns signal element and ARC1 together participate in downstream signal transmission.However,previous screening and identification of SRK interacting proteins have failed to isolate new signal elements other than ARC1.Therefore,the isolation and identification of MLPK interacting proteins is expected to be a breakthrough for obtaining new signal elements.Previous studies have suggested that PUB proteins may be involved in self-incompatibility signaling.Based on the functional domain analysis of MLPK,our team have successfully constructed the MLPK protein as bait protein and successfully detected the interaction between MLPK and ARC1 by yeast two-hybrid.Previously,based on the whole genome identification and expression analysis of the PUB gene family in Brassica oleracea,we used yeast two-hybrid to initially screen three candidate BoPUB proteins that may interact with MLPK.Based on the existing research,this study first cloned three-candidate PUB protein coding gene sequences and performed biological analysis.The tissue expression patterns of the three candidate PUB genes were analyzed by semi-quantitative RT-PCR.The MLPK mutant was constructed.The yeast two-hybrid and Bi FC technology were used to clarify the interaction between MLPK and PUBs,in order to lay the foundation for clarifying the role of MLPK in self-incompatibility signaling.The main research results obtained are as follows:(1)Gene cloning and bioinformatics analysis of three candidate PUB protein-coding sequencesUsing the highly self-incompatibility "A4" stigma c DNA of Brassica oleracea as a template,the gene sequences of protein encoding genes of BoPUB25,BoPUB43,and BoPUB75 were cloned by PCR technology with high fidelity enzymes.Bioinformatics analysis showed that BoPUB25,BoPUB43,and BoPUB75 were all hydrophilic proteins with no transmembrane structure,no signal peptide,and contained U-box and ARM functional domains.They belonged to the PUB family.Subcellular predictions indicate that the three were located in mitochondria,endoplasmic reticulum and endoplasmic reticulum.Protein structure predictions indicated that the three proteins were mainly alpha helixes.Tertiary structure prediction showed that all three proteins contained domains that regulated calcium ion transport.(2)MLPK sequence analysis and construction of MLPK mutantsAccording to previous studies,mutation of 112 th lysine to arginine or 194 th glycine to arginine in the MLPK protein sequence will cause MLPK to lose kinase activity.In this study,the MLPK kinase domain coding region sequence was specifically amplified.We mutated the 112 th lysine-coding base of the MLPK protein coding sequence to the arginine-coding base to construct an inactive mutant mlpk1.We mutated the 194 th glycine-coding base of the MLPK protein coding sequence to the arginine-coding base to construct an inactive mutant mlpk2.The sequencing results showed that the coding sequences of MLPK,mlpk1,mlpk2 genes were the same as expected.The mutation locis of mlpk1,mlpk2 were as expected,the gene sequences were correct,and the mutants were successfully constructed.(3)Expression patterns analysis of three candidate BoPUB genesWe used c DNAs of stigmas,anthers,petals,sepals,and leaves on the day of flowering of high self-incompatibility Brassica oleracea "A4" as metirals.Using PP2 A gene as an internal reference gene,the tissue expression patterns of the previous three candidate PUB genes were analyzed by semi-quantitative RT-PCR.The results showed that generally speaking,the four genes of BoPUB25,BoPUB43,BoPUB75 and ARC1 were all expressed in the stigma of Brassica oleracea,but unlike ARC1,the genes of BoPUB25,BoPUB43 and BoPUB75 were widely expressed in five tissues of Brassica oleracea.(4)Yeast two-hybrid and Bi FC verification of MLPK interaction with three candidate PUB proteinsWe used yeast two-hybrid method to detect the interaction of MLPK and its mutants with BoPUB25,BoPUB43 and BoPUB75.The results showed that the colonies of twelve combinations of MLPK and its two mutants and three PUBs and ARC1 could grow normally and turn blue.This indicated that MLPK and its inactive mutants might interact with the three PUB proteins and ARC1.In particular,the inactive mutants mlpk1 and mlpk2 might interact with the three PUB proteins and ARC1,indicating that the interaction between MLPK and the three PUB proteins and ARC1 might not completely depend on the kinase activity of MLPK.In order to further verify the detection results of yeast two-hybrid,we used Bi FC technology to detect the interaction between MLPK and its mutants and BoPUB25,BoPUB43 and BoPUB75.The results showed that there were obvious fluorescent signals in twelve combinations between MLPK and its mutants mlpk1,mlpk2 and BoPUB25,BoPUB43,BoPUB75 and ARC1,all of which were located on the cell membrane.It further proved that there was possible interactions between MLPK and its two mutants and BoPUB25,BoPUB43,BoPUB75 and ARC1 proteins,and the interaction might not completely depend on the kinase activity of MLPK.The interactions between MLPK and the three PUBs occured on the cell membrane.