Polymorphism Of DNA Coding For SRK-ARC1 Interaction Motif And Their Relationship With Compatibility Index In Cabbage

Self-incompatibility is an important genetic mechanism for floral plant to promote hybrid vigor, inhibit inbred decline. So far, most studies of self-incompatibility are focus on SCR-SRK-ARC1-Exo70Al-MOD signaling pathway. Among them, the interaction of SRK-ARC1 induced self-compatibility factor degradation is the most important step in the whole process.In our study, we chose different compatibility index or other different character cruciferae plants as materials. First, we defined SRK-ARC1 interaction domain and non-interaction domain, then amplified SRK-ARC1 interaction domains SRK kinase domain （SRKg） and ARC1 C terminal repeat arm domain （As） from gDNA. After sequencing, analysis by NCBI blastn and MEGA et al, found DNA sequence polymorphisms of SRKg and As, and the relationships between those sequence polymorphisms and compatibility index in Brassica oleracea. Among them, selected the SRKg and As which show significant relationship with compatibility index, detected the interaction between different SRKg and As by yeast two-hybrid assay. These results add some novel insights into the relationship between polymorphism of SI and compatibility index, and it also provides new evidence on identification plant compatibility by using DNA sequence polymorphisms. The main work and results are as follows: 1 The definition of interaction area and non-interaction area of SRK-ARC 1 in B. oleraceaIn this study we used bio-information technology to analysis the interaction motif between SRK and ARC1 in B. oleracea. （1） Protein primary structure sequence analysis demonstrated that BoSRK.6 kinase domain containing 391 amino acid residues and 27 potential phosphorylation sites, show weak hydrophobicity. Alignment SRK kinase domain of several different plant species, found conserved amino acids from E46 to A235, these amino acids can be divided into four sub-domains. BoARCl ARM repeat area containing 263 amino acid residues and 13 potential phosphorylation sites, show strong hydrophobicity. It can be divided in to five ARM based on distribution of hydrophobic amino acid sequence. （2） Secondary structure analysis shows that the proportion of random coil and a helix are different in BoSRK kinase domain and BoARCl arm repeat domain. BoSRK kinase domain contains 34.27% a helix and 33.76% random coil, comparatively, BoARCl arm repeat domain contains 56.65% a helix and only 18.63% random coil. （3） Tertiary structure analysis found that the energy is high and groove area is narrow at both ends area of BoSRK, display that substrate protein can hardly bind to these areas, while in the middle area the energy is lower and groove area is wider, show that substrate protein can easily bind to this area. The four spiral helix structures of BoARCl in D92-V253 area correspond to triplet spiral structure, otherwise, modeling spatial structure of this area found a wide groove. Those all point that the interaction area of SRK and ARC1 may present in SRK kinase E46-A235 and ARC 1 arm repeat domain D92-V253, other amino acid sequences are the non-interaction area.2 Cloning SRK-ARC1 interaction motifs, analyzing sequence polymorphism and the correlation of polymorphism and compatibility indexAmplified SRK-ARC 1 interaction motifs, SRK kinase domain SRKg and ARC1 C terminal repeat arm domain As, from gDNA. After sequencing, according definition interaction area and non-interaction area performed polymorphism analysis. （1） Analysis SRK-ARC1 interaction areas show that SRKg interaction area with different compatibility index can be divided into four types based on sequence differences. Among them, types exist the most different sequence, can be divided into strong self-incompatibility, Class Ib type, and stronger self-incompatibility, Class la type. Those four types of SRKg interaction area contain significant differentiated variants area, named SV1 （232-246bp）, SV2 （298-324bp）, SV3 （421-438bp） and SV4 （631-708bp）. All data indicated that there are relationship between sequence polymorphisms in different types and compatibility index. For As. they can be divided into three types based on sequence differences. Different haplotypes, compatibility index or other traits materials can distinguish by As interaction variants, named AV1 （370-381bp）, AV2 （514-549bp）,AV3（655-660bp） and AV4 （754-759bp）. All show that there are relationship between sequence polymorphisms in different types and compatibility index. （2） The analysis of non-interaction area with different compatibility index indicated that the similarly significant difference of SRKg introns 2/5/6, can be divided into five classes, while the differences of SRKg exons and SRKg introns 3/4 divided into two classes, which similar to the two of four types in SRKg interaction area. As non-interaction area shows weak sequence polymorphisms, based on sequence differences, they can be divided into three types, similar to the three types in As interaction area. （3） According to the relationship of sequence polymorphisms and compatibility index, selected four SRK kinase domain and two ARC1 arm repeat domain named E1 and m1. In four SRK kinase domain, three materials, C, m1 and 229, are strong self-incompatibility, belong to Class Ib type; K2 is stronger self-incompatibility, belongs to Class Ia type.3 Yeast two-hybrid reaction detected the interaction between SRK kinase domain and ARC1 C teminal from B.olereace with various compatibility indexSignificant different compatibility and polymorphism SRKc and As are used for construct preys PGADT7 vector SRKc （SRK_Cc、SRK_mlc、SRK₂29c、SRK_K2c） and bait pGBKT7 vector As （AsEl1、Asml）. And those recombinants were used to yeast two-hybrid reaction detection, they all could grow on QDO/x/A nutritional media with transcription activation of the reporter gene AUR1-C, MEL1, HIS3, ADE2, it indicated that there exists interaction between ARC1 and SRK from Brassica oleracea with various compatibility index, and the differences at amino acid level within SRK kinase domain or ARC1 may be not enough to change the conformation of the interaction motif. All above mentioned provides some novel insights into the successful interaction of SRK-ARC1 and the correlation between polymorphism and compatibility index in Brassica oleracea.