Study On The Interactions Of SCR-SRK In Brassica Oleracea L. And The Relational Model Of SCR-SRK Interaction Strengths And Yeast Growth

Self-incompatibility (SI) is a general concept for several genetic mechanisms in angiosperms, which prevent self-fertilization and encourage outcrossing. Self-incompatibility in Brassica species is controlled by a single S locus with multiple alleles, and it involves three highly polymorphic genes, S receptor kinase(SRK), S cystine rich(SCR) and S glycoprotein (SLG). The SRK and the SCR are the female determinant and the male determinant of the self incompatibility respectively.Binding of SCR to SRK then would elicit a signaling cascade in the papillar cell, leading to the rejection of self-pollen. The extracellular domain of SRK(eSRK) is responsible for SCR binding. However, The molecular mechanism of SRK-SCR action. is not well known up to now.For further study on the issue, the eSRK were amplified by nested PCR using Brassica oleracea L. D3, B3, E1, A1, H1,230,240,243, N1 and G1 with the trait of typical SI, while SCR were amplified by nested PCR from Brassica oleracea L. D3, E1 and G1. The interactions between eSRK and SCR were detected by the yeast two-hybrid system.The main contents are as the followings:1. Cloning and bioinformatics analysis of eSRKThe cDNA from stigma and pollen of Brassica oleracea L. D3, B3, E1, A1, H1, 230,240,243, N1 and G1 were used as templates for PCR amplification of eSRK. Sequence analysis showed that eSRKs of D3 and 240 are both 1303 nucleotides long and encodes a peptides of 434 amino acids respectively.The eSRKs of 230,243, E1, G1 and N1 all contain 1309 bp, which encodes a protein 436 amino acid respectively. The determined nucleotide and deduced amino acid sequences of eSRK A1, eSRK B3 and eSRK H1 are both 1312 bp and 437 amino acids, respectively. All eSRK proteins contain B-Lectin, SLG domain and PAN_APPLE domain. The a-helix and B-sheets of eSRK's Secondary Structure are rich. The eSRK is highly polymorphic and composed of three hyper-variable regions, but contain the twelve conserved cysteine residues. The eSRK is quick evolution and genetic branching period of the eSRK had great difference in different S haplotypes.2. Cloning and bioinformatics analysis of SCRTotal RNA was extracted from D3, E1 and G1, and their corresponding cDNAs were obtained through reverse transcription. Primers were designed to amplify SCR using 5'-end conserved amino acids and 3'-ploy(A). These cDNA sequences were 319 bp,311 bp and 377 bp in length respectively, containing 3'UTR, and could be translated into protein with 58,58, and 55 amino acids in the reading frame. SCRs of D3 and E1 have same sequences as SCR3 and SCR7. SCR of G1 is a new S-Haplotype. The gene and protein sequences of SCR-G1 and SCR-D3 have 69.5% and 63.8% similarity, respectively. SCRG1 and SCRE1 show 53.4% nucleotide similarity and 46.6% protein similarity. SCRD3 and SCRE1 show 55.7% nucleotide similarity and 48.3% protein similarity. The results showed that SCRGland SCRD3 are evolutionarily closely linked, whereas the SCR of G1, D3 and the SCR of E1 are more distant relatives. All three proteins have similiar structure and potential phosphorylation sites, contain one a-helix and three B-sheets, and are full of basic amino acids in hypervariable region.3.Bioinformatics analysis of recognition site of SCR in SRKThe eSRK and SCR sequences from Brassica. S3, S7, S8, S12, S13, S24, S29, S39 and S64 were used for domain analysis and binding recognition site analyses. The results showed that the phylogenetic tree of eSRK was similar to that of SCR. The HVⅠ, HVⅡand HVⅢof eSRK as a whole, whose phylogenetic tree was high similarity to that of eSRK. But as far as individual hypervariable region, the similarity ends there, especially HVC. The results showed that the hypervariable region of eSRK is the ecognition site of SCR. All HVI of eSRKs have binding sites of SCR, but no binding site in theβ-lectin domain.Different S-haplotypes eSRK have different binding site and amino acids, but Glu, Arg and Trp have the maximum probability in existence.4.Detection of Interactions between SCR and SRK in Brassica oleracea L. by Yeast Two-Hybrid System.For further study on mechanism of the mutual recognition between SRK and SCR in Brassica, the eSRK and SCR were amplified by nested PCR using Brassica oleracea L.'D3'and'El'with the trait of typical self-incompatibility (SI), and the interactions between eSRK and SCR was detected by the yeast two-hybrid system. Then the full-length eSRK and the SCR were fused to the Ga14 DNA activation domain (designated pGADT7eSRKD3 and pGADT7eSRKDE1) and the Ga14 DNA binding domain (designated pGBKT7SCRD3 and pGBKT7SCRE1) by the gene homologous recombination technique respectively. Then the pGADT7eSRKD3 and pGADT7eSRKDE1 plasmid were transformed into the Y187 yeast strain respectively, whereas pGBKT7SCRD3 and pGBKT7SCRE1 were transformed into the Y2HGold yeast strain respectively. The four transformed yeast strains did not exhibit autoactivation. The diploids pGADT7eSRKD3xpGBKT7SCR D3 and pGADT7eSRK E1xpGBKT7SCR E1, which were fused by the transformed Y2HGold yeast strain and the transformed Y187 yeast strain, grew on selective agar plates (SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA), and the resulting colonies were blue, which strongly indicated that eSRK and SCR could combine with each other. Whereas the diploids pGADT7eSRKD3xpGBKT7SCR E1 and pGADT7eSRK E1xpGBKT7SCR D3 could not grow on the same selective agar plates. The results showed that the SCR could bind to the ectodomain of its cognate "self" SRK and not bind nor activate "non-self" SRK.5. Study on the recognizing motif of SCR in SRKMany short eSRKs from different subdomains were amplified using Brassica oleracea L.'D3'. The multiple short eSRKs were fused to the Ga14 DNA activation domain (designated as pGADT7eSRKn(n=1,2......15)), then be transformed into the Y187 yeast strain respectively. All transformed yeast strains did not exhibit autoactivation. The diploids pGADT7eSRKnxpGBKT7SCRD3, which were fused by the transformed Y2HGold yeast strain and the transformed Y187 yeast strain, grew on selective agar plates (SD/-Ade/-His/-Leu/-Trp). When theβ-galactosidase were assayed by colony-lift filter using X-gal, the colonies pGADT7eSRK5xpGBKT7SCRD3,pGADT7eSRK6xpGBKT7SCRD3,pGADT7eSRK7 xpGBKT7SCRD3 and pGADT7eSRK10xpGBKT7SCRD3 were white. The results showed that HVⅡand HVⅢof eSRK have direct interaction with SCR. In order to test the result, HVⅡand HVⅢof eSRK was expressed in E. coli (BL21) using the expression vector pGEX-6P-3 (including a GST-tag), SCRD3 was expressed using vector pET-22b(+) (including a His-tag). Result by Western blot showed that SCR-His could combine with SRK-HVⅡ-HVⅢ-GST in vitro.6.Connection of interaction intensity between eSRK and SCR with yeast growth Three modules were estabolished about connection of SRK-SCR interaction with yeast growth using the diploids eSRKD3×eSCRD3, eSRK2×eSCRD3, eSRK3×eSCRD3, eSRK4×eSCRD3, eSRK8×eSCRD3, eSRK9×eSCRD3, eSRK11×eSCRD3, eSRK12×eSCRD3, eSRK13×eSCRD3, eSRK14×eSCRD3, eSRK15×eSCRD3 and eSRKE1×eSCRE1. Module 1:There was a significantly positive correlation between SRK-SCR interaction with yeast growth. Module 2:The module Y=0.0358+0.011X was very reasonable with SRK-SCR interaction as the independent variable. Module 3:The moduleY=-11.929+60.387X was very reasonable with yeast growth as the independent variable. The third model could reduce the cost of research, moreover, it might establish a new measuring method for Self-incompatibility of Brassica.