Construction Of SRK S-Domain Expression Vectors To Characterize SCR/SRK Interactions In Brassica Oleracea L.via A Yeast Two-Hybrid System

The multiple alleles of a single locus control the self-incompatibility of Brassica, which named as S-locus. It determines the specificity of the pollen recognition which locate in the stigma surface, control S receptor kinase gene(S-locus receptor kinase, SRK)and S locus glycoprotein gene (S-locus glycoprotein,SLG) are Two of the key proteins which control the self-incompatibility in Brassica:the SRK determine the self-incompatibility, SLG only the cofactor. The pollen S locus protein (S-locus cystein-rich protein,SCR) is SRK'S ligands, which is completely linked with two other genes.In this study, using yeast two-hybrid system,the pGADT7-eSRKs and pGBKT7-SCR srecombinant vector were constructed and transformed into yeast Y187 and Y2HGold separately. Our results showed that:1. The amplification of different Truncated fragments eSRKs and Sequence Analysis.The different Truncated fragments eSRKs were amplified from leaf genomic DNA in Brassica oleracea L.'B3'by PCR and using 1%(w/v) agarose gel electrophoresis, the fragments length are identical with the theory value of primer extension. Then subcloned into pGADT7 and constructed pGADT7-eSRKs plasmids. The DNA sequencing analysis showed that pGADT7-eSRKs plasmids contain eSRKs fragments, and the insertion site and the reading frame are correct.The analysis of BLAS online showed that.the eSRK encodes 421 amino acids, contains B-Lectin domain, SLG domain and PAN-APPLE domain. The eSRKl encodes the 5 to 427 amino acids, which contains three hypervariable region of eSRK sequence; eSRK2 encodes the amino acids between 139 and 427, which contains two hypervariable region of eSRK sequence; eSRK3 encodes the amino acids between 5 and 273, which contains two hypervariable region of eSRK sequence. eSRK2 encodes the amino acids between 5 and 140,which contains one hypervariable region of eSRK sequence.2.The toxicity and Autoactivation detection of recombinant AD plasmid pGADT7-eSRKs.The empty vector pGADT7 and pGADT7-eSRKs were transformed into Y187, the ransformants of Y187 [pGADT7] and Y187 [pGADT7-eSRKs] grew well on the SD/-Leu plates, while the yeast strain Y187 which was not transformed recombinant plasmid did not grow on SD/-Leu medium.It is suggested that recombinant plasmid pGADT7-eSRKs was successfully transformed into Y187 yeast cells and was not toxic to yeast cell Y187. In addition, the white clones of Y187 [pGADT7-eSRKs] grew well in SD/-Leu plates and the colonies did not turn blue on SD/-Leu/x-a-gal plates. The results indicate that recombinant plasmid pGADT7-eSRKs do not activate the reporter gene MEL1.3.The interaction detection between different truncated fragments of SCRs and eSRKs.The analysis of bioinformatics autodocktools software shows that SRKs can mutually recognize with SCRs in the space to form a fusion protein.The recombinant plasmids were success fully transformed into the diploid yeast mating cell, the two group of Y187 [pGADT7-eSRKl] x Y2HGold [pGBKT7-SCR2] and Y187 [pGADT7-eSRK4] xY2HGold[pGBKT7-SCR2] could emerge significantly blue clones, then remove blue clones on DDO/x/A plates to QDO/x/A plate, after four days, we can find the blue clones on the plate, which may indicate that activate the reporter gene AUR1-C, MEL1, ADE2 and HIS3. The resoults show that the yeast two-hybrid system could be used to study the interaction domain between eSRK and SCR. The results indicated that there existed two kinds of interactions among all interaction assays between SRK and SCR, whose interaction regions span 16-421 bp fragment of the first exon of SRK and 1876～2068 bp fragment of SCR.