Duble Crossing Test of a Yeast Alternatively Interacted on SCR,THL and SRK from Brassica oleracea L.

eSRK,SCR/SP11 and THL1 are 3 important components in self-incompatibility response of Brassica oleracea L.In our study,the coding sequences of SCR2(class-Ⅱ),eSRK2(class-Ⅱ),THL1,SRKJ(class-Ⅰ),eSRK28(class-Ⅰ)of Brassica oleracea L.were amplified by PCR,then SCR2 and THL1 were subcloned into pGBKT7 vector,eSRK2,eSRK28 and SRKJ were subcloned into pGADT7 vector.After sequencing,the recombinant bait plasmids were transformed into Y2HGold yeast,the recombinant prey plasmids were transformed into Y187.The experiments indicated that the recombinant vectors did not show autonomous activation and toxicity.The experimental group Y2HGold〔pGBKT7-SCR2〕×Y187〔pGADT7-eSRK2〕could grow on SD/-Trp-Leu-His/x-α-gal/AbA nutritional media with tranion activation of the reporter genes AUR1-C,MEL1,HIS3,except ADE2 gene.The experimental group Y2HGold〔pGBKT7-SCR2〕×Y187〔pGADT7-eSRK28〕could grow on SD/-Trp-Leu,but couldn't grow on TDO/x/A and indicated that there did not exsit interaction.The experimental group Y187〔pGADT7-SRKJ〕×Y2HGold〔pGBKT7-THL1〕could grow on QDO/x/A nutritional media with tranion activation of the reporter genes AUR1-C,MEL1,HIS3,ADE2.There was the interaction between the same haploids SRK and SCK,but the different haploids couldn't have that.Differences of the repoter genes expression in number and type may reflect the interaction intensity between class-ⅡSCR-SRK and class-ⅠSCR-SRK.It is clear that the interaction between class-ⅠeSRK and SCR is stronger than class-ⅡeSRK and SCR,and the interaction intensity between THL and SRK is strong.All above mentioned facts have provided some insights into the molecular mechanism of interaction.