甘蓝SCR、THL与SRK交替相互作用的酵母双杂交检测

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

eSRK(S位点受体激酶胞外域)、SCR/SP11(S位点富含半胱氨酸蛋白-S位点蛋白11)和THL1(类硫氧还蛋白)是甘蓝自交不亲和性信号传导过程重要的3个元件。利用PCR技术获得两种甘蓝SCR2(class-Ⅱ)、eSRK2(class-Ⅱ)、THL1、SRKJ(class-Ⅰ,SRK6激酶域)和eSRK28(class-Ⅰ)的编码序列,并分别构建以pGBKT7为载体的SCR2、THL1的重组诱饵质粒和以pGADT7为载体的eSRK2、eSRK28、SRKJ的重组猎物质粒。利用酵母双杂交系统验证eSRK2-SCR2、eSRK28-SCR2、SRKJ-THL1的相互作用。重组质粒在宿主细胞中未产生毒性及自激活作用,表明重组表达载体构建成功;组合Y2HGold〔pGBKT7-SCR2〕×Y187〔pGADT7-eSRK2〕在三缺平板(SD/-Trp-Leu-His/x-α-gal/AbA,TDO/x/A)上生长出蓝色克隆,激活了报告基因AUR1-C、MEL1、HIS3;组合Y2HGold〔pGBKT7-SCR2〕×Y187〔pGADT7-eSRK28〕能够在二缺平板(SD/-Trp-Leu)上生长,但在三缺平板上不生长,表明不同单倍型SRK-SCR可能不发生相互作用;组合Y187〔pGADT7-SRKJ〕×Y2HGold〔pGBKT7-THL1〕能够在四缺平板上生长,激活了4种报告基因(AUR1-C、MEL1、HIS3、ADE2)。相同的单倍型SRK与SCR之间能够相互作用,不同的单倍型之间不会发生相互作用;酵母中报告基因表达的数目和类型的差异反映了class-Ⅱ型内部的SCR-SRK和class-Ⅰ的SCR-SRK互作强度,Ⅰ型高于Ⅱ型;THL与SRK之间存在较高的互作强度。这为Ⅰ型和Ⅱ型不同的自交不亲和性表型所依赖的3个起始信号传导元件之间的识别程度差异提供了直接证据和新内容。

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.