甘蓝BoExo70A1与BoSEC3、BoExo84蛋白相互作用的酵母双杂交检测

Exo70A1是芸薹属作物柱头接受亲和花粉的必需因子,可能参与了干性柱头水分分泌和花粉水合过程。本文从自交不亲和甘蓝A1柱头c DNA中获取了Bo Exo70A1、Bo SEC3、Bo SEC10、Bo SEC15和Bo Exo84基因的编码序列,序列分析表明,Bo Exo70A1、Bo SEC3、Bo SEC10、Bo SEC15和Bo Exo84基因分别与At Exo70A1、At SEC3A、At SEC10、At SEC15B和At Exo84B基因的c DNA序列高度同源,其编码的5个蛋白均没有信号肽,Bo SEC3蛋白含有一个典型的EF-hand钙离子结合结构域。将Bo Exo84蛋白拆分为2个片段(Bo Exo84-N和Bo Exo84-C),并与Bo SEC3、Bo SEC10和Bo SEC15蛋白编码序列一起分别亚克隆至p GADT7质粒,Bo Exo70A1蛋白编码序列亚克隆至p GBKT7质粒,酵母双杂交检测结果表明,Bo Exo70A1蛋白能与Bo SEC3、Bo Exo84-N蛋白互作,但不能与Bo SEC10、Bo SEC15蛋白互作,这为深入探讨Bo Exo70A1蛋白乃至整个胞泌复合体在甘蓝干性柱头接受自花花粉排斥异花花粉过程中的作用机制提供了依据。

Interaction Analysis of Brassica oleracea L. BoExo70A1 with BoSEC3 and BoExo84 Proteins by Using Yeast Two-hybird System

Exo70A1 is necessary for Brassica stigma accepting the compatible pollen, and may play an important role in regulating the movement of water from the dry stigma to the pollen grain and compatible pollen hydration. Here, the coding sequences of BoExo70A1, BoSEC3, BoSEC10, BoSEC15, and BoExo84 genes were amplified from the stigma cDNA of the highly self-incompatible Brassica oleracea L. line A1. Sequence analysis showed that the cDNA sequences of BoExo70A1, BoSEC3, BoSEC10, BoSEC15, and BoExo84 genes were highly homologous to those of AtExo70A1, AtSEC3A, AtSEC10, AtSEC15B, and AtExo84B genes respectively. No signal peptide was found in their deduced protein sequences. BoSEC3 protein contained an EF-hand calcium-binding domain. The BoExo84 protein was divided into two fragments, BoExo84-N and BoExo84-C, and then their encoding sequences were subcloned into pGADT7 vector together with those of BoSEC3, BoSEC10, and BoSEC15 proteins respectively, whereas the encoding sequence of BoExo70A1 protein was subcloned into pGBKT7 vector for analysis of the proteins interaction by yeast two-hybid system. The results showed that BoExo70A1 protein interacted with BoSEC3 or BoExo84-N protein, but not with BoSEC10 or BoSEC15 protein.