关键词: 结球甘蓝, CaM12, S 位点受体激酶（SRK7）基因, 原核表达, 酵母双杂交

Abstract: In order to study the molecular mechanism and possible interaction domains between
pollen tube calmodulin（CaM） protein and S locus receptor kinase（SRK）from Brassica oleracea L. var.
capitata L. We got the full length sequence of CaM12 with 450 bp and SRK7 gene with 2 118 bp from
self-incompatibility of Brassica oleracea var. capitata E1，respectively，and got extracellular domain of
SRK（eSRK7） and intracellular kinase domain（iSRK7），then constructed prokaryotic expression vectors of pGEX-CaM12，pCold-eSRK7 and pCold-iSRK7，transformed into E. coli BL21（DE3）and checked the
interactions with purified expression products in vitro. The results showed that CaM12 protein and SRK7
could do interaction，and interactive domain is iSRK7 rather than eSRK7. In order to verify their
interaction furtherly，yeast two-hybrid system was used in this study，we constructed yeast expression
vectors pGBKT7-CaM12 ， pGADT7-eSRK7 ， pGADT7-iSRK7 and pGADT7-SRK7 ， transformed
into corresponding Y2HGold and Y187 yeast cells，and made sure that they did not appear the
self-activation and toxicity. The results was consistent with prokaryotic expression. Simultaneously，we
constructed yeast expression vectors pGADT7-CAM12-2-，pGADT7-CAM12-23-，pGADT7-CAM12-234-
and pGBKT7-iSRK7 with CaM12-2-，CaM12-23-，CaM12-234- mutants from three EF-hands of CaM12
and iSRK7，and tested their interactions. The results showed that all CaM EF-hands mutants CaM12-2-，
CaM12-23- and CaM12-234- cannot interact with iSRK7 in yeast two-hybrid system. CaM12 protein lost
the ability to combine Ca2+ and cannot interact with iSRK7 after EF-hands structure domains were
mutated．This study could provide a new reference for the mechanism of self-incompatibility in Brassica
oleracea.

Key words: Brassica oleracea, CaM12, SRK7, prokaryotic expression, yeast two-hybrid