关键词: 甘蓝, M–位点受体激酶, 基因克隆, 表达分析, 亚细胞定位, 基因编辑

Abstract: When cloning the BoMLPK gene from the Brassica oleracea，we obtained the reported MLPKf1/2 and a novel transcript（named BoMLPKn1）. The cDNA of BoMLPKf1/2 and BoMLPKn1 was 1 215，1 233 and 1 257 bp，encoding peptides with 404，410 and 418 amino acids，respectively. Compared to the BoMLPKf1/2，the BoMLPKn1 contained two fragment insertions：one was a 12 bp insertion located between 1 102 and 1 104 bp，and the other was a 30 bp insertion located between 1 152 and 1 182 bp. BoMLPKf1/2 gene was located on chromosome 3 and BoMLPKn1 gene on chromosome 4 of B. oleracea. The amino acid sequence alignment showed BoMLPKf1 and BoMLPKn1 had a similar structure，and both of them contained a typical plant myristoylation consensus sequence at their N terminus，while BoMLPKf2 had a hydrophobic region in the N-terminal. All transcripts contained a conservative Ser/Thr protein kinase domain. RT-PCR analysis displayed BoMLPKf2 had sharply increased expression within the initial 15 min after self-pollination. The expression levels of BoMLPKf1 and BoMLPKn1 showed no remarkable change after self-pollination. The subcellular localization analysis found that BoMLPKn1 localized to the plasma membrane. Then，the AtAPK1b gene，which is the BoMLPKn1 homologous gene in the Arabidopsis thaliana，was knocked out by using CRISPR-Cas 9 editing technology. Compared with the wild type，the mutants could blossom and bear fruit normally. The results showed that MLPKn1 gene is functionally conserved in Cruciferae and it did not participate in self-incompatibility in contrast to MLPKf1/2.

Key words: Brassica oleracea, M-locus protein kinase, gene cloning, expression analysis, subcellular localization, gene editing