甘蓝BoMLPKn1的克隆及其拟南芥同源基因AtAPK1b的功能研究

Cloning and Functional Analysis of BoMLPKn1’s Orthologs Gene AtAPK1b in Arabidopsis

在克隆甘蓝BoMLPK编码序列过程中,获得BoMLPKf1/2编码序列和1个新的转录文本(命名为BoMLPKn1)。BoMLPKf1/2和BoMLPKn1的c DNA大小分别为1215、1233和1257 bp,分别编码含404、410和418个氨基酸残基的多肽链。与BoMLPKf1/2相比,BoMLPKn1有两个插入序列,一个是在1102~1114 bp之间有12个碱基的插入,另一个是在1152~1182 bp之间有30个碱基的插入。BoMLPKf1/2定位在甘蓝3号染色体上,BoMLPKn1定位在4号染色体上。氨基酸序列比对发现,BoMLPKf1和BoMLPKn1的N端均含典型的豆蔻酰化基序,BoMLPKf2的N端含疏水结构域。3个转录文本均具有1个高度保守的Ser/Thr蛋白激酶结构域。RT-PCR检测发现自交不亲和甘蓝自花授粉15min后BoMLPKf2相对表达量急剧升高,BoMLPKf1和BoMLPKn1在自花授粉15 min后相对表达量无明显变化。亚细胞定位检测到BoMLPKn1定位在细胞膜上。利用CRISPR-Cas9植物编辑系统对BoMLPKn1拟南芥直系同源基因AtAPK1b基因组进行定点敲除,获得突变体植株。与野生型相比,突变体植株均能够正常开花结籽。结果表明MLPKn1基因在整个十字花科进化中是高度保守的,不参与自交不亲和反应,这与MLPKf1/2不同。

When cloning the BoMLPK gene from the Brassica oleracea,we obtained the reported MLPKf1/2 and a novel transcrip(tnamed BoMLPKn1).The c DNA of BoMLPKf1/2 and BoMLPKn1 was 1215,1233 and 1257 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 1102 and 1104 bp,and the other was a 30 bp insertion located between 1152 and 1182 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 AtAPK1 b 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.