红肉苹果丝裂原活化蛋白激酶激酶9基因的克隆及其在氮胁迫条件下的表达分析

Cloning of mitogen-activated protein kinase kinase 9 gene in the red-fleshed apple and its expression analysis under nitrogen stress

MKK9是丝裂原活化蛋白激酶(MAPK)级联信号转导途径中的重要成员。本研究从红肉苹果(Malus sieversii f.neidzwetzkyana)‘黛红’中克隆到MdMKK9基因(GenBank登录号为ON427820),其开放阅读框为945 bp,编码314个氨基酸的蛋白质,分子量35502 Da,等电点7.36。MdMKK9蛋白含有高度保守的特异结构域S/TxxxxxS/T激活基序及“DIK”活性位点,亚细胞定位显示MdMKK9存在于细胞核。‘黛红’组培苗在低氮培养过程中逐渐变红,MdMKK9基因上调表达,花青苷含量增加。构建CRISPR/Cas9 MdMKK9基因编辑载体,并成功转化‘王林’愈伤组织(CR),与转PRI101-MdMKK9过表达载体的‘王林’愈伤组织(OE)相比,随着培养基中氮素浓度降低,OE愈伤组织逐渐变红,花青苷含量升高,而CR愈伤组织颜色和花青苷含量均无变化。研究结果表明,MdMKK9基因通过介导植物对氮的吸收,调节红肉苹果组培苗和转基因愈伤组织在低氮胁迫时对花青素的合成。

MKK9 is an important member of the mitogen-activated protein kinase(MAPK)cascade signaling transduction pathway.In this study,MdMKK9 gene(GenBank accession number ON427820)was cloned from red-fleshed apple(Malus sieversii f.neidzwetzkyana)’Daihong’.Its open reading frame was 945 bp,which was encode a 314 amino acids residues polypeptide with molecular weight of 35502 Da and an isoelectric point(pI)of 7.36.MdMKK9 contained a highly conserved specific domain S/TxxxxxS/T activation motif and a“DIK”active site,and subcellular localization showed that MdMKK9 existed in the nucleus.In low nitrogen culture process,’Daihong’tissue culture seedlings gradually turned red,Md MKK9gene was up-regulated,anthocyanin content increased.The CRISPR/Cas9 MdMKK9 gene editing vector was constructed and successfully introduced into’Orin’calli(CR).Compared with the calli of’Orin’transfected with PRI101-MdMKK9 overexpression vector(OE),with the nitrogen content in the medium decreased,those OE calli gradually turned red,and the anthocyanin content increased,but the color and anthocyanin content of CR calli did not change.The results show that Md MKK9 gene regulates the synthesis of anthocyanin in red-fleshed apple tissue culture seedlings and transgenic calli under low nitrogen stress by mediating nitrogen uptake by plants.