黄瓜促分裂原活化蛋白激酶基因的生物信息学分析及原核表达

Bioinformatics Analysis and Prokaryotic Expression of MAPK in Cucumber

利用RT-PCR技术结合RACE技术,从NO3-胁迫下的黄瓜根中克隆出促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)基因的同源序列,命名为CsNMAPK,GenBank注册号为DQ812086。生物信息学分析表明,该基因全长1636bp,开放阅读框(ORF)1113bp,编码370个氨基酸;其编码蛋白可能定位于细胞质;该基因有一个强的跨膜螺旋结构;PlantCare分析结果显示该基因序列具有脱落酸诱导、茉莉酸诱导、赤霉素诱导、水杨酸诱导、伤害诱导等顺式作用元件。成功构建了原核表达载体,并转化E.coli BL21(DE3),SDS-PAGE检测结果显示表达了一个约46kD的蛋白。

A cucumber cDNA designated CsNMAPK （GenBank accession No. DQ812086） was isolated by RT-PCR and RACE. Bioinformatics analysis indicated that the full-length eDNA sequence was 1 636 bp, which contained an open reading frame of 1 113 bp and encoded a protein of 370 amino acid residues. The subcellular localization analysis predicted that the protein was in the cytoplasm and there was one strong inside to outside transmembrane helix. PlantCare analysis indicated that there were abscisic acid, MeJA, salicylic acid cis-acting responsive elements and gibberellin and wound-responsive elements in CsNMAPK. The CsNMAPK was cloned into pET-30a （ ＋ ） vector to construct recombination prokaryotic expression vector pET-CsNMAPK. After transformed to E. coli BL21 and induced by isopropyl-β-D-thiogalactopyranoside （IPTG）, recombinant protein about 46 kD was expressed in pET-CsNMAPK system and separated by SDS-PAGE electrophoresis.