木薯MeAHL31基因克隆及其在原核细胞中的表达与优化

Molecular Cloning of Cassava MeAHL31 Gene and Its Expression and Condition Optimization in Prokaryotic Cells

AT-hook是一类含有AT-hook基序的核定位蛋白,被命名为AHL蛋白(AT-hook nuclear localized proteins),在植物生长发育、器官建成以及对激素信号和逆境胁迫响应中起重要作用。本研究通过RT-PCR克隆获得木薯MeAHL31基因,全长为1 189 bp,其中开放阅读框(ORF)为1 014 bp,编码337个氨基酸,蛋白质分子量为35.69 k D,理论等电点为5.55,GRAVY为-0.74,是一个不稳定的亲水性蛋白,在128～264 aa区间存在一个AT-hook蛋白的保守结构域PPC domain,属于AT-hook蛋白家族。我们分别将全长MeAHL31(1～1 014)和MeAHL31 (370～742)基因片段构建到pET28a原核表达载体和含麦芽糖结合蛋白(maltose binding protein, MBP)标签的pET28a-MBP载体上,并转化至E. coli BL21 (DE3)中进行诱导表达和表达条件优化。结果表明,最佳诱导表达条件是OD600=0.6,IPTG浓度为1.0 mmol/L,15℃培养16 h;含MBP融合伴侣的pET28a-MBP载体更有利于Me AHL31蛋白的可溶性表达;在同种载体中预测可溶性高的MeAHL31(370～742)区段序列较MeAHL31 (1～1 014)全长基因的可溶性蛋白表达水平更高。本研究优化高效诱导表达条件获得MeAHL31融合蛋白,为探究MeAHL31基因的生物学功能提供理论依据。

AT-hook i s a class of nuclear localizing proteins containing AT-hook motif, named AHL protein(AT-hook nuclear localized proteins), which plays an important role in plant growth and development, organ formation, and response to hormone signaling and stress. In this study, the MeAHL31 gene of cassava was obtained by RT-PCR cloning. Sequence analysis showed that the sequence length was 1 189 bp with the AT-hook family gene sequence numbered Manes. 10 G027000 in the phytozome database, including the open reading frame(ORF)1 014 bp, which was encoded 337 amino acids, The PPC domain exists in the 128~264 aa region of the AT-hook protein. This is an unstable hydrophilic protein with a molecular weight of 35.69 kD, a theoretical isoelectric point of 5.55 and a GRAVY of-0.740. Firstly, full-length MeAHL31(1~1 014) and predicted higher soluble MeAHL31(370~742) gene fragments were constructed into pET28 a prokaryotic expression vector and maltose binding protein(MBP)-tagged pET28 a-MBP vector. Then, it was transformed into E. coli BL21(DE3) for induction expression and optimization of expression conditions. The results showed that the optimum expression was OD600=0.6, IPTG with a final concentration of 1.0 mmol/L and 16 h at 15℃, and pET28 a-MBP carrier containing MBP fused chaperone was more beneficial to the soluble expression of Me AHL31 protein, and the solubility of MeAHL31(370~742) in the homologous carrier was more soluble than that of MeAHL31(1~1 014) and the expression level of sex protein is higher. In this study, the optimal protocol for efficient induction and acquisition of MeAHL31 fusion protein was obtained, which was convenient for subsequent experiments and provide a theoretical basis for exploring the biological function of MeAHL31 gene.