玉米超高蛋氨酸18kD δ-醇溶蛋白基因dzs18克隆及其原核表达

Cloning and Prokaryotic Expression of dzs18 Gene Encoding Methionine Super-rich 18kD δ-zein of Maize

以玉米自交系吉818和富含蛋氨酸（Met）的BSSS53为材料,克隆编码超高蛋氨酸18k Dδ-醇溶蛋白（18kD δ-zein）的dzs18基因,并进行原核表达。结果表明,BSSS53中分离的目标DNA序列,其ORF内有8个碱基替换突变,第208个碱基C发生缺失,导致移码突变、产生截短的多肽链。从吉818中分离的目标DNA长度为741 bp,含有由648 bp构成的ORF,编码由215个氨基酸组成的超高蛋氨酸吉818-18kD-δ-zein。与参考序列相比,目标DNA序列中存在29个碱基替换突变、15个碱基的片段插入和3个碱基的片段缺失。吉818-18kD-δ-zein中Met残基数占27.9%,是10kDδ-zein的2倍,且比参考蛋白多20%,其中部蛋氨酸高密区（HMQR）内Met残基数占50.0%。玉米dzs18在原核细胞中的表达受菌株的影响,在大肠杆菌BL21（DE3）中不表达,在Rossetta（DE3）中正常表达。ITPG浓度在0.1-1.5 mmol/L范围内对目标蛋白的表达量没有明显影响;在1-5 h范围内,目标蛋白量随诱导时间的延长而增加,超过6 h,增加不明显。玉米dzs18基因在Rossetta（DE3）中表达的目标蛋白以包涵体即不溶性蛋白质颗粒形式存在。

Maize inbred lines Ji818 and methionine（Met）- rich BSSS53 as the materials, cloning and prokary-otic expression of dzs18 gene encoding Met-super-rich 18kD δ-zein were conducted. The results showed that thetarget DNA sequence from BSSS53 had 8 base substitution mutations and a single base deletion of the 208 th base Cwithin its ORF, resulting in frameshift mutation and producing truncated polypeptides. The target DNA sequencefrom Ji818 was 741 bp long with a 648bp-ORF encoding a Met-super-rich Ji818-18kD-δ-zein comprising of 215 amino acids, but involved 29 base substitution mutations, 15-base-fragment insertion and 3-base-fragment dele-tion compared with the reference sequence. Ji818-18kD-δ-zein had 60 Met residues accounting for 27.9%, and aHigh Met Quantity Region（HMQR） in its central section, in which Met quantity accounted for 50.0%. Ji818-18kD-δ-zein had 2 times as many Met residues as 10kD-δ-zein, and higher than the reference 18kD-δ-zein by 20%.The expression of maize dzs18 gene in prokaryotic cells was restricted by bacterial strains; it expressed in Rossetta（DE3）, but not in BL21（DE3）. The accumulation of the target protein in Rossetta（DE3） cells was affected little by IT-PG concentration from 0.1 to 1.5 mmol/L, whereas it increased with an increase in induction time from 1 to 5 h, butnot obviously when longer than 6h. The target protein of dzs18 gene expressed in prokaryotic cells existed in hard-soluble inclusion bodies.