玉米超高蛋氨酸18kD δ-醇溶蛋白基因dzs18克隆及其原核表达
以玉米自交系吉818和富含蛋氨酸的BSSS53为材料,克隆编码超高蛋氨酸18kD δ-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长度为728bp,其开放阅读框(ORF)内存在8个碱基替换突变且第208个碱基C发生缺失,导致移码突变、蛋氨酸高密区(HMQR)的丢失和产生截短的多肽链。从吉818中分离的目标DNA长度为741bp,含有由648bp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,序列一致性为93.2%。目的蛋白与参考蛋白相比,存在26个氨基酸变异,序列一致性为88.0%；目的蛋白含有60个蛋氨酸残基(Met),其中部由98个氨基酸组成的蛋氨酸高密区HMQR含有49个Met。吉818-18kD-δ-zein中Met数占27.9%、其HMQR内Met数占50.0%；吉818-18kD-δ-zein中Met数是10kD δ-zein的2倍,且比参考蛋白18kD-δ-zein多20%。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,但在Rossetta(DE3)中则能正常表达。ITPG浓度,在0.1～1.5mM/L范围内,对目标蛋白的表达量没有明显影响；在1～5小时范围内,目标蛋白量随诱导时间的延长而增加,但超过6小时,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。
英文摘要:
Maize inbreds Ji818 and methionine (Met) - rich BSSS53 as materials, cloning and prokaryotic expression of dzs18 gene encoding Met-super-rich 18kD δ-zein were conducted. The results showed that the length of target DNA sequence isolated from BSSS53 was 728bp with 8 base substitution mutations and a single base deletion of the 208th base C within its Open Reading Frame (ORF), resulting in frameshift mutation, missing its downstream High Met Quantity Region (HMQR) and producing truncated fragment of polypeptides. The length of target DNA sequence isolated from Ji818 was 741bp, involved a 648bp-ORF encoding a Met-super-rich 18kD δ-zein comprising of 215 amino acids. In comparison with reference sequence, the target DNA sequence had 29 base substitution mutations, 15-base-fragment insertion and 3-base-fragment deletion with the identity of 93.2%. The target protein encoded by Ji818-dzs18 had 26 amino acid mutations compared with reference protein with the identity of 88.0%, involved a HMQR comprising of 98 amino acids in its central section. Ji818-18kD-δ-zein had 60 Met residues, accounting for 27.9% of total amino acids; and its HMQR contained 49 Met residues, accounting for 50.0% of HMQR amino acids. The quantity of Met residues in Ji818-18kD-δ-zein was 2 times as many Met residues as 10kD-δ-zein, and higher than reference 18kD-δ-zein by 20%. The expression of maize dzs18 gene in prokaryotic cells was restricted by bacterial strains; it expressed in the strain Rossetta(DE3), but not in BL21(DE3). The concentration of ITPG varying from 0.1 to1.5mM/L revealed a little impact on expression of dzs18 gene in Rossetta(DE3) cells with no obvious difference in the target protein quantity, whereas the quantity of target protein increased with an increase in induction time from 1 to 5hrs, but not obviously when longer than 6hrs. The target protein of dzs18 gene expressed in prokaryotic cells existed in hard-soluble inclusion bodies.
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