Cloning and prokaryotic expression of hexokinase gene from Dimocarpus longan
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摘要:目的
克隆龙眼Dimocarpus longan己糖激酶（Hexokinase, HXK）基因，并对其进行生物信息学分析及原核表达分析.
方法以龙眼总RNA为模板，采用RT-PCR结合RACE法获得基因全长cDNA序列，构建pET-32a-DlHXK原核表达载体，转化到大肠埃希菌Escherichia coli Rosetta (DE3)中诱导表达.
结果和结论成功克隆龙眼己糖激酶基因的cDNA全长序列，命名为DlHXK，登录号为KF776906.1.龙眼DlHXK序列全长2 101 bp，包括1个1 488 bp的开放阅读框，预测编码495个氨基酸；进化树和相似性分析发现，该基因与温州蜜柑Citrus unshiu的相似性最高，达到了82%；荧光定量表达分析表明，DlHXK基因随着果实的发育成熟表达量逐渐上升；经IPTG诱导和SDS-PAGE检测，所构建的原核表达载体表达的融合蛋白与预期蛋白大小相吻合.由此可见，利用RT-PCR结合RACE方法成功克隆了龙眼的己糖激酶基因，构建原核表达载体，并在大肠埃希菌Rosetta (DE3)中获得高效表达.
Abstract:ObjectiveHexokinase gene (named DlHXK) was cloned from longan, Dimocarpus longan cv. Shixia, fruit. The bioinformatics and prokaryotic expression of DlHXK were analyzed.
MethodTotal RNA extracted from longan fruit was used as a template. The full length cDNA of the gene was cloned using a combination of RT-PCR and RACE-PCR. Prokaryotic expression vector pET-32a-DlHXK was constructed and transformed into Escherichia coli Rosetta (DE3) to induce expression.
Result and conclusionThe full length cDNA of DlHXK was cloned and its NCBI GeneBank accession number was KF776906.1, which has 2 101 bp nucleotides including a 1 488 bp ORF. It was predicted to encode 495 amino acids. It had the highest homology with the Citrus unshiu (82%) through the NCBI and evolutionary tree analysis. The results of quantitative RT-PCR showed that DlHXK gene expression increased gradually during the longan fruit maturation. Induced by IPTG and determinated by SDS-PAGE, the inducing expressed recombinant protein from prokaryotic expression vector was consistent with the putative protein of DlHXK. Therefore, the full length of DlHXK has been cloned and its prokaryotic expression vector has been constructed successfully. It was induced to express effectively in E. coli Rosetta (DE3).
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Keywords:
- Dimocarpus longan /
- hexokinase /
- gene cloning /
- quantitative RT-PCR /
- prokaryotic expression
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图 1 琼脂糖凝胶电泳结果
M：DNA maker DL2000；1、2：总RNA；3：保守区扩增；4：3′RACE的第2轮扩增；5：5′RACE的扩增；6：ORF扩增.
Figure 1. Results of agarose gel electrophoresis
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图 2 DlHXK基因与其他植物中HXK基因编码的氨基酸序列相似性比较
P1：Phosphate 1；Con 1：Connect 1；P2：Phosphate 2；Con2：Connect 2；L1：Loop1；L3：Loop3；L4：Loop4.基因序列登录号CsHXK：温州蜜柑Citrus unshiu AAG28503.1；CmHXK：甜瓜Cucumis melo ACJ04704.1；SlHXK：番茄Solanum lycopersicum NP_001234710.1；NtHXK：烟草Nicotiana tabacum AAS60195.1；AtHXK：拟南芥Arabidopsis thaliana NP_179576.1；EjHXK：枇杷Eriobotrya japonica ADZ96378.1；DlHXK：本文克隆.
Figure 2. The amino acid sequence alignment of DlHXK compared with HXK genes of other species
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图 3 DlHXK与其他物种HXK基因编码的氨基酸序列进化树分析
Figure 3. The phylogenetic tree of amino acid sequences based on DlHXK and other species with HXK gene
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图 4 龙眼果实发育期间假种皮DlHXK基因表达的变化
Figure 4. Changes on DlHXK gene expression during the development of longan aril
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图 5 PCR扩增及pET-32a载体双酶切电泳图
M：DNA marker DL2000；1：pET-32a的质粒双酶切；2：龙眼DlHXK基因ORF扩增；3：pET-32a-DlHXK菌液PCR鉴定.
Figure 5. Agarose gel electrophoresis of PCR amplification and pET-32a digested by BamH Ⅰ and Sal Ⅰ
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图 6 龙眼DlHXK基因重组蛋白在大肠埃希菌中的表达
M：蛋白质相对分子质量标准；1：Rosetta（DE3）；2：pET-32a未诱导；3：pET-32a诱导；4：pET-32a-DlHXK未诱导；5 ~ 8：分别为pET-32a-DlHXK经0.4 mmol·L-1 IPTG诱导1、3、5、7 h.
Figure 6. Expression of recombinant pET-32a-DlHXK protein in Rosetta (DE3)
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