苎麻纤维素合酶BnCesA1高变区蛋白的原核表达与条件优化
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Prokaryotic Expression of the NH2-terminal Hypervariable Region of Cellulose Synthase Protein in Boehmeria nivea (BnCesA1) and the Expression Condition Optimization
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    摘要:

    高等植物纤维素合酶是含有多个功能结构域的糖苷转移酶,催化合成β-1 ,4葡萄糖苷链,即高等植物细胞壁的重要成分之一纤维素。在同一物种例如在拟南芥中比对纤维素合酶家族各成员的序列信息,发现其蛋白序列中存在两个高变异区域(HVR),其中第一个HVR靠近NH2端(NHVR),富含酸性氨基酸。本研究克隆了苎麻纤维素合酶基因(BnCesA1) 的NHVR编码序列,并以正确的阅读框亚克隆至含有6×His接头的pQE-N1原核表达载体,构建了pQE-N1-NHVR重组表达载体。在大肠杆菌BL21(DE3)中表达的重组蛋白His-tag-NHVR经Western-blotting验证后,正交优化得到该蛋白小量表达的最优条件组合为:所挑取的2号菌落在37 ℃下,IPTG的诱导浓度为0.1 mmol/L, 诱导表达4 hr。本研究结果为纯化His-tag-NHVR融合蛋白,制备其抗体及进一步研究苎麻纤维素合酶局部功能或其组织特异性作用打下基础。

    Abstract:

    Cellulose synthases of advanced plants have multiple functional domains and belong to the family of glycosyltransferases. They act as the catalyst for the transfer of the β-1, 4-glucan, and construct the cellulose molecule which is one of the important cell wall components of advanced plants. Comparison among cellulose synthase family members within one species such as Arabidopsis demonstrated that there are two hypervariable regions (HVR) in its protein, The first HVR lies at the NH2-terminal (NHVR) and is rich of acidic amino acid residues. In this study, we cloned the NHVR coding sequence of the Boehmeria nivea (ramie) cellulose synthase gene (BnCesA1). Then the gene fragment was subcloned into the prokaryotic expression vector pQE-N1 after its 6×His tag in the right reading frame to construct pQE-N1-NHVR recombinant expression vector. After the confirmation of the expression of the fusion protein His-tag-NHVR by western-blotting, the expression conditions had been optimized using orthogonal array testing in E. coli BL21(DE3). The results showed that the optimal combination of small-scale expression conditions is: colony No. 2, with concentration of IPTG at 0.1 mmol/L, inducing time for 4 h and temperature at 37℃. Altogether, our results would benefit the subsequent purification of His-tag-NHVR fusion protein and the preparation of its antibody, and support the further study to regional functions of BnCesA1 and its tissue specific functions in ramie.

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谢庆宁,揭雨成,周清明,等.苎麻纤维素合酶BnCesA1高变区蛋白的原核表达与条件优化[J].植物遗传资源学报,2013,14(6):1130-1136.

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  • 收稿日期:2013-03-24
  • 最后修改日期:2013-03-26
  • 录用日期:2013-09-24
  • 在线发布日期: 2013-09-24
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