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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2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 h。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。
Higher plant cellulose synthase is a glycosyltransferase containing multiple functional domains that catalyzes the synthesis of beta-1,4 glucosidase chains, one of the important constituents of higher plant cell walls. In the same species, for example, in Arabidopsis, the sequence information of each member of the cellulose synthase family was compared. It was found that there were 2 hypervariable regions (HVRs) in its protein sequence. The first HVR was close to the NH2 side (NHVR) Acidic amino acids. In this study, we cloned the NHVR coding sequence of BnCesA1 gene and subcloned it into the pQE-N1 prokaryotic expression vector containing 6 × His linker in the correct reading frame to construct pQE-N1-NHVR recombinant expression vector . The recombinant protein His-tag-NHVR expressed in Escherichia coli BL21 (DE3) was verified by Western-blotting. The optimum conditions for the orthogonal design of the protein were as follows: The number 2 colonies picked at 37 ℃ The induced concentration of IPTG was 0.1 mmol / L and induced for 4 h. The results of this study lay the foundation for purifying His-tag-NHVR fusion protein, preparing its antibody and further studying the local function of ramie cellulose synthase or its tissue-specific effect.