【摘要】 以杂交水稻汕优63的根组织cDNA为模板,采用引物设计引入酶切位点和RT-PCR方法,扩增出了籼稻姊妹染色单体粘着基因OsRad21-i(AY318757)开放阅读框(ORF),命名为Eqfr,两端分别带有BamHⅠ和SalⅠ限制性位点。然后克隆到pGEM-T载体,将重组克隆载体pGEM-T-Eqfr与表达载体pQE30分别同时进行BamHⅠ和SalⅠ双酶切,连接酶切后的Eqfr和pQE30,并转化DH5α感受态细胞,获得了DH5α[pQE30-Eqfr]重组克隆。再以其重组质粒转化M15[pREP-4]感受态细胞,筛选出M15[pQE30-Eqfr,pREP-4]重组克隆。通过诱导表达,检测到了相对分子质量约为116 000的重组蛋白表达,在诱导后的1~3 h里表达量较高,之后表达量开始下降。用实验证实了理论推测ORF的正确性,所获得的表达蛋白及其表达体系为下一步的蛋白质实验奠定了基础。更多还原

【Abstract】 OsRad21-i(GeneBank accession number AY318757) was cloned from the root of Shanyou 63,the famous hybrid rice variety(Oryza sativa ssp.indica).It belongs to cohesin-like family,which mediates rice sister chromatid cohesion during mitosis and meiosis.The complete ORF of OsRad21-i,containing the restriction sites of BamHI and SalI introduced by primers,was named Eqfr and cloned into pGEM-T vector.The recombinant clone,DH5α[pGEM-T-Eqfr],was screened and identified using PCR.The plasmids of pGEM-T-Eqfr and pQE30 were respectively digested with BamHI and SalI at the same time.After the segregation of electrophoresis,the linear pQE30 vector and digested Eqfr were ligated by T4 DNA ligase at 4 ℃ overnight.The competent cell of M15[pREP-4] was transformed by the diluted ligation product.The recombinant clones of M15[pQE30-Eqfr,pREP-4] were identified.All of the plasmids in these recombinant clones were respectively extracted and digested with both endonucleases BamHI and SalI.The restriction pattern was in agreement with the predicted pattern.It showed that the target clone containing pQE30-Eqfr was obtained.In addition,the target clones were cultivated and induced to express the desired protein by IPTG.The molecular mass of expressed protein was about 116 000.The expression quantity was high in the first to third hour after induction,but declined after the third hour.This revealed that the ORF of OsRad21-i theoretically deducted at before was right.The expressed protein and system would be beneficial to next step experiment.更多还原