青稞脱水素基因dhn4的克隆与原核表达

Cloning and procaryotic expression of the dehydrin dhn4 gene from Tibetan hulless barley

用RT-PCR的方法从重要的高原作物青稞(Hordeum vulgareL.var.nudumHook.f.)中克隆到了脱水素基因dhn4,其编码区长为678 bp,所编码的蛋白质DHN4为YSK2型脱水素.经预测,该蛋白二级结构易形成α-螺旋,并无固定的三级结构.将dhn4基因cDNA编码区定向克隆到载体PET30-c上,获得了重组质粒PET-dhn4,该重组质粒再转化到大肠杆菌BL21菌株,37℃经1.5 mmol/L IPTG诱导获得了大量分子量约为35 kD的融合蛋白.然后用亲和层析的方法对该蛋白进行纯化,得到了纯化的脱水素DHN4蛋白.

A dehydrin gene （dhn4） cDNA fragment has been obtained via RT-PCR from Tibetan hulless barley（Hordeum vulgate L. var. nudum Hook. f. ）. It indicated that dhn4 encoded a YSK2 type dehydrin （DHN4）. The secondary structure of DHN4 predicated with software Anthepro 5.0 is prone to α-helix, and the tertiary structure predicated by SWISS-PROT indicated intrinsically unstructured. The coding region of the dhn4 cDNA recombinated into PET30-c vector was transformed into the Eschrichia coli overexpression strain BL21, and the protein DHN4 was then produced. The fused 35 kD proteins induced with 1.5 mmol/L IPTG at 37 ℃ for 4 hours were overexpressed and then purified by Ni-NTA Purification method. The properties were visualized by SDS-PAGE and identified by Western-blotting analysis. These experiments described above made advantage of the following researches on the physiological and biochemical functions of dehydrins.