小麦Ra3基因编码区的克隆、原核表达及纯化

【目的】克隆小麦ramosa3(Ra3)基因完整编码区,利用原核表达系统表达并纯化获得重组的小麦RAMOSA3(RA3)。【方法】利用RT-PCR技术,从普通小麦"中国春"幼穗中扩增小麦Ra3,将其重组到原核表达载体pET-41a,并在T7Express competentE.coli菌株中进行诱导表达;利用Ni-NTA亲和层析柱纯化获得重组蛋白,并通过Western印迹技术对其进行鉴定。【结果】获得了长度为1080bp的cDNA片段,该片段包含小麦Ra3完整编码区,编码产物长度为360个氨基酸残基,属于TPP酶家族成员;SDS-PAGE结果显示,菌体总蛋白中出现约66ku的预期条带,其表达量占总蛋白的44.6%;抽提液中加入体积分数0.3%的Triton X-100,可显著提高重组蛋白的溶解度;纯化后的重组蛋白呈单点纯,Western印迹结果进一步确证其为目标蛋白。【结论】利用原核系统高效表达并纯化获得了重组的小麦RA3。

Cloning,prokaryotic expression and purification of Ra3's encoding region in wheat

Objective] The study cloned the encoding region of wheat ramosa3 and obtained this protein by expression and purification in prokaryotic system.Method] The cDNA of wheat Ra3 was amplified from young spike of common wheat Chinese Spring by RT-PCR;expression vector pET-41a was constructed,and fusion protein was expressed in E.coli strain T7 Expression;the recombinant protein was purified by Ni-NTA resin column and identified by western blot analysis.Result] A length of 1 080 bp of cDNA fragment was obtaine...