| 摘要: |
| 【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T7 Express 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1 080 bp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66 ku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。 |
| 关键词: 小麦 ramosa3 同源克隆 重组表达 亲和纯化 |
| DOI： |
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| 基金项目:国家转基因专项“高产转基因小麦新品种培育”（2008ZX08002-003）；西北农林科技大学青年科学基金项目（QN2009070）；西北农林科技大学青年学术骨干支持计划项目 |
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| Cloning,prokaryotic expression and purification of Ra3’ s encoding region in wheat |
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| Abstract: |
| 【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 obtained,which contained the complete encoding region of wheat Ra3.It encoded 360 amino acid residues,belonging to TPP enzyme family.SDS PAGE indicated the expected 66 ku band appeared,and in the total protein the percentage of fusion protein was up to 44.6%,adding 0.3% Triton X-100 in lysis buffer can significantly increase the solubility of recombinant proteins.Purified recombinant protein showed a single homogeneous band,and it was further confirmed by western blot.【Conclusion】 Wheat RAMOSA3 can be expressed and purified in prokaryotic system efficiently. |
| Key words: Triticum aestivum ramosa3 homologous cloning recombinant expression affinity purification |
