冰核细菌Erwiniaananas110冰核基因iceA的原核表达及冰核活性分析.PDF

39 4 () Vol.39 No.4 2013 8 Journal of Hunan Agricultural University (Natural Sciences) Aug 2013 DOI:10.3724/SP.J.1238.2013.00354 冰核细菌 Erwinia ananas 110 冰核基因 iceA 的 原核表达及冰核活性分析 1,2 1,2 * (1. 410128; 2. 410128) 摘 要Erwinia ananas 110 iceA pMD19–T DH5αpET–23a(+) DH5αBL21(DE3)pLysSIPTG SDS–PAGE iceA 180 000 BL21(DE3)pLysS/pET–ice Erwinia ananas 110 –5–4 –3–2 ℃ 关 键 词 中图分类号S433 Q81 文献标志码A 文章编号1007–1032(2013)04–0354–05 Prokaryotic expression of iceA gene from ice nucleation active bacteria Erwinia ananas 110 and analysis of ice nucleation activity YAO Run-xian1,2, YUAN Zhe-ming1,2* (1.Hunan Provincial Key Laboratory for Germplasm Innovation and Utilization of Crop, Changsha 410128, China; 2.Hunan Provincial Key Laboratory for Biology and Control of Plant Diseases and Insect Pests, Changsha 410128, China) Abstract: To obtain recombinant strain with high ice nucleation activityiceA gene were amplified by PCR from ice nucleation active bacteria Erwinia ananas 110 and cloned into vector pMD19–T which was transformed into E.coli DH5α. The recombinant clones were screened by single and double digestion before sequenced. From the positive recombinant strainiceA gene was subcloned into prokaryotic expression vector pET–23a(+)resulting in recombinant plasmid pET–23a(+)-ice which was transformed into E.coli BL21(DE3)pLysS and induced by IPTG. SDSindicated that ice nucleation active protein was expressed as inclusion bodies with molecular weight of about 180 000. Ice nucleation activity test showed there was no difference in ic