截短的小麦冷胁迫反应蛋白基因片段TA3-13的融合表达、纯化与诱导抗TMV功能研究

His-tag fusion expression,purification and TMV resistance induction of truncated wheat cold shock protein gene fragment TA3-13

TA3-13是小麦中一个冷胁迫蛋白WCSP1编码基因的5′端截短的DNA序列。它与水稻白叶枯病菌Harpin蛋白的编码基因hpa1_(X∞)(又称hrf1,hrfA)具有59%的一致性。为了研究TA3-13蛋白的生物学性状和功能及其与Harpin蛋白的关系,本研究以pET30a(+)为载体,构建了His融合表达重组体,转化大肠杆菌后在异丙基硫代-β-D-半乳糖苷(IPTG)和阿拉伯糖的诱导下获得了融合蛋白的表达。利用镍柱对融合蛋白进行了纯化研究,结果表明:融合蛋白在200 mmol·L^(-1)咪唑浓度下能够被有效地洗脱下来,电泳显示具有单一条带。烟草花叶病毒(TMV)接种试验显示:纯化的TA3-13蛋白喷雾烟草后仍具有较高的诱导烟草抗TMV的作用。

TA3-13 is a 5＇truncated sequence of a wheat gene that encodes the cold shock protein WCSP1.TA3-13 shares 59% identity with hpa1_（Xoo）,a Harpin encoding gene of Xanthomonas oryzae pv.oryzae.To study the biological function of TA3-13,we made a His-tag fusion expression construct that could express the fusion protein under the induction of isopropylthio-beta-D-galactopyranoside （IPTG） and arabinose.The fusion protein was purified by nickel chromatograph and eluted with 200 mmol·L^（-1） imidazole. When applied to tobacco by spraying plant leaf,the purified TA3-13 protein induced resistance to tobacco mosaic virus.