小麦类蛋白激酶基因片段TA50-10的克隆及其TMV抗性诱导功能

Cloning and TMV resistance induction of wheat protein kinase like gene fragment TA50-10

TA50-10是小麦中的一个截短的cDNA序列,与水稻白叶枯病菌harpin蛋白基因hpa1Xoo(又称hrf1,hr-fA)具有59%的一致性,它编码的多肽有类蛋白激酶催化域(Protein kinase like domain)。为了研究TA50-10蛋白质的生物学性状和功能以及与harpin蛋白的关系,用PCR法从小麦cDNA中扩增到TA50-10基因,并以pET30a(+)为载体构建了原核表达重组体pET30-TA50-10。将其转化大肠杆菌后在异丙基硫代-!-D-半乳糖苷(IPTG)的诱导下获得了表达蛋白质,并研究了该表达蛋白质诱导烟草抗烟草花叶病毒(TMV)的作用。结果表明:原核表达的TA50-10蛋白质具有热稳定性;用热处理后部分纯化的TA50-10蛋白质溶液喷施烟草叶片能够诱导三生烟草局部抗TMV,并且诱抗效果在处理后10 d内没有明显改变。

TA50-10,a truncated cDNA sequence of wheat（Triticum aestivum L.）,encodes a protein kinase-like domain.TA50-10 shares 59% similarity with hpa1Xoo,which is also named hrf1 or hrfA,a harpin encoding gene of Xanthomonas oryzae pv.oryzae.To study the biological function of TA50-10,TA50-10 fragment was cloned from cDNA of wheat using polymerase chain reaction（PCR） amplification.The procaryotic expression construction was made to express TA50-10 under the induction of isopropylthion-beta-D-galactopyranoside（IPTG）.The induced resistance against TMV infection by the expressed protein was determined.The results showed that the expressed TA50-10 protein was heat stable.Partially purified TA50-10 protein induced persistent resistance of tobacco to tobacco mosaic virus（TMV） infection when sprayed on plant leaf.The disease resistance mediated by TA50-10 lasted for 10 d without obvious reduction.