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从热处理的番茄叶cDNA文库中分离到一个全长为2213-bp的ftsH基因。该基因包括一个2019-bp的读码框,推测的蛋白前体定位到叶绿体中,序列中存在AAA结构域和Zn2+结合结构域等已知的金属蛋白酶FtsH家族的特征结构域。在已克隆的基因中,该ftsH与拟南芥ftsH6最近源,被命名为LeftsH6(Lycopersiconesculentumfilamentationtemperature-sensitiveH6)。体外蛋白酶活性分析结果表明,纯化的FtsH具有蛋白水解活性,能降解酪蛋白但不降解BSA;突变的FtsH(Zn2+结合结构域中的谷氨酸Glu472突变为谷氨酰胺Gln)失去了体外蛋白酶活性。Southern杂交结果表明,该基因在番茄基因组中是单拷贝;Northern和Western杂交均表明该基因表达被热诱导,但其表达不被低温、干旱、盐胁迫、高光等胁迫调节。首次证明了高等植物中存在能被热诱导表达的ftsH基因。
A full-length ftsH gene of 2213-bp was isolated from heat-treated tomato leaf cDNA library. The gene contains a 2019-bp reading frame. The putative protein precursor is located in the chloroplast. The sequences include the characteristic domains of the known FtsH family of metalloproteases, such as the AAA domain and the Zn2 + binding domain. Among the cloned genes, this ftsH is the closest source to ftsH6 of Arabidopsis and is named LeftsH6 (Lycopersicon esculentumfilamentationtemperature-sensitiveH6). In vitro protease activity analysis showed that the purified FtsH had proteolytic activity and degraded casein but did not degrade BSA. Mutant FtsH (glutamic acid Glu472 in Zn2 + binding domain mutated to glutamine Gln) lost protease activity in vitro . Southern blot showed that the gene was single copy in tomato genome. The Northern and Western hybridization showed that the gene was induced by heat, but its expression was not regulated by low temperature, drought, salt stress and high light stress. For the first time, it was demonstrated that ftsH gene can be induced by heat in higher plants.