番茄金属蛋白酶基因LeftsH6的克隆和分子特性(英文)

Cloning and Molecular Characteristic of the Metalloprotease Gene LeftsH6 from Tomato

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。

The full-length 2 213-bpftsH （filamentation temperature-sensitive H） cDNA was cloned from the cDNA library of heat-shocked tomato leaves. According to an open reading frame of 2 019-bp, the deduced protein precursor was predicted to target chloroplast. The putative AAA （ATPases associated with diverse cellular activities） domain and the Zn^2＋-binding domain, characteristic of FtsH metalloproteases family, were found in the FtsH-like protein. Most similar to the FtsH6 of Arabidopsis thaliana, the tomato fisH-like gene was named as Lycopersicon esculentum filamentation temperature-sensitive 146 （LefisH6）. Purified FtsH degraded casein but not BSA in vitro, whereas a FtsH mutant with the Glu472 in the zinc-binding motif replaced by Gln had lost the protease activity. A single copy of LeftsH6 was detected in tomato genome by Southern blot analysis. Northern and Western blot analyses revealed consistently the heat-inducible character of the LeftsH6 gene. No LefisH6 expression was detected after cold, salt, drought or light stress. The results provided the first experimental evidence of the existence of heat-inducible fisH gene in higher plants.