番茄金属蛋白酶基因LeftsH6的克隆和分子特性

从热处理的番茄叶cDNA文库中分离到一个全长为2213-bp的fisH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Zn^2＋结合结构域等已知的金属蛋白酶PtsH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LefisH6（Lycopersicon esculentum filamentation temperature-sensitive H6）。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（Zn^2＋结合结构域中的谷氨酸Glu^472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。

Cloning and Molecular Characteristic of the Metalloprotease Gene Lefts H6 from Tomato

The full-length 2 213-bp ftsH (filamentationtemperature-sensitive H) cDNA was cloned from thecDNA library of heat-shocked tomato leaves. Accordingto an open reading frame of 2 019-bp, the deduced pro-tein precursor was predicted to target chloroplast. Theputative AAA (ATPases associated with diverse cellularactivities) domain and the Zn2+-binding domain, charac-teristic of FtsH metalloproteases family, were found inthe FtsH-like protein. Most similar to the FtsH6 ofArabidopsis thaliana, the tomato ftsH-like gene wasnamed as Lycopersicon esculentum filamentation tem-perature-sensitive H6 (LeftsH6). Purified FtsH degradedcasein but not BSA in vitro, whereas a FtsH mutant withthe Glu472 in the zinc-binding motif replaced by Gln hadlost the protease activity. A single copy of LeftsH6 wasdetected in tomato genome by Southern blot analysis.Northern and Western blot analyses revealed consistentlythe heat-inducible character of the LeftsH6 gene. NoLeftsH6 expression was detected after cold, salt, droughtor light stress. The results provided the first experimen-tal evidence of the existence of heat-inducible ftsH genein higher plants.