Joshi L, St Leger R J, Roberts D W
Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853, USA.
Gene. 1997 Sep 15;197(1-2):1-8. doi: 10.1016/s0378-1119(97)00132-7.
Reverse transcription differential display PCR (RT-DD-PCR) was used to identify genes that are specifically expressed by Metarhizium anisopliae when it contacts the host insect cuticle. Using a homology-based subtilisin-like protease primer we identified a hitherto unsuspected differentially expressed subtilisin-like protease (Pr1B) encoding gene. The deduced amino acid sequence shows 54% similarity to the well characterized Pr1A subtilisin of M. anisopliae and karyotype analysis revealed that Pr1A and Pr1B are located on separate chromosomes. Like Pr1A, Pr1B is synthesized as a large precursor (1158 nucleotides; deduced molecular mass = 40031 Da) containing a signal peptide, a propeptide and the mature protease (283 aa; deduced molecular mass = 28714 Da). However, Pr1B possesses several substitutions in the highly conserved sequences comprising the active sites of subtilisins. In particular, the substitution of Thr220 by serine is unique to Pr1B. Substitution of Asn155 by glycine is also very unusual, and we discuss the likely effects these changes will have on the catalytic efficiency of Pr1B.
逆转录差异显示 PCR（RT-DD-PCR）被用于鉴定绿僵菌接触宿主昆虫表皮时特异性表达的基因。使用基于同源性的枯草杆菌蛋白酶样蛋白酶引物，我们鉴定出一个此前未被怀疑的差异表达的枯草杆菌蛋白酶样蛋白酶（Pr1B）编码基因。推导的氨基酸序列与已充分表征的绿僵菌 Pr1A 枯草杆菌蛋白酶显示出 54%的相似性，核型分析表明 Pr1A 和 Pr1B 位于不同的染色体上。与 Pr1A 一样，Pr1B 以大的前体形式合成（1158 个核苷酸；推导分子量 = 40031 Da），包含一个信号肽、一个前肽和成熟蛋白酶（283 个氨基酸；推导分子量 = 28714 Da）。然而，Pr1B 在构成枯草杆菌蛋白酶活性位点的高度保守序列中存在几个替换。特别是，Pr1B 特有的苏氨酸 220 被丝氨酸替换。天冬酰胺 155 被甘氨酸替换也非常不寻常，我们讨论了这些变化可能对 Pr1B 催化效率产生的影响。
