Yeboah N A, Arahira M, Nong V H, Zhang D, Kadokura K, Watanabe A, Fukazawa C
Genetic Engineering Laboratory, Ministry of Agriculture Forestry and Fisheries, Ibaraki, Japan.
Plant Mol Biol. 1998 Feb;36(3):407-15. doi: 10.1023/a:1005921104288.
A soybean chitinase which has an apparent molecular mass of 28 kDa by SDS-PAGE, and has chitinase specific activity of 133 units per mg protein at pH 5.2 and an apparent pI of 5.7, was purified from mature dry seeds. Based upon the selected part (the residue positions 10-17) of the determined N-terminal 38 amino acid sequence, a 23-mer degenerate oligonucleotide was synthesized and used for the PCR cloning of the chitinase cDNA. The resulting 1340 bp cDNA was comprised of a 5'-untranslated region of 39 bases, a coding region corresponding to a 25 amino acid signal sequence, followed by a mature 308 amino acid sequence (calculated molecular mass 34,269, calculated pI 4.7), and a 235 nucleotide 3'-terminal untranslated region including 24 bases of the poly(A) tail. By comparing the deduced primary sequence with those of plant chitinases known to date, this enzyme was more than 50% identical to every class III acidic chitinase, but has no significant similarity to other families of chitinases. The comparison also showed that the C-terminal region of this chitinase is markedly extended, by at least 31 residues. Northern blot analysis demonstrated that this mRNA species is remarkably transcribed from the early stage until the late middle stage of seed development, whilst it is hardly expressed in the leaves and the stems of soybean. Spatial and temporal expression of this single gene imply that this class III chitinase is mainly devoted to the seed defense, not only in development but also in dormancy of soybean seed. This is the first reported isolation and cDNA cloning of a class III acidic endochitinase from seeds. According to the chitinase nomenclature we propose that this enzyme would be classified into a new class of chitinase PR-8 family, together with a Sesbania homologue.
从成熟干种子中纯化出一种大豆几丁质酶，经SDS - PAGE分析其表观分子量为28 kDa，在pH 5.2时几丁质酶比活性为每毫克蛋白质133单位，表观pI为5.7。根据所测定的N端38个氨基酸序列的选定部分（第10 - 17位残基），合成了一个23聚体简并寡核苷酸，并用于几丁质酶cDNA的PCR克隆。所得的1340 bp cDNA由39个碱基的5'非翻译区、对应25个氨基酸信号序列的编码区、其后是成熟的308个氨基酸序列（计算分子量34269，计算pI 4.7）以及包括24个聚腺苷酸尾碱基的235个核苷酸的3'末端非翻译区组成。通过将推导的一级序列与迄今已知的植物几丁质酶序列进行比较，该酶与每一种III类酸性几丁质酶的同源性超过50%，但与其他几丁质酶家族没有明显的相似性。比较还表明，这种几丁质酶的C端区域明显延长，至少延长了31个残基。Northern印迹分析表明，这种mRNA在种子发育的早期到中期后期都有显著转录，而在大豆的叶和茎中几乎不表达。这个单基因的时空表达表明，这种III类几丁质酶不仅在大豆种子发育过程中，而且在种子休眠期间都主要参与种子防御。这是首次报道从种子中分离和克隆III类酸性内切几丁质酶。根据几丁质酶命名法，我们建议将这种酶与一种田菁属同源物一起归入几丁质酶PR - 8家族的一个新类别。
