总状毛霉(Mucor racemosus)甲壳素脱乙酰酶全长cDNA的克隆及序列分析

用保守的特异引物进行PCR扩增,结合RACE技术,分离和克隆到了总状毛霉(Mucorracemosus)甲壳素脱乙酰酶(CDA)的全长cDNA,并进行了全序列测定,提交GenBank登陆号DQ538514。研究结果表明:(1)总状毛霉CDA基因全长为1506bp,包括67bp5'非翻译区,1344bp阅读框以及95bp3'非翻译区,3'非翻译区包含Poly(A)加尾信号AATAAA。总状毛霉的CDA基因共编码448个氨基酸,在该基因中部还包含一个144氨基酸的多糖脱乙酰酶结构域,约占CDA基因全长的32%。(2)总状毛霉CDA基因与其它相近种米根霉(Rhizopusoryzae)、卷柄根霉(Rhizopuscircinans)的CDA1和CDA2、鲁氏毛霉(Mucorrouxii)、卵形孢球托霉(Gongronellabutleri)、匍枝根霉(Rhizopusstolonifer)、布拉克须霉(Phycomycesblakesleeanus)和酿酒酵母(Saccharomycescerevisiae)的CDA1和CDA2的基因序列同源性分别为:75%、58%、56%、56%、48%、39%、39%、17%和16%;相应的氨基酸序列的同源性分别为:69%、57%、59%、55%、47%、30%、32%、18%和21%。表明CDA基因在不同的真菌中有着不同的亲缘关系。(3)根据总状毛霉CDA基因的氨基酸序列构建的不同真菌的系统树,与采用经典分类法构建的系统树基本一致。(4)通过生物信息学的方法,预测该基因所编码的蛋白质三级结构,验证了该蛋白质具有甲壳素脱乙酰酶完整的功能性结构,并包含一个多糖脱乙酰酶结构域,两者具有相似的空间结构。

Cloning and Sequence Analysis of Complete Chitin Deacetylase(CDA) cDNA from Mucor racemosus

A complete chitin deacetylase (CDA) cDNA from Mucor racemonus was cloned and sequenced by RT-PCR and RACE with special conserved primers. The cDNA sequence was submitted to GenBank (DQ538514). The results showed: (1) The complete cDNA was with length of 1 506 bp containing 67 bp 5'-untranslated region, an open reading frame of 1 344 bp and 95 bp 3'-untranslated region including tailing site AATAAA. The gene encoded a sequence of 448 amino acid residues and consisted of core nucleotides encoding a polysaccharide deacetylase domain which covered 32% of the entire sequence. (2) The CDA gene shared different sequence homology with those of fungi including Rhizopus oryzae (75%), CDA1(58%) and CDA2(56%) of Rhizopus circinans, Mucor rouxii(56%), Gongreonella bulteri(48%), Rhizopus stolonifer(39%), Phycomyces blakesleeanus(39%), CDA1(17%) and CDA2(16%) of Saccharomyces cerevisiae. The corresponding homology of the deduced amino acid sequences was 69%, 57%, 59%, 55%, 47%, 30%, 32%, 18% and 21%, respectively. (3) Phylogenetic analysis according to the deduced amino acid sequences was matched with the classical taxonomic classification of the fungi. (4) The 3-D-structure of this protein was predicted. The protein had a whole CDA functional domain and a polysaccharide deacetylase domain.