水稻几丁质酶基因克隆RCH8的DNA结构分析

DNA Structure Analysis of Chitinase Gene Clone RCH8 from Rice

用DNA外切核酸酶Ⅲ和S1核酸酶生成连续缺失突变体，用Sanger双脱氧链终止法对该克隆进行双向DNA顺序测定，测序全长2049个碱基，初步确定了1057bp的5'端上游顺序，966bp不含内含子的完整编码区和可能的TATAbox等。所编码的322个氨基酸包括N-端20个氨基酸的信号肽，其后40个氨基酸长度的含8个半胱氨酸的hevein结构域和一个催化结构域。

RCH8 is a basic chitinase genomic clone isolated from rice (var. IR36) genomic library using a bean chitinase gene fragment as a probe. The complete nucleotide sequence was determined in both directions by the dideoxy chaintermination method. Deletions of the DNA chain for sequencing were created by exonuclease Ⅲ and S1 nuclease (Fig. 1 ). The 2049 bp clone contains a 1057 bp upstream sequence and a single, complete open reading frame of 966 bp with no introns (Fig. 2 ). The 5' side sequence contains possible regulatory elementS (Table 1 ). The ORF encodes a polypeptide of 322 amino acids with a putative 20 amino acid Nterminal signal peptide followed by a 40 amino acid cysteine-rich domain contuining 8 cystein residues and a chitinase catalytic domain. The deduced amino acid sequence shares 88. 5%, 80. 4 % and 78. 9% identity with those of three other published basic chitinase genes from rice(Table 2,Fig. 3). In addition, the hevein domain sequence homologies of nucleic acids and amino acids were compared between RCH8 and several other proteins(Table 3, Fig.4 ).