甜瓜属野生种铝胁迫诱导基因ChAI的克隆及序列分析

应用同源序列克隆策略从铝盐胁迫处理的甜瓜属野生种(Cucumis hystrix Chakr.) 叶片中获得了612bp的片段,通过在EST数据库进行同源检索,发现一条甜瓜EST序列（GenBank登录号：AM724963.2）与之高度同源,据此设计引物并经RT-PCR扩增和序列拼接,最后获得了铝诱导基因cDNA序列全长,命名为ChAI (GenBank登录号：FJ968438 )。序列分析表明该基因全长967bp,其中开放读码框(ORF)长711bp编码236个氨基酸组成的多肽,5′ 和3′ 端非编码区长度各为15bp和241bp。DNAMAN同源性分析表明该基因与葡萄、棉花、羊乳（轮叶党参）、白骨壤（真红树植物）、拟南芥等植物的铝诱导蛋白的氨基酸序列具有78.6%以上的同源性。运用半定量RT-PCR技术对ChAI 基因的转录水平进行分析,根据该基因在铝胁迫下大量表达推测ChAI 与耐铝盐胁迫响应相关,但具体耐铝胁迫机制还有待深入研究。

CLONING AND SEQUENCE ANALYSIS OF ALUMINUM-INDUCED GENE ChAI FROM THE WILD CUCUMIS SPECIES (Cucumis hystrix Chakr.)

Degenerated primers were designed based on conserved amino acids region of aluminum-induced protein from other plants, and a 612bp special fragment was amplified from the wild Cucumis species(Cucumis hystrix Chakr.) leaves under aluminum stress through homologous strategy. Using this fragment sequence data as a querying probe, one highly homologous melon EST sequence (Accession: AM724963.2) was detected. Primers designed based on this EST sequence, the full-length of aluminum-induced gene, designated as ChAI (GenBank Accession no: FJ968438 ), was obtained by RT-PCR method and sequence assembling. Sequence analysis showed that ChAI was 967bp in length, which encompassed an 711bp open reading frame (ORF) encoding 236 amino acid residues, and adjacent non-translation 15bp of 5′-terminal region and 241bp of 3′-terminal region, respectively. The results of DNAMAN homologous analysis demonstrated that ChAI had more than 78.6% identity of the amino acids residue with Vitis vinifera, Gossypium hirsutum, Codonopsis lanceolata, Avicennia marina and Arabidopsis thaliana. The transcription analysis of ChAI by semi-quantitative-PCR revealed the high expression under Al stress, the expression of ChAI was predicted to be related to anti-Al stress responsive mechanism, but its accurate function and anti-Al stress mechanism were still elusive and needed to be further investigated.