根据已知的大豆1-氨基环丙烷-1-羧酸合酶（1-aminocyclopropane-1-carboxylatesynt- hase ，ACC合酶）基因序列（GenBank Accession No:dq273840）设计三个基因特异的反向引物CTA798 sp1，CTA798 sp2，CTA798 sp3，分别与4个简并引物配对，通过热不对称PCR (thermal asymmetric interlaced PCR,TAIL-PCR) 扩增，获得了该基因上游约600bp的序列。利用5'-cDNA末端快速扩增（5' Rapid Amplification of cDNA ENDs,5'-RACE）实验，确定转录起始位点(TTS) 位于起始密码子上游62 bp 处，由此获得了大豆ACC合酶基因上游长约328bp的启动子序列。用PLACE和PLANTCARE 软件分析此序列，发现该序列具有启动子的基本元件TATA-box和CAAT-box；此外发现三个胁迫诱导元件：光应答元件Box1，Box4和诱导物应答元件W-box。以pBI121为基础，进一步构建了由ACC合酶基因上游调控序列启动的gus基因植物表达载体，通过农杆菌介导的叶盘法转化烟草叶片，瞬时表达结果表明GUS基因在该片段的调控下获得了表达，该片段具有启动子活性。
Based on the previously reported sequence of soybean 1-aminocyclopr- opane-1-carboxylate synthase gene (GenBank Accession No.dq273840), three gene-specific reverse primers, CTA798 sp1, CTA798 sp-2, CTA798 sp3 were designed, and a DNA fragment about 600bp was obtained by TAIL-PCR. The transcription start site was localized at 62bp upstream of the start ATG identified by 5′-RACE. The obtained sequence was analyzed by PLACE and PLANTCARE program and showed putative ACC synthase gene promoter features: it contains the basic core elements of TATA-box and CAAT-box, as well as stress-induced elements: light-responsive element Box1, Box4 and elicitor-induced element W-box. Based on the binary vector pBI121, a plant gene expression vector carrying gusA gene under the putative ACC synthase gene promoter was constructed and introduced into Agrobacterium tumefaciens EH105. The transient expression of gusA gene was then observed by histochemical staining in tobacco leaves through the leaf disc transformation mediated by Agrobacterium tumefaciens, which revealed the obtained sequence has promoter activity.
赵燚,杨君,安利佳. 大豆1-氨基环丙烷-1-羧酸合酶基因启动子克隆与分析[J]. 中国生物工程杂志, 2008, 28(专刊): 93-97.
. Cloning and Characterization of Soybean 1-aminocyclopropane-1-carboxylate synthase Gene Promoter. China Biotechnology, 2008, 28(专刊): 93-97.
https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2008/V28/I专刊/93
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