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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CTA798sp1,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164 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。
Three gene-specific reverse primers were designed according to the known 1-aminocyclopropane-1-carboxylate synthase gene sequence (GenBank Accession No: dq273840) CTA798sp1, CTA798 sp2 and CTA798 sp3 were respectively paired with four degenerate primers and amplified by thermal asymmetric interlaced PCR (TAIL-PCR) to obtain a sequence of about 600 bp upstream of the gene. Using 5’Rapid Amplification of cDNA ENDs (5’-RACE) assay, the transcription start site (TTS) was determined to be 164 bp upstream of the start codon, thereby obtaining soybean ACC The promoter sequence approximately 328 bp upstream of the synthase gene. This sequence was analyzed by PLACE and PLANTCARE software and found to contain the basic elements of promoter TATA-box and CAAT-box. In addition, three stress inducing elements: light-responsive elements Box1, Box4 and inducer response element W-box were found. Based on pBI121, the plant expression vector of GUS gene, which was initiated by the upstream regulatory sequence of ACC synthase gene, was further constructed. Agrobacterium-mediated leaf disc method was used to transform tobacco leaves. The transient expression results showed that the GUS gene was obtained under the control of this fragment Expression, this fragment has promoter activity.