重叠延伸PCR克隆拟南芥ACCase基因和植物表达载体构建

植物的乙酰辅酶A羧化酶（ACCase）催化乙酰辅酶A羧化生成丙二酸单酰辅酶A，是脂肪酸合成的第一步，该步骤是种子脂肪酸合成与含油量形成的关键调控步骤。拟南芥中的真核形式ACCase基因cDNA长约7000bp，难以直接克隆。本研究先通过常规PCR将其cDNA分成八个重叠的片段分别扩增出来，再用重叠延伸PCR将得到的片段逐步拼接成长度为6890bp的完整的基因序列，测序证实克隆序列正确无误，最后将其克隆到农杆菌转化植物的载体上。序列分析发现ACCase基因的开放阅读框长度为6765bp，编码一个含2254个氨基酸残基的多肽链，推测其分子量约为250kDa。ACCase基因的成功克隆为利用该基因调控油料作物的含油量奠定了良好基础，同时为克隆长度较大的基因提供了一种经济可行的方法。

An overlap extension PCR method used in isolation of long cDNAs of acetyl-CoA carboxylase from Arabidopsis

Acetyl-CoA carboxylase （ACCase） is a biotin-dependent enzyme that catalyzes the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA, which is the key regulation step for fatty acid synthesis and oil accumulation. The cDNA sequence for Arabidopsis ACCase gene was about 7 000bp in length and is very difficult to clone by conventional method. In this study, we first amplified 8 overlap fragments covering the whole ACCase sequence and then spliced them into an integrated sequence by the overlap extension PCR technique. The whole ACCase sequence was cloned into the Ti plasmid used for plant transformation after sequencing. Sequence analysis indicated that the obtained sequence consisted of a 6 765 bp open reading frame encoding a 2 254 amino acid poly-peptide with a molecular weight about 250kDa. The successful cloning of ACCase gene makes it possible to utilize the gene to improve seed oil content in oilseed crops. It also provides an economic and feasible method to clone long-strand DNA or cDNA sequence.