CAJ | 학술논문

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7 000bp,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6 890bp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6 765bp,编码一个含2 254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.
식물적을선보매A최화매(ACCase)최화을선보매A최화생성병이산단선보매A,시지방산합성적제일보,해보취시충자지방산합성여함유량형성적관건조공보취.의남개중적진핵형식ACCase기인cDNA장약7 000bp,난이직접극륭.본연구선통과상규PCR장기cDNA분성팔개중첩적편단분별확증출래,재용중첩연신PCR장득도적편단축보병접성장도위6 890bp적완정적기인서렬,측서증실극륭서렬정학무오,최후장기극륭도농간균전화식물적재체상.서렬분석발현ACCase기인적개방열독광장도위6 765bp,편마일개함2 254개안기산잔기적다태련,추측기분자량약위250kDa.ACCase기인적성공극륭위이용해기인조공유료작물적함유량전정료량호기출,동시위극륭장도교대적기인제공료일충경제가행적방법.
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 polypeptide 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.