用重叠PCR合成植物甜蛋白brazzein基因  

Synthesize the Plant Sweet Protein Brazzein Gene with Overlapping PCR

作　　者：陈波[1] 

机构地区：[1]西南科技大学生命科学与工程学院,四川绵阳621010

出　　处：《生物技术》2007年第4期43-45,共3页Biotechnology

摘　　要：目的:为在泡盛曲霉(Aspergillus awamori)中进行表达,采用重叠PCR合成了植物甜蛋白brazzein基因。方法:根据非洲热带植物Pentadiplandra brazzeana产生的天然甜蛋白brazzein的氨基酸序列及泡盛曲霉糖化酶基因glaA的密码子偏爱性,设计并化学合成了2对3’-端互补的寡聚核苷酸,通过PCR延伸获得2条末端有部分重叠的双链核苷酸片段,再通过重叠PCR扩增,合成了用于泡盛曲霉表达的植物甜蛋白brazzein基因。结果:将brazzein基因克隆到pMD18-T载体,随机挑取6个重组质粒测序,结果1个重组质粒有连续4个碱基缺失,3个重组质粒各有1个碱基缺失,2个重组质粒携带的brazzein基因核苷酸序列完全正确。结论:合成的brazzein基因大小162 bp,编码54个氨基酸,推断的氨基酸序列与Pentadiplandra brazzeana产生的天然brazzein完全一致,表明植物甜蛋白brazzein基因成功合成。Objective： For expression in Aspergillus awamori,the plant sweet protein brazzein gene was synthesized with overlapping PCR.Methods： According to the amino acid sequence of the native brazzein protein produced by the African plant Pentadipandra brazzeana and the codon usage of Aspergillus awamori glucoamylase gene （glaA） ,two pairs of oligonuclecotides with 3＇ - ends complementary were designed and synthesized to obtain two end- overlapped fragments of brazzein gene through PCR polymerization. The full- length brazzein gene was synthesized using the two fragments as template through overlapping PCR and cloned into pMD18 - T vector.Results： Sequencing results showed,of the six recombinant plasmids randomly picked carrying brazzein gene insert,four were 4- base consecutively deleted,three were 1 - base deleted,and two were completely correct in nucleotide sequence. Conclusion. The synthetic brazzein gene has the size of 162 base pairs,coding for 54 amino acid residues, the deduced amino acid sequence of which is identical with that of the native brazzein produced by Pentadiplandra brazzeana.

关 键 词：植物甜蛋白 BRAZZEIN基因 合成 重叠PCR 泡盛曲霉 

分 类 号：Q781[生物学—分子生物学]