胡萝卜抗冻蛋白AFP基因的克隆与序列分析

Cloning and sequence analysis of anti-freeze protein gene from carrot

以胡萝卜幼苗为材料,提取基因组DNA,根据GenBank中的已知序列设计引物,利用聚合酶链式反应技术(PCR)体外扩增获得胡萝卜抗冻蛋白基因(AFP),将其连接到pGEM-Teasy vector载体上,并对其进行生物学信息分析.结果表明:克隆的AFP基因片段全长1 206bp,其中编码区长1 026bp,共编码341个氨基酸,蛋白质分子量为38.048ku,等电点为5.43,该蛋白为亲水性分泌型蛋白,有信号肽存在;其蛋白质二级结构以α-螺旋、β-折叠和无规卷曲为主.与GenBank中(A91926.1)的基因序列和氨基酸序列比较,同源性分别为99.4%和97.6%,表明AFP基因克隆成功.与不同地方品种胡萝卜AFP基因做同源性比较,结果发现不同品种间的AFP基因保持着很高的内同源性.

According to the known sequence in GenBank to design primer,genomic DNA was extracted form the seeding of carrot,the carrot antifreeze protein（AFP） was cloned by polymerase chain reaction（PCR）.Then connected it into the pGEM-T easy vector and its bioinformation analysis was carried out.The result indicated that the full-length cDNA was 1 206 bp and contained 1 026 bp open reading frame encoding 341 amino acids.The protein molecular weight was 38.048 ku and theoretical isoelectric point was 5.43.According to the amino acid sequence and structural analysis,it showed that the protein encoding by AFP was hydrophilicity and secretory protein,and there was signal peptide.the secondary structure of AFP protein mainly included α-helix,β-sheet and random coil.Compared the nucleotides sequence and amino acid sequence of cloned gene AFP with GenBank（A91926.1）,it have high homology of 99.4% and 97.6% respectively.It showed the gene AFP was cloned successfully.Comparing the AFP gene with different local varieties,it has existed high inner homology.