修饰合成冬鲽抗冻肽基因的设计和克隆

动物界和植物界密码子使用频率不同，冬Ｄｉｅ抗冻肽中丙氨酸占６０％而且偏爱ＧＣＣ，３８个Ａｌａ密友子中２３个为ＧＣＣ。我们设计合成抗冻肽基因时采用了植物基因常用密码子，用甘薯信号肽序列取了抗冻肽的ｐｒｅ序列，省略了Ｐｒｏ序列，并 信号肽下游二、四、八拷贝正向串联的成太的修饰基因，经测序证实与设计完成一致。利用ＱＩＡ表达系统，在大肠杆菌中实现了ＤＨＦＲ－成熟抗冻肽单体和二体的融合表达。

Design and Cloning a Modified Synthetic Gene for Flounder Antifreeze Peptide

Codon usage varies throughout the animal and plant kingdoms.The flounder antifreeze peptide (AFP) is Ala rich (60%) and the Ala codons of the fish cDNA reveal a strong bias toward GCC (23/38).We have taken the opportunity to modify the codon selection for all the amino acids to be consistent with normal usage in plant genes.The pre sequence of AFP was replaced by the pre sequence of sweet potato Sporamin A (EMBL X13 509),and the pro sequence was not included.The modified genes coding 2,4,8 tandem mature AFP were constructed and confirmed by DNA sequencing.Using QIA express system,expression of DHFR mature AFP constructs in E.coli suggests the modified synthetic AFP gene could be work in plants.