甜菜夜蛾GOBP2基因的克隆及序列测定

 比较已报道的 ５种昆虫的 ＧＯＢＰ２基因序列，设计了一对简并引物，以甜菜夜蛾 Ｓｐｏｄｏｐｔｅｒａ ｅｘｉｇｕａ的触角ｃＤＮＡ为模板进行ＰＣＲ扩增，获得了一条４００ｂｐ左右的特异性条带。将以上片段克隆到Ｔ－ｅａｓｙ载体上，获得重组子ＧＯＢＰ２Ｓｅｘｉ，序列测定和结构分析结果表明，ＧＯＢＰ２Ｓｅｘｉ阅读框全长４２６ｂｐ，编码１４１个氨基酸残基，预测分子量和等电点分别为１６．０７ｋｕ和５．０９。另外，序列中有６个保守的半胱氨酸位点，具有气味结合蛋白的典型特征。同时以基因组 ＤＮＡ为模板进行ＰＣＲ扩增，得到了一条 ２ｋｂ左右的特异性条带，同样克隆到 Ｔ－ｅａｓｙ载体上进行测序，结果表明，在该蛋白的第２２和第２３个氨基酸之间及第８２和第８３个氨基酸之间分别插入了一个１６０ｂｐ和１４０３ｂｐ的内含子。Ｎｏｒｔｈｅｒｎ杂交结果表明，ＧＯＢＰ２基因在甜菜夜蛾触角中特异性表达，并且在雌雄蛾触角中表达水平相当。该基因已在ＧｅｎＢａｎｋ／ＥＭＢＬ中登记，序列号为ＡＪ２９４８０９。

Cloning and Sequencing of a Gene Encoding GOBP2 in the Antenna of Spodoptera exigua

A pair of degenerate primers was designed, based on the comparison of five insects' GOBP2 gene sequences reported previously. A specific band(about 400bp in length) was amplified from cDNA of Spodoptera exigua antenna and another specific band(about 2kb in length) was amplified from genomic DNA. The two segments were cloned into T-easy vector, respectively. Results of sequencing and struc- tural analysis phowed that the full-length of GOBP2Sexi ORF is 426bp,141 amino acid residues were en- coded. The predicted MW and pI are 16. 07ku and 5. 09, respectively. There are six conservative Cys locus in the sequence, which is the typical characteristic of OBPs. GOBP2Sexi gene was inserted by two introns between amino acid residue 22 and 23 and between 82 and 83. The length of two introns is 160bp and 1403bp. Results of Northern blot showed that GOBP2 gene expressed specifically in the antenna of SPodoPtera exigua, and the expression level is consistent in the antenna of male and female moths. The se- quence was deposited in GenBank/EMBL and the accession number is AJ294809.