Eisenia fetida中蚯蚓纤溶酶P-Ⅲ组分序列多态性

Polymorphism of the earthworm fibrinolytic enzyme P-Ⅲ gene in Eisenia fetida

为了探讨蚯蚓纤溶酶P-Ⅲ组分序列多态性及其与蚓种关系。从Eiseniafetida中提取总RNA,利用P-Ⅲ组分序列两端的保守性设计引物,通过RT-PCR获得P-Ⅲ组分基因,并构建该组分质粒(pUC18)文库,经PCR筛选后进行序列测定及分析。在原已获得的Efp-Ⅲ-1和Efp-Ⅲ-3的基础上,利用中间引物从文库中筛选到Efp-Ⅲ-2基因。序列分析表明,该基因编码序列为720bp,编码239个氨基酸,与GenBank报道的Efp-Ⅲ-2五条序列整体相似性高达99.02%。通过序列比对,发现Efp-Ⅲ-1、2和3具有完全相同的结构域,差异位点零星分布在序列中间;Lumbricus rubellus的六条序列同样具有Eisenia fetida这个特点,并无明显差异。序列比对证明它们是蚯蚓体内P-Ⅲ组分基因多态性的表现,而且在Lumbricidae科中与蚓种无关。

The purpose of the experiment to study the polymorphism of the P- Ⅲ gene and its relation with species of earthworm.. The total RNA from Eisenia fetida was extracted and the P -Ⅲ gene was amplified by RT- PC, R, which used the primers designed based on the homology of the two sides of the P- Ⅲ sequences, then inserted into pUC18 vector. The recombinant plasmid was identified by PCR in library and then the gene was sequenced and analyzed. Based on the sequence of genes Efp- Ⅲ- 1 and Efp- Ⅲ - 3 acquired by our lab,Efp -Ⅲ- 2 gene was found by using the middle primer. DNA sequence analysis revealed that the encoding fragment was 720bp, which encoded 239 amino acid residues. The sequences showed 98.47% similarity to the Efp- Ⅲ - 2 gene reported in GenBank. Sequence alignment indicated that Ⅲ - 1,2 and 3 had the same structural domain and a few polymorphism sites distributed in some areas; Six sequences from Lumbricus rubellus had the same characteristics, and there were no significant differences among them. Three kinds of P- Ⅲ gene were cloned from Eisenia fetida for the first time in our lab. According to the sequence alignment, polymorphism of the P-Ⅲ gene in earthworm was proved,and it had no relation with species of earthworm.