Eisenia fetida中蚯蚓纤溶酶P-Ⅲ组分序列多态性

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Eisenia fetida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报道的点Efp-Ⅲ-2五条序列整体相似性高达99．02％。通过序列比对，发现Efb-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。

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

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.