海湾扇贝g型溶菌酶基因cDNA的克隆与分析

通过EST和锚定PCR技术，从海湾扇贝(Argopecten irradians)中克隆得到一种溶菌酶基因的全长cDNA(全长659bp，编码200个氨基酸)。BLASTx分析的结果显示，它和脊椎动物g型溶菌酶相似性较高，却不同于无脊椎动物中已发现的c型和i型溶菌酶。在其编码的氨基酸序列中发现了g型溶菌酶的活性中心(Glu82，Asp97，Asp108)，同时6个保守的半胱氨酸也与鸟类和鱼类的g型溶菌酶相一致。结合BLASTX分析的结果，可以确认所获得的cDNA序列是一种在无脊椎动物中首次发现的g型溶菌酶的编码序列。采用Clustalw软件，对多种脊椎动物c型、g型溶菌酶和无脊椎动物c型、i型溶菌酶以及本研究发现的无脊椎动物g型溶菌酶进行了分析，结果发现，无脊椎动物c型、i型溶菌酶和脊椎动物c型溶菌酶同源性较高，而海湾扇贝g型溶菌酶和脊椎动物g型溶菌酶同源性较高。由此说明c型、g型溶菌酶从无脊椎到脊椎动物的进化是平行发生的，这对进一步研究溶菌酶及其分子进化具有重要意义。

Molecular cloning and characterization analysis of cDNA encoding g-type lysozyme from scallop (Argopecten irradians)

Expressed sequence tagging techniques were used to identify the partial sequences of lysozymes from bay scallop (Argopecten irradians), and then the full length of the lysozyme cDNA was cloned through the approach of Anchored-PCR with the specific primers based on the sequence of the EST. The full length of the lysozyme cDNA from A. irradians was of 659 bp and encoded 200 amino acids. BLAST analysis revealed that the lysozyme gene from A. irradians shared high identity with the g-type lysozyme genes from vertebrate, while different from the c or i-type lysozyme in invertebrate. Three catalytic residues (Glu 82, Asp 97, Asp 108) conserved in g-type lysozyme were detected in the amino acid sequence from A. irradiant and these 6 cysteines involved in the formation of disulfide linkage were also the same as that in the g-type lysozymes from bird and fish. The results indicated that the cDNA sequence cloned from A. irradians was a member of g-type lysozyme family. A phylogenetic tree was constructed based on the amino acid sequences of g-type lysozyme from the scallop and a number of known lysozymes. The c and i-type lysozymes from invertebrate were clustered together with the c-type from vertebrate, while g-type lysozyme from scallop was clustered together with the g-type from vertebrate. It suggested that g-type and c-type lysozymes co-existed in both vertibrate and invertibrate animals and they could evolve from invertibrate to vertibrate in parallel routes.