短沟对虾和斑节对虾酚氧化酶原基因的克隆和序列分析

cDNA CLONING AND SEQUENCE ANALYSIS OF PROPHENOLOXIDASE IN PENAEUS SEMISULCATUS AND PENAEUS MONODON

采用RT PCR原理和长片段扩增技术克隆短沟对虾和斑节对虾酚氧化酶原基因。设计合成特异引物、提取血细胞总RNA ,反转录后扩增获得的特异片段回收并克隆到pGEM TEasyVector系统的T载体上 ,重组子进行碱基序列测定。结果表明 ,所克隆的短沟对虾酚氧化酶原基因含起始密码子ATG到终止密码子TGA的 2 0 5 5bp ,编码 6 84个氨基酸。斑节对虾酚氧化酶原基因含ATG到TGA的 2 0 6 1bp ,编码 6 86个氨基酸。短沟对虾酚氧化酶原推算分子量为 781 5 3Da ,等电点pI为 6 2 5 ;斑节对虾则为 785 81Da ,pI为 5 83。用DNA分析软件分析显示两种对虾酚氧化酶原基因具有高度的同源性 ,二者的碱基和推测的氨基酸序列同源性分别为 93 %和 92 %;对虾与其他节肢动物酚氧化酶原基因的同源性的高低与种类间亲缘关系相关 ;不同种类间酚氧化酶活性中心重要组成部分的 2个铜结合位点、位于铜结合位点内的6个组氨酸以及与铜结合位点相邻的氨基酸序列高度保守。

High quality RNA were isolated from hemocytes of Penaeus semisulcatus and P.monodon by SV total RNA isolation system.Specific primers for PCR were designed and synthesized according to the sequence of prophenoloxidase (proPO) of Penaeus monodon reported by Sritunyalucksana et al (1999) (GenBank Accession No.AF099741).Using the total RNA as templates,specific DNA fragments for both shrimps were obtained by reverse transcription PCR (RT- PCR).The DNA fragments were purified and cloned into T vector of pGEM-T Easy Vector system.Recombinants were selected and confirmed by restriction enzyme analysis.Nucleotide sequences analyses of the recombinants show that the cloned cDNA of prophenoloxidase of P.semisulcatus contains the open reading frame 2055bp,from initiation codon ATG to termination codon TGA,which encodes 684 amino acids.While the cloned cDNA from P.monodon includes the open reading frame of 2061bp which encodes 686 amino acids.The molecular masses of the deduced amino acids sequences for P.semisulcatus and P.monodon are 78153Da and 78581Da,and their estimated pI are 6.25 and 5.83 respectively.Alignment analyses show that the nucleotide sequence and deduced amino acid sequence of prophenoloxidase of P.semisulcatus has high similarity to that of P.monodon (93% and 92% respectively).Both the nucleotide sequences and deduced amino acid sequences of prophenoloxidase of P.monodon cloned are highly similar to that of P.monodon,AF099741 (above 99%).And there are only 4 different nucleic acids in the 2kb sequence between the two sequences,which existing in the non-structural regions,so it is supposed that such differences would not influence the gene function.Homology of prophenoloxidase among shrimps and other arthropods are connected with their relative relationship.The prophenoloxidases of the compared species can be divided into three main groups.Three shrimps (P.monodon,P.semisulcatus and P.leniusculus) and one prawn (Macrobrachium japonicus) can be classified into the same subgroup.The two cloned prophenoloxidases,as well as that of the other arthropods species,contain two highly conserved putative copper binding sites and sequence around these sites.Such sites combining with two copper atoms would be part of the activity center of the enzyme.