Cloning And Sequencing Of The Penaeus Monodon Fabricius Ribosome Gene

Penaeus monodon Fabricius belongs to Order Decapoda,Suborder Dendrobranchiaia Bate,Family Penaeidae,Genus Penaeus Fabricius.It is the largest species in Genus Penaeus,and distributed in Pakistan,South Africa,Malaysia,and India,and Australia,Sri Lanka,northern Thailand,Japan,and China's Fujian,Taiwan,Zhejiang,North Sea of Guangxi,West of Guangdong,Hainan Island,and so on.Of which North Sea of Guangxi and Hainan Island are the main distributions.Penaeus monodon become one of the most productive shrimp species and has the largest artificial breeding area in the world.At present,the research on Penaeus monodon mainly concentrated in the production of aquaculture industry,the influence and control of bacterial infection,artificial breeding of triploid and tetraploid,ovarian growth and microstructure observation,and so on.There is little research on molecular biology of Penaeus monodon,especially about its ribosomal DNA(rDNA).18SrDNA exists in all eukaryotic cells with highly conservative and easy to amplificate using general primers.It is recognized in the world as the evidence of biological evolution.In this paper,Penaeus monodon from Lingshui,Wanning and Wenchang in Hainan were collected and using kit assay to extract DNA of Penaeus monodon.18SrDNA was cloned and sequenced,and sequence alignment was conducted with other species in GenBank.Phylogenetic tree of Genus Penaeus was constructed.We analyzed different genera and families in shrimps,and different genetic regularities and evolutionary differences.The results of the study are as follows:1.Phylogenetic tree of 18SrDNASequencing results of Penaeus monodon rDNA showed that:The lengths of 18SrDNA f Penaeus monodon from Lingshui Xincun,Wanning Gangbei and Wenchang Gaolong Bay are 911 bp-1264bp,944bp-1628bp,and 938bp-972bp,respectively.Analysis of phylogenetic tree indicated that different species from same genus first clustered together,meaning that is the distance between genus is greater than the distance between species.Protozoa was in a seperate branch,explaining its relatively primitive characteristic.Penaeus monodon,Litopenaeus vannamei,and Penaeus semiculcatus belong to the same family Penaeidae,and they clustered together and had the closest genetic relationship.The overall situation shows the same result with traditional taxonomy(Phylum,Class,Order,Family,Genus).2.Sequence alignment of Penaeus monodon from three different geographical areasBy PCR amplifications and sequencing of 18SrDNA of Penaeus monodon from three different geographical areas(Lingshui,Wanning,Wenchang)using one primer,No.4,No.18 and No.26 of Penaeus monodon were selected for sequence alignment.It is concluded that the lengths of 18SrDNA in No.4,No.18 and No.26 are 969 bp,977 bp,and 972 bp,respectively.In different regions of 18SrDNA replacements of bases appear in individual sites.The similarity of nucleotide sequences are 97.94%between No.4 and No.18,99.28%between No.4 and No.26,and 97.74%between No.26 and No.18,indicating that in different geographical areas,there are no obvious differences in Penaeus monodon rDNA.3.Sequencing results of 18SrDNA from different areas using three primersSequencing results of Penaeus monodon showed that species from three different areas first classified.Penaeus monodon from same seawaters first clustered and had a closer genetic relationship.Penaeus monodon from Lingshui clustered with Penaeus monodon from Wenchang,which means a higher similarity in Penaeus monodon from Lingshui and Wenchang.The causes might be the changes of the surrounding waters environment and human factors such as sewage discharge and aquaculture wastewater pollution.The changes can result in partial variation in Penaeus monodon(The bases of 18SrDNA are changed,replaced,or missing).Designed primers did not influence the clone and sequencing of ribosomal gene,meaning that in the same region 18SrDNA of Penaeus monodon three are the same.Interspecific differences are larger than geographical differences.