Characterization Of Active LTR Retrotransposons In Nosema Bombycis

Microsporidia are a group of unicellular eukaryotes which develop as obligate intracellular parasites.They can infect a wide variety of animals ranging from invertebrates to vertebrates,including human.By now,more than 1,200 microsporidia species belonging to 150 genera have been reported.As a member of them,Nosema bombycis is known as a pathogen of pebrine,which usually prevails in sericulture.Thus, the prevention and treatment of pebrine is still an important and difficult problem for sericulture.Transposable elements(TEs)are ubiquitous in most organisms and have emerged as one of the key forces driving the evolution of their genomes.They can be grouped into two major classes(â… andâ…¡)based on their sequence organization and mechanism of transposition.Classâ… elements(retrotransposons)transpose through an RNA intermediate using reverse transcriptase(RT),while Classâ…¡elements(DNA transposons)move strictly from DNA to DNA.One categorie of retrotransposons,LTR retrotransposons,which are flanked by long terminal repeat(LTR)at their ends and contain two internal open reading frames encoding Gag and Pol polyproteins,are similar both in structure and retrotransposition mechanism to retroviruses.So far,TEs have been found in several prokaryotes and all higher eukaryotes analyzed.However, different situations have been reported in EncepHalitozoon cuniculi genome,which had suggested that Microsporidia lack of mobile elements.Nevertheless,recently,eight intact LTR retrotransposons(Nbr1-Nbr8)from Nosema bombycis are identified in our lab,indicating that microsporidian genome are varied and not extremely reduced and compacted as reported.By calculating the Ka/Ks,a deduction that active LTR retrotransposons might exist in the genome of Nosema bombycis was proposed.In order to confirm it,we have further analyzed all the EST and proteomics data,and seven active LTR retrotransposons have been identified in this paper.(1)Identification of four Pol polyproteins with peptide mass fingerprinting by MALDI-TOF-MSWe set up the protein database according to the predicted coding sequence based on the whole genome database of Nosema bombycis and 71 protein spots have been detected by MALDI-TOF-MS,which 28 proteins identified with known function.Four among them are pol polyproteins,designated Nbr5(reported),Nbr9,Nbr10 and Nbr11, respectively.They have shown different distribution among genome with the length of 625aa,165aa,372aa and 267aa and the molecular weight of 34kD,19kD,49kD and 31kD,respectively.Each protein is a transposase and encoded by one fragment of Pol ORF.(2)Further identification of four Pol polyproteins using RT-PCRThough four Pol polyproteins are identified by MALDI-TOF-MS,their amino acid sequences can not be confirmed yet.Thus,we further analyzed their transcriptional activity of the four proteins through RT-PCR.Total RNA was acquired from midgut collected from infected silkworm,and then single-stranded cDNA was synthesized as templet.The target PCR fragments were amplified by specific primers of Pol,indicating that four proteins are transcribed.(3)Identification of three Pol polyproteins in EST databaseThe total RNA was extracted from purifed spores of Nosema bombycis and cDNA library was constructed.Three EST sequences among this database were identified as Pol polyproteins by BLASTP(E-value of＞5e-72,identities of＞87%and length of more than 300bp)and christened as Nbr12,Nbr13 and Nbr14,respectively.Thus,another three active Pol polyproteins were retrieved.(4)Bioinformatics analysis for the seven active LTR retrotransposonsIntact LTR retrotransposons were identified via extending both ends of sequences of active Pol polyproteins.Both the LTRs and domains of the retrotransposons are varied:the lengths of LTRs range from 26 to 306bp;Head of the Nbr11 ORF have been lost and Gag,Pro and part of RT are absent;the Pro domain of Nbr10 is in absence and RT of Nbr9 is incomplete too;we also found a 1026bp DNA segment inserted in the RT domain of Nbr12.Despite incomplete,some conserve motifs have existed in retrotransposons via multiple sequence alignments yet.Nbr10 and Nbr13 possess the Cys motif(CX₂CX₄HX₄C),while Nbr12 contain a similar Cys motif(CX₂HX₄HX₄C) and a cysteine is substituted for a histidine.Alignment of the RHaseH domains shows the DAS motif found in Nbr9,Nbr10,Nbr11,Nbr12 and Nbr14,while Nbr5 and Nbr13 possess a similar motif with the amino acid sequence of DAC.The function of these resemble motifs is unkown and need more studies.The conserve D(T/S)G motif is found in the protease domains of Nbr12-Nbr14 and YXDD motif is detected from RT domains of all seven Nbr.All of seven retrotransposon families have low genomic copy numbers(1-10)but high heterogeneities(e.g.,Pi of Nbr14 is 0.4280),inferring that their transposition incidents have taken place long ago and some copies might be lost in the genome of Nosema bombycis.However,some copies of each Nbr families except Nbr13 with high identity were found(＞92%of nucleotide level),indicating that recent transposition incidents have happened while which also is a support for their activities.However,big heterogeneities were shown in some copies in spite of low copy numbers(Pi of Nbr14 is 0.4280),inferring that could allow the existence and variation of retrotransposon.Only one copy was found in Nbr13 and presumed that others were not identified due to their high degree of heterogeneities.Like eight Nbrs reported,all seven active LTR retrotransposons possess the feature of Ty3/Gypsy group,which the location of INT domain at the downstream of RT,and was supported by phylogenetic tree.Two clades of total 14 Nbrs were also defined by this neighbour joining tree.One clade contained Nbrs of unknown activity had close relationship to Ty3 retrotransposon from Saccharomyces cerevisiae,while the other activity was relative to Yoyo of Cerattitis capitata.Previous documents thought that most of Nbr were clustered with other TEs or between two synteny blocks,which did not interfere the assignment of genome. However,in this paper,Nbr11 was found to insert into the interior of a synteny block and lead to its inversion.Ka/Ks ratio of this LTR retrotransposon was greater than 1.0 and indicating positive selection had been acted on it in genome,conjecturing that both its transposition and the inversion of genome blocks were adapted in genome and retained.Thus,these results suggest that retrotransposons might play an important role in reshuffling and evolution of Nosema bombycis genome.In conclusion:Seven active Pol polyproteins,which belong to one part of intact LTR retrotransposons,were identified through MALDI-TOF-MS,RT-PCR and EST.By a series of bioinformatics analysis,they were determined to belong to Ty3/Gypsy group and possessed low genomic copy numbers but high degree of heterogeneities among family members.These LTR retrotransposons were regarded as the significant factors influencing the increasing,reshuffling and evolution of genome.