The Study Of Molecular Biology Of The Pieris Rapae Granulovirus

Pieris rapae granulovirus (PiraGV) is a member of genus Granulovirus inBaculoviridae. PiraGV only infects the insect Pieris rapae in nature andfunctions as an important biological agent to control the population of Pierisrapae in the ecosystem. Although PiraGV has been registered as a commercialbiopesticide to control Pieris rapae for several decades, research on this virushas mainly been limited on the basic biology and very little is known about thegenetic and molecular information of this virus. In this work, a study on themolecular characterization of PiraGV was performed that included the followingaspects:1. Morphology, pathogenecity and in vitro cloning of PiraGV: The originalgranulovirus isolate was isolated from a single died larva cadaver of Pierisrapae with syndromes of typical granulovirus infection, collected in the field.Typical occlusion bodies of granulovirus were found under electron microscopein the samples prepared from the infected Pieris rapae larva. Infection of Pierisrapae larvae with the virus preparation was successful. The original PiraGV wassubjected to three rounds of in vivo cloning by consecutive infection of the virus from larva-to-larva to ensure the homogenized genotype. Restriction fragmentanalyses with multiple enzymes conformed that cloned PiraGV-E3 was a singlegenotype.2. Physical map of PiraGV-E3: The total size of the PiraGV genome wasestimated to be 109 kbp by restriction analysis. Digestion of PiraGV-E3 withEcoRI, SalI, HindlII, and KpnI yielded 17, 23, 14, and 14 fragments,respectively. A physical map of the PiraGV-E3 genome was constructed basedon the terminal sequences of the cloned fragments from the KpnI-library,Hindâ…¢-library, EcoRI-library and the SalI-library and single or double digestionof the cloned fragments with various enzymes. Comparison of physical map ofPiraGV-E3 to those of PiraGV isolates mapped previously revealed a fewrestriction site differences. Of 17 EcoRI sites in PiraGV-E3, 14 match with thoseof ArGV1 (a England isolate of PiraGV) that has 19 EcoRI sites with anestimated genome of 110 kb. The difference include 5 deletions and 3 additionsfor PiraGV E3.3. Characterization of PiraGV DNA polymerase gene (dnapol): The DNApolymerase gene (dnapol) of the PiraGV-E3 was found to locate between73.1-76 m.u. on the PiraGV genome. Its open reading frame (ORF) contains3135 nucleotides with 35% G-C content and encodes 1045 amino acids with apredicted molecular mass of 122.16 kDa. Homology analysis indicated thatPiraGV dnapol had 28-70% amino acid identity to that of other knownbaculoviruses. Comparative sequence analyses demonstrated that the PiraGV dnapol gene contains conserved 3'-5' exonuclease motifs and DNA bindingfunctional domains of the DNA polymerase enzyme found in all knownbaculovirus dnapols. Northern blot results showed that in infected Pieris rapaelarvae the PiraGV dnapol gene was transcribed as a predominant 3.7 kb mRNA.5' and 3' RACE indicated that the PiraGV dnapol transcript was initiated fromthe thymine residue located at-378 nt upstream from the ATG start codon andterminated at the polyadenylation signal AATAAA. Phylogenetic analysis ofdnapol sequences suggests that the PiraGV dnapol is more closely related totortricid-infecting Cydia pomonella GV (CpGV), Choristoneura occidentalisGV (ChocGV), and Cryptophlebia leucotreta GV (CrleGV) than to those ofother baculoviruses.4. Complete genome sequence analysis of PiraGV: The genome of thePiraGV-E3 was completely sequenced by a short gun method. The gennome was108 476 bp in length with 68.8% A+T content and contained 125 potential ORFs.Of these, 29 ORFs were conserved in all fully sequenced baculovirus genomes,30 were GV-specific, and 6 were unique. Four homologous regions (hrs)/repeatregions, lacking typical NPV hr palindromes were identified. PiraGV hrs weresimilar to each other but not to other GV hrs. A 785 kb repeat region with a highA+T content and multiple repeats was found between ORF23 and 24. This arearesembled the non-homologous region origin of DNA replication (non-hr ori)identified in CpGV, CrleGV and ChocGV. Based on the mean amino acididentities of homologous proteins, PiraGV was closest to fully sequenced genomes ChocGV (57.4 %), CpGV (54.4%) and CrleGV (54.4%). The GeneParity Plot analysis showed that PiraGV genome shared the highest collinearityof gene order with tortricid-infecting CpGV, ChocGV and CrleGV than to GVsthat infect other lepidopteran families and NPVs.