Molecular Cloning And Expression Patterns Of Serine Proteinase Genes In Pieris Rapae

Serine proteinases spread widely in insects. When organisms were infected by pathogens, extracelluar serine proteinases proteolysis cascades activated the key enzymes of Toll pathway and hemolymph melanization which would induce organisms producing cytotoxin such as antimicrobial peptides,quinone to kill invaders. Pieris rapae (Lepidoptera:Pieridae) is an impotant brassicaceous vegetable pest of the world, Pteromalus puparum (Hymenoptera:Pteromalidae) is dominant pupae parasitic wasp of P. rapae. The venom derived from P. puparum could block hosts'innate immunity by inhibiting the serine proteinases cascades. To define the roles of serine proteinases in P. rapae immunity and the mechanism of the venom suppression on the activity of serine proteinases, in this study, i amplified serine proteinases cDNA from P. rapae pupae fatbody or hemolymph. These proteinases' tissues distribution,different devolopmental stages and larvae instars distribution,transcripts and expressions patterns were studies.1) Full length of 4 serine proteinases cDNA were cloned and the sequences characters were analysised. Seven cDNA fragments of serine proteinases gene, screened by RT-PCR using degenerated primers, were all about 450 bp long. Full length of 4 cDNA cloned using RACE strategy were named as Pr-SP1,Pr-SP2,Pr-SP3,Pr-SP4 respectively, the length of which were 1489,1794,149,1330 bp, open reading frame involed in cDNA were 1059,1230,1245,1206 bp long respectively. Analysis of sequences characteristic could predicted Pr-SP1,Pr-SP2,Pr-SP3,Pr-SP4 all possesses signal peptides, and integrity catalytic triad residues in C-terminus, Pr-SP1 possessed a clip domain in N-terminus. Multiple sequences alignment result revealed that this four serine proteinases genes share high identities with its homologos in other insect species.2) I defined the distribution of 4 serine proteinases genes in P. rapae tissuses. At 1 h post E. coli-injection, we dissected P. rapae pupae, extracted mRNA and collected total protein from different tissuses to test the transcipts and expression levels of Pr-SP1,Pr-SP2,Pr-SP3,Pr-SP4, the results indicated that Pr-SP1,Pr-SP2 primely trascript in granulocytes, and the protein product of Pr-SP1,Pr-SP2 was detected mainly in plasma; Pr-SP3 primely trascript in plasmatocytes, and the protein product of Pr-SP3 was detected mainly in plasma; Pr-SP4 trascript in all tissuses, and the protein product of Pr-SP4 was detected mainly in plasma.3) I defined the trascipts and expressions levels of 4 serine proteinases genes in different developmental stages and instars P. rapae. The trascipts and expressions levels of Pr-SP1,Pr-SP2,Pr-SP3,Pr-SP4 were tested in eggs,1st,3rd,5th instars larvae and pupae of P. rapae which had undergone immune challenge, the results indicated that Pr-SP1,Pr-SP2 got similar viariety trends:Both of them expressed in all stages, the lowest and highest expression levels appeared in eggs and 5th instars larvae respectively. The expression levels come down significatively in pupae stage compared to 5th instar larvae; the transcripts of Pr-SP3 in eggs was very low, and the protein product was not detected. The transcrpts level increased accompanied with increase of instars in larvae stage, The expression levels come down significatively in pupae stage compared to 5th instar larvae; the transcripts of Pr-SP4 in eggs was very low, and the protein product was not detected. The transcrpts levels didn't change significatively in larvae stage, and The expression level raised significatively in pupae stage compared to 5th instar larvae.4) I defined transcripts and expressions levels of 4 serine proteinases genes after different challenges at different sampling time points. I injected beads,E. coli,Pichia pastor is and Micrococcus luteus into P. rapae pupae, injection-PBS was done as control group. I found that compared to control group, the expression levels of Pr-SP1 of E. coli,P. pastoris and M. luteus injection groups increased significatively; beads could not induce the expression in all time points. Beads could induce the expression levels of Pr-SP2 in early stages of immune responses (1 h), while E. coli,P. pastoris and M. luteus could do it in 8 h and 24 h. Beads,E. coli,P. pastoris and M. luteus could induce the expression levels of Pr-SP3, and the level of the E. coli-injection group at 8 h post-injection was significatively higher than all of other groups. At 1 h after post immune challenge, I found that compared to control group, the expression level of Pr-SP1 of E. coli,P. pastoris and M. luteus injection groups increased significatively, while the expression levels descended at 8 h and 24 h post immune challenge. This four serine proteins may participate in different innate immune responses.5) I defined transcripts and expressions levels of 4 serine proteinases genes post venom-injection derived from P. puparum or parasitisms. I injected beads,bead+venom or paratisms, injection-PBS was done as control group. I found that post beads injection, the transcripts and expressions levels of Pr-SP1 were as much as control group, venom didn't inhibit Pr-SP1 expression. One hour post beads injection, transcripts and expressions levels of Pr-SP2,4 increased signicatively compared to control group. When venom and beads exsited simultaneously or parasitism, the transcripts and expression were inhibited compared to beads-injection alone, the levels were close to control group. Eight hour post beads injection, transcripts levels of Pr-SP3 increased signicatively compared to control group. When venom and beads exsited simultaneously or parasitism, the transcripts and expression were not inhibited, the levels were close to beads-injection group. Pr-SP2,Pr-SP4 may participate in resisting parasitism, and their transcripts and expression could be regulated by venom.Taken together, these results indicted that Pr-SP1,Pr-SP2,Pr-SP3,Pr-SP4 are involved in innate immunity of P. rapae, and transcripts and expressions of Pr-SP2,Pr-SP4 could be regulated by venom derived from P. puparum. I did groudwork for serine proteinases' function study in P. rapae, and also provided target proteins candidator for pest immunue inhibitors.