EST Analysis Of Nosema Pernyi, NpSWP1Gene Cloning And Immune Response In Antheraea Pernyi

The Chinese oak silkworm (Antheraea pernyi Guerin-Meneville,1855) belongs to Lepidoptera:Saturniidae which is the most well known wild silkmoths. The annual output of silkworm cocoons from China is about7×104t accounts for90%of world production. Nosema pernyi (Wen et Ding) is a lethal pathogen of microsporidiosis in Antheraea pernyi which is the only quarantine disease in sericultural production. This disease is widely distributed in most sericultural areas and cost economic losses. In recent years, microsporidiosis occurring and spreading rate rised. Carry on the systematics research, obtaining genes information, cloning spore wall protein gene and the pattern of genetic expressions in midgut of A. pernyi after N. pernyi infection, these researches have significant role in studing biological characteristics of N. pernyi and guarantee the healthy development of sericultrual industry.This research observed purified spores of N. pernyi using light and electron microscope; total RNA was extracted for cDNA library construction using SMART technology and ESTs were analysed; phylogenetic trees were constuctied based on tubulin genes; one spore wall protein of N. pernyi was cloned and prokaryotic expression was successfully induced; Realtime PCR technology were used to investigate the genes of ApTPl and ApHSC70expression patterns in the midgut of A. pernyi after N. pernyi infection. The results were as follows:1. Morphological study of N. pernyi spores. Purified spores of N. pernyi can be obtained using differential centrifugation and Percoll density gradient centrifugation (discontinuous density gradient Percoll:25%,50%,75%and100%) at15000r-min-1for30min. Using light microscope and CCD system, we tested the size of spores. Purified spores were surrounded by blue-fluorescence due to refractivity under light microscope. Uniformity spores were oval like, and the mean length and width measurements for N. pernyi were4.36±0.42μm and1.49±0.19μm, respectively. Using a scanning electron microscope, the spots have a smooth surface. Transmission electron microscopy of longitudinal sections and cross-sections of spores revealed that the spore wall consisted of exospore, endospore and plasma membrane. The diplokaryotic nuclei occupied the center of the spore and were surrounded by10-11turns of the polar tube.2. Phylogenetic study of N. pernyi. Using RT-PCR,3’RACE technology, α,β and y-tubulin genes of N. pernyi were cloned. Neighbour-Joining and Maximum Likelihood were used to construct a phylogenetic tree. Phylogenetic analysis shows that the microsporidia are a single group in the fungi that are most closely related to the Entomophthoromycota, and are sistergroups to the Basidiomycota, Chytridiomycota, Zygomycota, Glomeromycota and some of the Ascomycota. N. pernyi, together with other Nosema spp., belongs to one of two groups in the microsporidia.3. cDNA library of N. pernyi. Using SMART (switching mechanism at5’end of RNA transcript) technology, we constructed a full-length cDNA library of N. pernyi. The identification results of library construction quality showed that the titer of primary library was4.2×106pfu/mL, the library capacity was1.0×107pfu with98.39%recombinant. PCR amplification of32randomly picked clones revealed that the inserted cDNA fragments ranged from750to3000bp with an average length of1000bp. These data indicated that a successful cDNA library of N. pernyi has been constructed in this research. From864randomly selected and sequenced clones,626high-quality ESTs were generated. These ESTs were assembled into197Unigenes including64Contigs (38.49%) and133Singlets (61.51%). The redundancy rate is68.53%. Among these Unigenes which had homology with submitted sequences in the NCBI database, about146Unigenes were similarity to known mRNAs of Nosema ceranae, Nosema bombycis, and Encephalitozoon cuniculi with BLASTn and BLASTx analysis. A full-length gene encode Sporulation Protein (named NpSP-1) has got from the cDNA library. This cDNA contained an ORF of1014bp coding for a protein of337amino acids. The predicted molecular weight and isoelectric point of this protein was39.2kD and5.71, respectively.4. NpSWPl cloning and prokaryotic expression. From the constructed cDNA library of N. pernyi, an EST of annotated spore wall protein was seletcted. Using RT-PCR and3’RACE technology, a spore wall protein gene named NpSWPl (GenBank accession number: KJ573111) from N. pernyi were cloned by screening the EST information and analyzing homological genes. This cDNA contained an ORF of837bp coding for a protein of278amino acids. The predicted molecular weight and isoelectric point of this protein was32.02kD and7.02, respectively. Thought Blastp analysis, the deduced amino acid sequence of NpSWP1shared79%identity with NbSWP1(GenBank accession number:EOB12097) in Nosema bombycis, and inferred NpSWP1to be an endosporal protein. Recombinant plasmid of NpSWP1-E1Vector was transferred into Transetta (DE3) and the target protein was expressed. According to SDS-PAGE result, the molecular weight of the target protein was32kD under the condition of IPTG for5h. Western-blot showed that the target gene was about32kD, and it has been successfully expressed in E. coli.5. Expression pattern analysis of ApHS70. From the transcriptomic research of midgut tissues from healthy larvae and N. pernyi infected larvae, HSP70gene was seletcted. Using RT-PCR technology, a HSP70gene of A. pernyi was cloned (named ApHSC70, GenBank accession numbers:KJ437496). The ORF is1959bp and encodes652amino acids with predicted molecular mass of71.49kD and theoretical isoelectric point of5.38, which contains cytoplasmic characteristics motif. Quantitative Real time PCR (qRT-PCR) was employed to further identify the expression pattern of ApHSC70gene in the midgut of A. pernyi after N. pernyi infected. qRT-PCR indicated that ApHSC70gene content in the midgut of A. pernyi little changed during0-12h, content begin rised at15h and reached the maximum expression at21h.6. Expression pattern analysis of ApTP1. Under the condition of artificial feeding N. pernyi, a trysin-like serine proteases protein expression pattern was studied. Using RT-PCR, ApTP1(GenBank accession number:KF779933) was cloned. This gene contained an ORF of774bp coding for a protein of257amino acids. The predicted molecular weight and isoelectric point of this protein was27.7kD and10.5, respectively. ApTP1had the common characteristics of trypsin-like serine proteases with the catalytic active centers of histidine, aspartic acid and serine. The predicted protein constructed of six conservative cysteines and a20-residue signal peptide sequence. Thought Blastp analysis, the deduced amino acid sequence of ApTP1shared930identity with Antheraea yamamai, the homology of other species ranged from30%to70%. Recombinant plasmid of ApTP1-E1Vector was transferred into E. coli and the expressed protein was28kD the same as forecast. Semiquantitative PCR detection indicated that ApTP1is expressed in all stage and tissues of5th larvae, but the highest expression was the instar of larvae, the tissues of midgut has the highest expression in5th instar. qRT-PCR indicated that ApTP1changed little from1to3h, and6h reach the highest level.