| 1. Development and morphology of Cotesia vestalis teratocytesSerosal membrane at two ends of parasitoids embryos dissociated into free teratocytes at36h after Plutella xylostella larvae parasitized by C. vestalis. After dissociation, teratocytes become dispersed in the hemolymph of the host at48h after parasitization when the parasitoid eggs hatched.Plutella xylostella were dissected2days after they were monoparasitized by C. vestalis and94±7teratocytes were detected from one P. xylostella larva. The cells increased to the maximum number of144±13in5days after parasitism and followed by a decrease of its number until parasitoid larvae finished their development in host body. The diameter of teratocytes dissected from P. xylostella2days after monoparasitized was15.05±0.73μm and increased to57.49±5.38μm at7days after parasitism. The examination by scan electron microscopy (SEM) and light microscopy shew that C. vestalis teratocytes were nearly spherical in shape and increased in size as parasitoid larvae developed. The teratocytes were covered by microvilli which also increased both in size and number as parasitoid larvae developed.When teratocytes were in vitro cultured,96±8cells dissociated from one parasitoid embryo2days after parasitism, and cell numbers increased to151±10at6days after parasitism and were not significantly changed then after. The diameter of in vitro cultured teratocytes was15.05±0.73μm at2days after parasitism and not significantly changed as parasitoid larvae developed. The examination by scan electron microscopy (SEM) and light microscopy shew that in vitro cultured teratocytes were irregular in shape and not significantly changed in size as parasitoid larvae developed; a few microvilli covered on the surface of in vitro cultured teratocytes. 2. Transcriptome sequencing and analysis of Cotesia vestalis teratocytesSequencing of C. vestalis teratocyte transcriptome resulted in6,967,361reads,233,765contigs,38,706scaffolds and24,165unigenes. Distribution analysis shew that most sequences of contigs, scaffolds and unigenes were shorter than400bp. Based on Nr annotation, the E-value of BLASTx of79%unigenes ranged from1.0E-5to1.0E-50; the similarity of44%unigenes against homologous sequences ranged from60%to80%; and47%homologous sequences were from Drosophila melanogaster, Apis mellifica, Anopheles gambiae and Nasonia vitripennis.Based on Nr annotation, six groups of functional genes were identified from teratocyte transcriptome:(1) parasitoid larvae nutrient uptake related genes, e.g., trehalase, matrix metalloproteinase14, fatty acid binding protein and hexamerin;(2) host regulations related genes, e.g., gamma-glutamyl cyclotransferase-like venom protein, juvenile hormone esterase, teratocytes secreted protein14, venom protein8, endochitinase and cysteine-rich/pacifastin venom protein;(3) host immunosuppression related genes, e.g., venom protein Vn4.6, venom protein Vn50and C-type lectin;(4) toxin related genes, e.g., venom allergen5and venom acid phosphatase A2;(5) anti-microbial peptide, e.g., defensin;(6) microvilli construction related genes, e.g., ezrin-radixin-moesin, supervillin and fimbrin.3. Digital gene expression (DGE) profile of Cotesia vestalis teratocytesSequencing of three DGEs, i.e.,1-day old (teratocytes-Id),3-day old (teratocytes-3d) and5-day old (teratocytes-5d) teratocytes, resulted in5,895,449,6,272,972and6,214,769reads, respectively. The three DGEs were hinted to14,504,11,772and13917sequences in transcriptome database, respectively. GO annotation of three DGEs shew that genes from ontologies of cell killing and protein tag were expressed only in3-day old teratocytes (teratocytes-3d), genes from ontology of nutrient reservoir activity expressed in3-day old and5-day old teratocytes (teratocytes-3d and teratocytes-5d), and genes from other ontologies expressed in all DGEs.The analyses of the gene expression profiles of functional genes shew that among the genes involved in parasitoid larvae nutrient uptake, trehalase had a very high expression level in the early stage of cell development and decreased as the cells developed; metrix metalloproteinase14expressed only in the later stage, and hexamerin increased its expression level as the cells developed. The genes involved in host regulation were expressed in3patterns:the genes related to host hormone regulation mostly expressed in the early stage of cell development; venom protein8and cysteine-rich/pacifastin venom protein expressed only in the later stage; and endochitinase and gamma-glutamyl cyclotransferase-like venom protein had a similar expression level in each stage of cell development. The genes related to host immunosuppression, anti-microbial peptide and toxin were mostly expressed in the later stage of cell development. Genes involved in microvilli construction mostly expressed in the early and middle stages of cell development.4. Cloning and expression of genes related to nutrient uptake of parasitoid larvaeExpression profiles of four genes were verified by qPCR. The results shew that unigene19276(trehalase) expressed mainly in2-day old teratocytes; unigene5800(metrix metalloproteinase14) expressed mostly in5-day old teratocytes; unigene10893(hexamerin) increased its expression level as the cells developed; and unigene20417(fatty acid binding protein) maintained an expression level during first4days and decreased in5-day old teratocytes.The cDNA of unigene5800were cloned with its full length2,052bp, named as MMP-14. It contained a1,791bp open reading frame (ORF) and encoded a66.35kD protein; a signal anchor sequence and3conserved domains (PG binding1s, ZnMc_MMP and HX) were predicted from its amino acid sequence; recombinant vector pET-28-MMP-14was constructed and a recombinant protein (more than70kD) was expressed in BL21(DE3).5. Cloning and expression of genes related to host regulationExpression profiles of six genes were verified by qPCR. The results shew that the expression level of4760(gamma-glutamyl cyclotransferase-like venom protein) in last two days was significently higher than that of first two days; the expression levels of unigene14814(juvenile hormone esterase) and unigene17429(teratocytes secreted protein14) in the first day were significently higher than those of other stages; unigene20419(venom protein8), unigene20917(endochitinase) and unigene18359(cysteine-rich/pacifastin venom protein) mostly expressed in5-day old teratocytes.Full length cDNAs of unigene4760and unigene17429were cloned and named as TSVP-GGCT and TSP-13, respectively. TSVP-GGCT had a full length of665bp and contained a558bp ORF, encoding a21.37kD protein; a conserved domain of GGCT_like was predicted from its amino acid sequence but no signal peptide present; recombinant vector pET-28-TSVP-GGCT was constructed and a26kD recombinant protein was expressed in BL21(DE3);a polycolonal antibody prepared by this recombinant protein had an immune reaction against venom proteins of C. vestalis. TSP-13had a full length of558bp, contained a342bp ORF and encoded a12.9kD protein;a signal peptide predicted from its amino acid sequence but no conserved domain present; recombinant vector pET-32-TSP-13was constructed but no recombinant protein was expressed.6. Cloning and expression of genes related to host immunosuppressionExpression profiles of unigene20181(venom protein Vn4.6), unigene23309(venom protein Vn50) and unigene20101(C-type lectin) were verified by qPCR and the results revealed that all of the three genes expressed mainly in5-day old teratocytes.Full length cDNAs of unigene20181, unigene23309and unigene23761were cloned and named as TSVP-8, TSVP-42, TSVP-SEP, respectively. TSVP-8had a full length of454bbp and contained a216bp ORF, enconding a7.97kD protein. TSVP-42had a full length of1314bp and contained a1143bp ORF, enconding a41.94kD protein. TSVP-SEP had a full length of1389bp and contained a1116bp ORF, encoding a40.68kD proteins. No conserved domain was predicted from amino acid sequence of TSVP-8while a conserved domain of Tryp_SPc was predicted from amino acid sequences of both TSVP-8and TSVP-SEP. Signal peptides were predicted from amino acid sequences of TSVP-8and TSVP-SEP but no signal peptide was present in TSVP-42. Recombinant vector s of pET-32-TSVP-8and pET-28-TSVP-42was constructed and recombinant proteins (larger than26kD and smaller than43kD) were expressed in BL21(DE3), and polycolonal antibodies prepared by these two recombinant proteins had an immune reaction against venom proteins of C. vestalis. An further study confirmed that TSVP-8could inhibit the melanization of hemolymph of P. xvlostella larvae.7. Cloning and expression of toxin-related genesExpression profiles of unigene19070(venom allergen5) and unigene8816(venom acid phosphatase A2) were verified by qPCR and the results shew that these two genes expressed mostly in5-day old teratocytes.The cDNA of unigene8816was cloned and as TSVP-allergen, which had a full length of1085bp and contained a ORF of825bp, conding a30.8kD protein; a signal peptide was predicted at N end of amino acid sequence of TSVP-allergen; one conserved domain of SCP superfamily was predicted; recombinant vector pET-28-TSVP-allergen was constructed and a recombinant protein of TSVP-allergen was expressed in BL21(DE3);and polyclonal antibody prepared by this protein reacted against venom protein of C. vestalis. |
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