基于转录组分析的菜蛾盘绒茧蜂畸形细胞调控寄主的分子机制研究
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摘要
1菜蛾盘绒茧蜂畸形细胞的发育和形态
菜蛾盘绒茧蜂寄生寄主小菜蛾36h后,其胚胎两端的浆膜层开始解离形成畸形细胞,并在寄生48h后寄生蜂卵孵化时扩散到寄主血淋巴中。寄生2d后解剖,每头寄生后的小菜蛾获得畸形细胞94±7个,寄生5d后达到最大值144±13个,此后开始下降直至幼蜂在寄主体内发育结束；初产生的细胞直径15.05±0.73μm,并持续增大,至寄生后7d后达到最大值57.49±5.38gm,寄生8d后略有减小；光学显微镜和扫描电镜的观察结果显示畸形细胞在不同的发育时期均为近球形,体积不断增大,微绒毛分布规则且密度持续增大。离体培养时,寄生2d后每个寄生蜂胚胎产生畸形细胞96±8个,寄生6d后达到最大值151±10个并维持这一水平直至寄生8d后；初产生的畸形细胞直径16.14±0.98μm,并在发育过程中维持这一水平；光学显微镜和扫描电镜的观察结果显示离体培养的畸形细胞初产生时近球形,发育过程中形状逐渐变得不规则,体积增长趋势不明显,细胞表面微绒毛较为稀疏,分布杂乱。
2菜蛾盘绒茧蜂畸形细胞的转录组测定和分析
菜蛾盘绒茧蜂畸形细胞的转录组测序共产生总碱基数为1,254,124,980的6,967,361条reads、233,765个contig、38,706个scaffold和24,165个unigene。分别对contig、scaffold和unigene长度的分布进行分析发现绝大多数序列长度小于400bp。Nr注释结果显示共有63.5%的unigene(15,340个)比对上数据库中的15,043个已知基因。可注释序列中79%的比对结果E值介于1.0E-50到1.0E-5之间；相似度的分析发现44%的序列与比对上序列的相似度介于60%-80%之间；对比对上的已知序列47%来自于黑腹果蝇、西方蜜蜂、冈比亚按蚊和丽蝇蛹集金小蜂。
根据基因注释的结果,畸形细胞转录组测序所得序列鉴定得到以下类群功能基因：①寄生蜂幼虫营养摄取的相关基因,主要包括trehalase、matrixmetalloproteinase14、fatty acid binding protein和hexamerin等；②对寄主生理调控的相关基因,如gamma-glutamyl cyclotransferase-like venom protein、juvenile hormone esterase、 teratocytes secreted protein14、venom protein8、endochitinase和cysteine-rich/pacifastin venom protein等；③抑制寄主免疫的相关基因,主要包括venom protein Vn4.6、venom protein Vn50、C-type lectin等；④毒素基因,主要包括venom allergen5和venom acid phosphatase A2;⑤抗菌肽基因,包括definsin等；⑥微绒毛组装的相关基因,主要包括ezrin-radixin-moesin、supervillin和fambrin等。
3菜蛾盘绒茧蜂畸形细胞的数字基因表达谱测序
1、3、5日龄畸形细胞(teratocytes-1d、teratocytes-3d和teratocytes-5d)的数字基因表达谱测序分别得到5,895,449、6,272,972和6,214,769条reads,比对上转录组中的序列数分别为14,504、11,772和13,917个。相关性分析发现teratocytes-1d和teratocytes-5d相关性最小。GO注释结果显示,比对结果为cell killing、protein tag的基因只在3日龄畸形细胞中表达,比对结果为nutrient reservoir activity的基因只在3日龄和5日龄畸形细胞中表达,比对上其他功能类别的基因在不同发育时期的表达较为类似。
功能基因表达模式研究发现,寄生蜂幼虫营养摄取相关基因中trehalase前期表达量很高,并随着细胞的发育逐渐降低；matrix metalloproteinase14基本在细胞发育后期表达,fatty acid binding protein则细胞发育早期表达量较高,并逐渐降低；hexamerin基因随细胞发育表达量逐渐增加。涉及对寄主发育调控的6类基因有3种表达模式,juvenile hormone esterase和teratocytes secreted protein14在细胞发育前期有较高的表达,其他发育阶段表达量较低；venom protein8和cysteine-rich/pacifastin venom protein只在细胞发育末期表达；gamma-glutamyl cyclotransferase-like venom protein和endochitinase在细胞发育不同时期表达水平相差不多。抑制寄主免疫相关基因、抗菌肽和毒素基因主要在细胞发育后期表达。涉及微绒毛组装的基因细胞发育前期和中期表达水平较高,后期有所降低。
4寄生蜂幼虫营养摄取相关基因的克隆和表达
用qPCR对unigene19276(trehalase)、unigene5800(matrix metalloproteinase14)、unigene20417(fatty acid binding protein)和unigene10893(hexamerin)的表达模式进行验证,发现unigene19276主要在前期(2日龄畸形细胞)表达,而unigene5800则主要在后期(5日龄畸形细胞)表达,unigene10893的表达量呈逐渐缓慢增加的趋势,unigene20417在发育早期(1-4日龄畸形细胞)发育过程中表达较为稳定,后期(5日龄畸形细胞)略有降低,。
克隆得到unigene5800的全长cDNA序列,命名为MMP-14,基因序列全长2,052bp,1,791bp的开放阅读框编码一个66.35kD的蛋白；N末端有一个信号锚序列；氨基酸序列上分布有3个保守功能域,依次为PG_binding_1s,ZnMc_MMP和HX；构建表达载体pET-28-MMP-14并原核表达得到一个大于70kD的蛋白。
5调控寄主发育相关基因的克隆和表达
用qPCR对unigene4760(gamma-glutamyl cyclotransferase-like venom protein)、unigene14814(juvenile hormone esterase)、 unigene17429(teratocytes secreted protein14)、unigene20419(venom protein8)、unigene20917(endochitinase)和unigene18359(cysteine-rich/pacifastin venom protein)的表达模式进行验证。Unigene4760在4、5日龄时表达量显著高于1、2日龄；unigene14814和unigene17429在1日龄时的表达量均显著高于其它时期,unigene17429在4日龄时也有一个表达高峰；unigene20419、unigene20917和unigene18359在5日龄时的表达量显著高于其它时期。
对unigene4760和unigene17429进行克隆,得到两个全长分别为665bp和585bp的基因序列,命名为TSVP-GGCT和TSP-13。TSVP-GGCT包含一个558bp的开放阅读框,编码分子量21.37kD的蛋白；无信号肽；含有一个GGCT like功能域；构建表达载体pET-2S-TSVP-GGCT并原核表达得到一个26kD左右的蛋白,制备的多克隆抗体可以与菜蛾盘绒茧蜂毒液蛋白发生免疫反应。TSP-13基因342bp的开放读码框编码一个12.9kD的蛋白质,有信号肽序列,无保守功能域,构建表达载体pET-32-TSP-13但没有表达出相应的蛋白。
6抑制寄主免疫相关基因的克隆和表达
qPCR对unigene20181(venom protein Vn4.6)、unigene23309(venom protein Vn50)和unigene20101(C-type lectin)的基因表达模式进行验证发现主要在细胞发育后期表达。
克隆得到unigene20181、unigene23309、unigene23761等3个基因的cDNA全长序列,分别命名为TSVP-8、TSVP-42和TSVP-SEP。TSVP-8的cDNA全长为454bp,含有一个216bp的开放读码框,编码分子量为7.97kD的蛋白质；有信号肽序列；无保守功能域。TSVP-42的cDNA全长为1314bp,含有一个1143bp的开放读码框,编码分子量为41.94kD的蛋白质；无信号肽序列；包含一个Tryp_SPc保守功能域。TSVP-SEP的cDNA全长为1389bp,含有一个1116bp的开放读码框,编码分子量为40.68kD的蛋白质,有信号肽序列,包含一个Tryp_SPc保守功能域。构建表达载体pET32-TSVP-8和pET28-TSVP-42分别表达出一个小于26kD和大于43kD的蛋白质,pET28-TSVP-SEP则未表达出相应的蛋白质。重组蛋白制备多克隆抗体可以与菜蛾盘绒茧蜂的毒液蛋白发生免疫反应。进一步研究发现TSVP-8原核表达蛋白可以抑制寄主血淋巴的黑化。
7毒素基因的克隆和表达
用qPCR对unigene8816(venom allergen5)和unigene19070(venom acid phosphatase A2)的基因表达模式进行验证,发现这两个基因均以细胞发育后期表达为主。
克隆unigene8816的cDNA得到一个全长1085bp的序列,命名为TSVP-allergen,含有一个长825bp的开放读码框,编码分子量为30.8kD的蛋白质,有信号肽序列,包含一个属于SCP超家族的保守功能域。构建表达载体pET-TSVP-allergen并原核表达得到一个55kD左右的蛋白质。蛋白质纯化后制备得到的多克隆抗体可以与菜蛾盘绒茧蜂的毒液蛋白发生免疫反应。
1. Development and morphology of Cotesia vestalis teratocytes
Serosal 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 teratocytes
Sequencing 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 teratocytes
Sequencing 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 larvae
Expression 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 regulation
Expression 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 immunosuppression
Expression 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 genes
Expression 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.
菜蛾盘绒茧蜂寄生寄主小菜蛾36h后,其胚胎两端的浆膜层开始解离形成畸形细胞,并在寄生48h后寄生蜂卵孵化时扩散到寄主血淋巴中。寄生2d后解剖,每头寄生后的小菜蛾获得畸形细胞94±7个,寄生5d后达到最大值144±13个,此后开始下降直至幼蜂在寄主体内发育结束；初产生的细胞直径15.05±0.73μm,并持续增大,至寄生后7d后达到最大值57.49±5.38gm,寄生8d后略有减小；光学显微镜和扫描电镜的观察结果显示畸形细胞在不同的发育时期均为近球形,体积不断增大,微绒毛分布规则且密度持续增大。离体培养时,寄生2d后每个寄生蜂胚胎产生畸形细胞96±8个,寄生6d后达到最大值151±10个并维持这一水平直至寄生8d后；初产生的畸形细胞直径16.14±0.98μm,并在发育过程中维持这一水平；光学显微镜和扫描电镜的观察结果显示离体培养的畸形细胞初产生时近球形,发育过程中形状逐渐变得不规则,体积增长趋势不明显,细胞表面微绒毛较为稀疏,分布杂乱。
2菜蛾盘绒茧蜂畸形细胞的转录组测定和分析
菜蛾盘绒茧蜂畸形细胞的转录组测序共产生总碱基数为1,254,124,980的6,967,361条reads、233,765个contig、38,706个scaffold和24,165个unigene。分别对contig、scaffold和unigene长度的分布进行分析发现绝大多数序列长度小于400bp。Nr注释结果显示共有63.5%的unigene(15,340个)比对上数据库中的15,043个已知基因。可注释序列中79%的比对结果E值介于1.0E-50到1.0E-5之间；相似度的分析发现44%的序列与比对上序列的相似度介于60%-80%之间；对比对上的已知序列47%来自于黑腹果蝇、西方蜜蜂、冈比亚按蚊和丽蝇蛹集金小蜂。
根据基因注释的结果,畸形细胞转录组测序所得序列鉴定得到以下类群功能基因：①寄生蜂幼虫营养摄取的相关基因,主要包括trehalase、matrixmetalloproteinase14、fatty acid binding protein和hexamerin等；②对寄主生理调控的相关基因,如gamma-glutamyl cyclotransferase-like venom protein、juvenile hormone esterase、 teratocytes secreted protein14、venom protein8、endochitinase和cysteine-rich/pacifastin venom protein等；③抑制寄主免疫的相关基因,主要包括venom protein Vn4.6、venom protein Vn50、C-type lectin等；④毒素基因,主要包括venom allergen5和venom acid phosphatase A2;⑤抗菌肽基因,包括definsin等；⑥微绒毛组装的相关基因,主要包括ezrin-radixin-moesin、supervillin和fambrin等。
3菜蛾盘绒茧蜂畸形细胞的数字基因表达谱测序
1、3、5日龄畸形细胞(teratocytes-1d、teratocytes-3d和teratocytes-5d)的数字基因表达谱测序分别得到5,895,449、6,272,972和6,214,769条reads,比对上转录组中的序列数分别为14,504、11,772和13,917个。相关性分析发现teratocytes-1d和teratocytes-5d相关性最小。GO注释结果显示,比对结果为cell killing、protein tag的基因只在3日龄畸形细胞中表达,比对结果为nutrient reservoir activity的基因只在3日龄和5日龄畸形细胞中表达,比对上其他功能类别的基因在不同发育时期的表达较为类似。
功能基因表达模式研究发现,寄生蜂幼虫营养摄取相关基因中trehalase前期表达量很高,并随着细胞的发育逐渐降低；matrix metalloproteinase14基本在细胞发育后期表达,fatty acid binding protein则细胞发育早期表达量较高,并逐渐降低；hexamerin基因随细胞发育表达量逐渐增加。涉及对寄主发育调控的6类基因有3种表达模式,juvenile hormone esterase和teratocytes secreted protein14在细胞发育前期有较高的表达,其他发育阶段表达量较低；venom protein8和cysteine-rich/pacifastin venom protein只在细胞发育末期表达；gamma-glutamyl cyclotransferase-like venom protein和endochitinase在细胞发育不同时期表达水平相差不多。抑制寄主免疫相关基因、抗菌肽和毒素基因主要在细胞发育后期表达。涉及微绒毛组装的基因细胞发育前期和中期表达水平较高,后期有所降低。
4寄生蜂幼虫营养摄取相关基因的克隆和表达
用qPCR对unigene19276(trehalase)、unigene5800(matrix metalloproteinase14)、unigene20417(fatty acid binding protein)和unigene10893(hexamerin)的表达模式进行验证,发现unigene19276主要在前期(2日龄畸形细胞)表达,而unigene5800则主要在后期(5日龄畸形细胞)表达,unigene10893的表达量呈逐渐缓慢增加的趋势,unigene20417在发育早期(1-4日龄畸形细胞)发育过程中表达较为稳定,后期(5日龄畸形细胞)略有降低,。
克隆得到unigene5800的全长cDNA序列,命名为MMP-14,基因序列全长2,052bp,1,791bp的开放阅读框编码一个66.35kD的蛋白；N末端有一个信号锚序列；氨基酸序列上分布有3个保守功能域,依次为PG_binding_1s,ZnMc_MMP和HX；构建表达载体pET-28-MMP-14并原核表达得到一个大于70kD的蛋白。
5调控寄主发育相关基因的克隆和表达
用qPCR对unigene4760(gamma-glutamyl cyclotransferase-like venom protein)、unigene14814(juvenile hormone esterase)、 unigene17429(teratocytes secreted protein14)、unigene20419(venom protein8)、unigene20917(endochitinase)和unigene18359(cysteine-rich/pacifastin venom protein)的表达模式进行验证。Unigene4760在4、5日龄时表达量显著高于1、2日龄；unigene14814和unigene17429在1日龄时的表达量均显著高于其它时期,unigene17429在4日龄时也有一个表达高峰；unigene20419、unigene20917和unigene18359在5日龄时的表达量显著高于其它时期。
对unigene4760和unigene17429进行克隆,得到两个全长分别为665bp和585bp的基因序列,命名为TSVP-GGCT和TSP-13。TSVP-GGCT包含一个558bp的开放阅读框,编码分子量21.37kD的蛋白；无信号肽；含有一个GGCT like功能域；构建表达载体pET-2S-TSVP-GGCT并原核表达得到一个26kD左右的蛋白,制备的多克隆抗体可以与菜蛾盘绒茧蜂毒液蛋白发生免疫反应。TSP-13基因342bp的开放读码框编码一个12.9kD的蛋白质,有信号肽序列,无保守功能域,构建表达载体pET-32-TSP-13但没有表达出相应的蛋白。
6抑制寄主免疫相关基因的克隆和表达
qPCR对unigene20181(venom protein Vn4.6)、unigene23309(venom protein Vn50)和unigene20101(C-type lectin)的基因表达模式进行验证发现主要在细胞发育后期表达。
克隆得到unigene20181、unigene23309、unigene23761等3个基因的cDNA全长序列,分别命名为TSVP-8、TSVP-42和TSVP-SEP。TSVP-8的cDNA全长为454bp,含有一个216bp的开放读码框,编码分子量为7.97kD的蛋白质；有信号肽序列；无保守功能域。TSVP-42的cDNA全长为1314bp,含有一个1143bp的开放读码框,编码分子量为41.94kD的蛋白质；无信号肽序列；包含一个Tryp_SPc保守功能域。TSVP-SEP的cDNA全长为1389bp,含有一个1116bp的开放读码框,编码分子量为40.68kD的蛋白质,有信号肽序列,包含一个Tryp_SPc保守功能域。构建表达载体pET32-TSVP-8和pET28-TSVP-42分别表达出一个小于26kD和大于43kD的蛋白质,pET28-TSVP-SEP则未表达出相应的蛋白质。重组蛋白制备多克隆抗体可以与菜蛾盘绒茧蜂的毒液蛋白发生免疫反应。进一步研究发现TSVP-8原核表达蛋白可以抑制寄主血淋巴的黑化。
7毒素基因的克隆和表达
用qPCR对unigene8816(venom allergen5)和unigene19070(venom acid phosphatase A2)的基因表达模式进行验证,发现这两个基因均以细胞发育后期表达为主。
克隆unigene8816的cDNA得到一个全长1085bp的序列,命名为TSVP-allergen,含有一个长825bp的开放读码框,编码分子量为30.8kD的蛋白质,有信号肽序列,包含一个属于SCP超家族的保守功能域。构建表达载体pET-TSVP-allergen并原核表达得到一个55kD左右的蛋白质。蛋白质纯化后制备得到的多克隆抗体可以与菜蛾盘绒茧蜂的毒液蛋白发生免疫反应。
1. Development and morphology of Cotesia vestalis teratocytes
Serosal 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 teratocytes
Sequencing 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 teratocytes
Sequencing 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 larvae
Expression 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 regulation
Expression 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 immunosuppression
Expression 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 genes
Expression 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.
引文
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