蝶蛹金小蜂毒液蛋白质组与四个毒液蛋白生理功能的分析
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摘要
寄生蜂是农业害虫重要的天敌,在害虫生物防治中发挥重要作用。寄生蜂在产卵的同时,将毒液、多分DNA病毒(PDV)或类病毒颗粒(VLP)等寄生因子一同注入寄主体内,抑制寄主免疫反应或调控寄主生长发育以保护其子代能够成功完成发育。毒液等寄生因子可以用来开发新型免疫抑制剂或用于转基因作物的开发,具有广阔的应用前景。因此,寻找寄生蜂寄生因子,明确寄生因子的功能可以为下一步的开发利用奠定关键基础。目前,仅对少数几个寄生蜂毒液进行了研究,获得的毒液基因也很有限,功能的研究则更少。因此,本研究以菜粉蝶Pieris rapae的蛹期优势内寄生蜂蝶蛹金小蜂Pteromalus puparum为研究对象,采用毒腺转录组和毒液蛋白质组的分析方法解析了蝶蛹金小蜂毒液蛋白的组分。同时选取四个毒液蛋白基因为重点,研究并明确了其基因序列、表达模式与生物学功能。
1.蝶蛹金小蜂毒液蛋白质组分析
首先测定了蝶蛹金小蜂雌蜂毒腺／非毒腺组织的转录组,对转录组数据进行了分析。在利用shotgun蛋白质组方法对蝶蛹金小蜂毒液进行分析的基础上,对获得的蛋白质组质谱信息与蝶蛹金小蜂毒腺／非毒腺组织转录组数据库和后生动物门蛋白质数据库作比对分析,鉴定明确了蝶蛹金小蜂毒液蛋白组分中的60个蛋白,其中可分为蛋白酶类、蛋白酶抑制剂、识别或结合蛋白、其他及功能未知蛋白5大类。
2.蝶蛹金小蜂毒液钙网蛋白的功能及其与寄主菜粉蝶钙网蛋白关系的分析
蝶蛹金小蜂毒液钙网蛋白氨基酸序列可分为N、P和C三个区域,并且对序列进行进一步分析首次发现在C末端有一个Coiled-coil结构域,原核表达并纯化蝶蛹金小蜂毒液钙网蛋白或缺失Coiled-coil结构域的突变体,制备了兔多克隆抗体。定量PCR检测到蝶蛹金小蜂毒液钙网蛋白基因在各个组织都有表达,在毒腺中表达量最高,而在羽化后第二天的毒腺表达量最高,后表达量降低。Western blotting结果表明钙网蛋白存在于雌蜂各个组织和毒液中,羽化0-6天毒液中都存在,并且差异不明显。融合表达的毒液钙网蛋白和缺失Coiled-coil结构域的突变体能够结合到蝶蛹金小蜂卵表面。融合表达的毒液钙网蛋白能够进入寄主菜粉蝶血细胞,而缺失Coiled-coil结构域的突变体只能少量进入寄主菜粉蝶血细胞。融合表达的毒液钙网蛋白在体外能够抑制寄主菜粉蝶血细胞的延展和包囊,缺失Coiled-coil结构域的突变体也能够抑制寄主菜粉蝶血细胞的延展和包囊,但效果不如野生型融合蛋白。将融合表达的毒液钙网蛋白注射到菜粉蝶体内,定量PCR检测到菜粉蝶血细胞钙网蛋白和清道夫受体基因的表达受到抑制。菜粉蝶钙网蛋白能够被酵母和微珠显著诱导表达,能够促进血细胞的的包囊反应,并且蝶蛹金小蜂寄生后菜粉蝶钙网蛋白表达量降低。
3.蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂的分离纯化及功能分析
利用RP-HPLC对蝶蛹金小蜂总毒液进行分离,获得了单个毒液多肽组分,其中2号峰经质谱及N端测序鉴定为Pacifastin丝氨酸蛋白抑制剂。设计引物克隆了蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂的基因,全长365bp,其中开放阅读框225bp,编码74个氨基酸。原核表达Pacifastin丝氨酸蛋白抑制剂并进行纯化,同时化学合成了多肽,利用原核表达纯化的蛋白制备了兔多克隆抗体。定量PCR分析发现蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂基因特异性的在毒腺中表达,在毒腺中以羽化后第2天表达量最高；VWestern blotting结果表明蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂特异性的存在于毒液中,毒液中Pacifastin丝氨酸蛋白抑制剂在羽化后第三天达到最高且持续到第六天。体外测定了融合表达的和化学合成的蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂对适应性寄主和非适应寄主血淋巴中酚氧化酶(PO)和酚氧化酶前体激活(PPO activation)的影响。结果表明,蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂对寄主菜粉蝶和柑橘凤蝶蛹血淋巴PO活性无影响,但显著抑制了2个寄主血淋巴的PPO激活。蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂对非寄主昆虫家蚕的PO活性无影响,抑制血淋巴PPO激活。蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂对非寄主昆虫二化螟血淋巴PO活性及PPO激活均无影响。而有趣的是,蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂能够促进烟草天蛾Manduca sexta血淋巴PPO激活。通过对丝氨酸酶活性的测定,发现蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂能够显著抑制胰蛋白酶trypsin活性,而对胰凝乳蛋白酶chymotrypsin和proteinase K活性无影响。
4.蝶蛹金小蜂毒液Kazal丝氨酸蛋白酶抑制剂的功能分析
利用转录组所获得的序列设计引物克隆了蝶蛹金小蜂毒液Kazal丝氨酸蛋白酶抑制剂的基因,全长398bp,开放阅读框全长210bp,编码70氨基酸。原核融合表达并纯化了毒液Kazal丝氨酸蛋白酶抑制剂,同时化学合成了毒液Kazal丝氨酸蛋白酶抑制剂,利用原核表达纯化的蛋白制备了兔多克隆抗体。定量PCR检测发现毒液Kazal丝氨酸蛋白酶抑制剂基因在毒腺中特异性表达,且在羽化后第2天表达量最高；VWestern blotting检测发现毒液Kazal丝氨酸蛋白酶抑制剂只存在于毒液中,从羽化到羽化后第6天毒液中都存在。体外证实了融合表达的和化学合成的毒液Kazal丝氨酸蛋白酶抑制剂对寄主菜粉蝶和柑橘凤蝶血淋巴PO活性无影响,对菜粉蝶和柑橘凤蝶血淋巴PPO激活有抑制作用。
5.蝶蛹金小蜂毒液普通气味结合蛋白的特性分析
克隆了蝶蛹金小蜂毒液普通气味结合蛋白的基因,全长517bp,开放阅读框为396bp编码332个氨基酸。原核表达毒液普通气味结合蛋白,纯化后制备兔多克隆抗体。定量PCR结果表明蝶蛹金小蜂毒液普通气味结合蛋白基因在毒腺中特异性高表达,在羽化后第二天表达量最高,后表达量逐渐降低；Western blotting检测发现毒液普通气味结合蛋白存在于毒液和毒腺中,在羽化0-6天毒液中毒液普通气味结合蛋白量比较稳定。另外,交配对蝶蛹金小蜂毒液普通气味结合蛋白基因的表达没有影响；而喂食和寄生则能够促进蝶蛹金小蜂毒液普通气味结合蛋白基因的表达。利用毒液普通气味结合蛋白抗体免疫组织化学检测发现蝶蛹金小蜂毒液普通气味结合蛋白均匀的分布在毒腺内。
本研究通过结合转录组和蛋白质组技术,鉴定了蝶蛹金小蜂毒液的组分,获得了60个毒液蛋白,并选取了毒液钙网蛋白、Pacifastin丝氨酸蛋白抑制剂、Kazal丝氨酸蛋白酶抑制剂和毒液普通气味结合蛋白进行了表达模式及功能研究。本研究增加了对寄生蜂毒液组分的认识,扩展了寄生蜂毒液的作用模式,为下一步拓展利用寄生蜂毒液蛋白用于害虫控制具有指导意义。
Parasitoids are natural enemies of numerous insects that as important biological control agent to achieve pest status in agriculture system. Its deposit their eggs within the host insect along with virulence parasitic factors such as venom, polydnaviruse (PDV), virus-like particle (VLP), and teratocyte to interfere with host's immune response or to disrupt host's development to ensure their offspring successful development of in the hemocoel. The parasitic factors such as venom would be potentially valuable resources on developing new environmentally safe insect immune-suppression agents or transgenetic crops with modern biotechnologies. So, searching the parasitoid virulence parasitic factors and determining the functions of parasitic factors would help the next development. Up to date, only some parasitoids venom protein has been documented and only little venom protein has been studied. In this study, we chose Pteromalus puparum (Hymenoptera:Pteromalidae), a predominate pupal endoparasitoid of Pieris rapae (Lepidoptera:Pieridae) as object, combined venom gland transcriptome and venom proteomic method to analysis the constituents of Pteromalus puparum venom. And we selected some venom protein to expression in bacterial, and prepared rabbit polyclonal antibodies, studied their expression pattern and verified their biological function on its host.
1. The constituents of Pteromalus puparum venom
We sequenced the transcriptome of P. puparum venom gland and other tissue firstly, and analysed the transcriptome data. Then we used shotgun proteomic method to analysis P. puparum venom protein, the obtained data blasted with the database of P. puparum transcriptome and Metazoa database. And we obtained60venom protein from P. puparum venom, divided them into5types, including enzymes, proteinase inhibitors, recognition or binding proteins, unknown proteins and other.
2. Functional analysis of calreticulin from Pteromalus puparum venom and its relationship with calreticulin of Pieris rapae
We found a Coiled-coil domain in C terminal, and expressed the wild-type and the Coiled-coil domain deleted mutant P. puparum CRT (PpCRT) in bacterial and prepared the polyclonal antibody against PpCRT. RT-PCR results showed PpCRT gene expressed highest in venom gland, and the highest expression level in venom gland at after emergency2days. Western blotting analyses showed that PpCRT not only existed in venom but also in all tissues, and PpCRT lasted in venom after emergency. Recombinant His-tag wild-type and mutant PpCRT could unequally bind to P. puparum egg surface. PpCRT entered Pieris rapae hemocytes, and the Coiled-coil domain was important for entering hemocytes. Recombinant PpCRT inhibited host P. rapae hemocyte spreading and cellular encapsulation in vitro, and the Coiled-coil domain of PpCRT affected PpCRT exerting function. Injection PpCRT into P. rapae pupa, real time PCR results showed PpCRT inhibited P. rapae encapsulation-related genes (CRT, scavenger receptor) expression. PrCRT mRNA in hemocytes was significantly induced after injection of yeast or beads, but did not change noticeably after injection of Escherichia coli or Micrococcus lysodeikticus. Recombinant PrCRT enhanced cellular encapsulation by P. rapae hemocytes in vitro, and the N-domain of PrCRT was required for encapsulation. RNAi of PrCRT by dsRNA injection impaired the ability of hemocytes to encapsulate beads. After parasitization by P. puparum, PrCRT mRNA and protein levels in P. rapae pupal hemocytes were significantly suppressed compared to non-parasitized control.
3. Isolation and functional analysis of Pacifastin type serine protease inhibitor from Pteromalus puparum venom
We isolated the Pteromalus puparum venom by RP-HPLC, and obtained a single venom component. After MS/MS and N sequence, we identified the No.2peak as P. puparum venom Pacifastin type serine protease inhibitor (PpPI). We cloned the full sequence of PpPI, expressed in bacterial and purified, prepared the rabbit polyclonal antibody. RT-PCR results indicated PpPI gene special expression in venom gland and the highest expression level in venom gland at after emergency2days. Western blotting results showed PpPI special existed in venom protein and the highest level in venom at after emergency3days. We tested the recombinant or chemical synthesis PpPI effect on host or non-host hemolymph PO activity or PPO activation. Results showed PpPI had no effect on hemolymph PO activity, and inhibited the hemolymph PPO activation of host Pieris rapae and Papilio xuthus. PpPI also had no effect on hemolymph PO activity, inhibited the hemolymph PPO activation of non-host Bombyx mori. PpPI had no effect on hemolymph PO activity and PPO activation of non-host Chilo supperssalis. Interesting, PpPI enhanced the hemolymph PPO activation of non-host Manduca sexta. Through serine proteinase assay, PpPI inhibited trypsin activity, but had no effect on chymotrypsin and ptoteinase K activity.
4. Functional analysis of Kazal type serine proteinase inhibitor from Pteromalus puparum venom
We cloned the full sequence of P. puparum venom Kazal type serine proteinase inhibitor (PpKazal), expressed in bacterial and purified, prepared the rabbit polyclonal antibody. RT-PCR results showed PpKazal gene special expression in venom gland and the highest expression level in venom gland at after emergency2days. Western blotting results showed PpKazal special existed in venom protein, and lasted in venom through adult period. PpKazal had no effect on hemolymph PO activity, and inhibited the hemolymph PPO activation of host Pieris rapae and Papilio xuthus.
5. Characterization of general odorant binding protein from Pteromalus puparum venom
We cloned the full sequence of P. puparum venom general odorant binding protein (PpGOBP), expressed in bacterial and prepared the rabbit polyclonal antibody. PpGOBP gene special expression in venom gland and the highest expression level in venom gland at after emergency2days. PpGOBP existed in venom gland and venom, and its expression stable in venom at adult stage. Compared with non-mated, PpGOBP gene expression in venom gland after mated had no significant difference. After feeding honey or parasitized, PpGOBP gene expression in venom gland was siginificant promoted. Using PpGOBP polyclonal antibody, we detected PpGOBP located in venom gland.
Combined transcriptome and ptoteomic methods, we idenfified the constituents of Pteromalus puparum venom and obtained60venom proteins. We selected calreticulin, Pacifastin type serine protease inhibitor, Kazal type serine proteinase inhibitor and general odorant binding protein to study their expression pattern and biological functions. This study made a great contribution to gain insights into understanding parasitoid venom components and made the foundation for exploiting venom protein for agricultal insect control.
1.蝶蛹金小蜂毒液蛋白质组分析
首先测定了蝶蛹金小蜂雌蜂毒腺／非毒腺组织的转录组,对转录组数据进行了分析。在利用shotgun蛋白质组方法对蝶蛹金小蜂毒液进行分析的基础上,对获得的蛋白质组质谱信息与蝶蛹金小蜂毒腺／非毒腺组织转录组数据库和后生动物门蛋白质数据库作比对分析,鉴定明确了蝶蛹金小蜂毒液蛋白组分中的60个蛋白,其中可分为蛋白酶类、蛋白酶抑制剂、识别或结合蛋白、其他及功能未知蛋白5大类。
2.蝶蛹金小蜂毒液钙网蛋白的功能及其与寄主菜粉蝶钙网蛋白关系的分析
蝶蛹金小蜂毒液钙网蛋白氨基酸序列可分为N、P和C三个区域,并且对序列进行进一步分析首次发现在C末端有一个Coiled-coil结构域,原核表达并纯化蝶蛹金小蜂毒液钙网蛋白或缺失Coiled-coil结构域的突变体,制备了兔多克隆抗体。定量PCR检测到蝶蛹金小蜂毒液钙网蛋白基因在各个组织都有表达,在毒腺中表达量最高,而在羽化后第二天的毒腺表达量最高,后表达量降低。Western blotting结果表明钙网蛋白存在于雌蜂各个组织和毒液中,羽化0-6天毒液中都存在,并且差异不明显。融合表达的毒液钙网蛋白和缺失Coiled-coil结构域的突变体能够结合到蝶蛹金小蜂卵表面。融合表达的毒液钙网蛋白能够进入寄主菜粉蝶血细胞,而缺失Coiled-coil结构域的突变体只能少量进入寄主菜粉蝶血细胞。融合表达的毒液钙网蛋白在体外能够抑制寄主菜粉蝶血细胞的延展和包囊,缺失Coiled-coil结构域的突变体也能够抑制寄主菜粉蝶血细胞的延展和包囊,但效果不如野生型融合蛋白。将融合表达的毒液钙网蛋白注射到菜粉蝶体内,定量PCR检测到菜粉蝶血细胞钙网蛋白和清道夫受体基因的表达受到抑制。菜粉蝶钙网蛋白能够被酵母和微珠显著诱导表达,能够促进血细胞的的包囊反应,并且蝶蛹金小蜂寄生后菜粉蝶钙网蛋白表达量降低。
3.蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂的分离纯化及功能分析
利用RP-HPLC对蝶蛹金小蜂总毒液进行分离,获得了单个毒液多肽组分,其中2号峰经质谱及N端测序鉴定为Pacifastin丝氨酸蛋白抑制剂。设计引物克隆了蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂的基因,全长365bp,其中开放阅读框225bp,编码74个氨基酸。原核表达Pacifastin丝氨酸蛋白抑制剂并进行纯化,同时化学合成了多肽,利用原核表达纯化的蛋白制备了兔多克隆抗体。定量PCR分析发现蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂基因特异性的在毒腺中表达,在毒腺中以羽化后第2天表达量最高；VWestern blotting结果表明蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂特异性的存在于毒液中,毒液中Pacifastin丝氨酸蛋白抑制剂在羽化后第三天达到最高且持续到第六天。体外测定了融合表达的和化学合成的蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂对适应性寄主和非适应寄主血淋巴中酚氧化酶(PO)和酚氧化酶前体激活(PPO activation)的影响。结果表明,蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂对寄主菜粉蝶和柑橘凤蝶蛹血淋巴PO活性无影响,但显著抑制了2个寄主血淋巴的PPO激活。蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂对非寄主昆虫家蚕的PO活性无影响,抑制血淋巴PPO激活。蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂对非寄主昆虫二化螟血淋巴PO活性及PPO激活均无影响。而有趣的是,蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂能够促进烟草天蛾Manduca sexta血淋巴PPO激活。通过对丝氨酸酶活性的测定,发现蝶蛹金小蜂毒液Pacifastin丝氨酸蛋白抑制剂能够显著抑制胰蛋白酶trypsin活性,而对胰凝乳蛋白酶chymotrypsin和proteinase K活性无影响。
4.蝶蛹金小蜂毒液Kazal丝氨酸蛋白酶抑制剂的功能分析
利用转录组所获得的序列设计引物克隆了蝶蛹金小蜂毒液Kazal丝氨酸蛋白酶抑制剂的基因,全长398bp,开放阅读框全长210bp,编码70氨基酸。原核融合表达并纯化了毒液Kazal丝氨酸蛋白酶抑制剂,同时化学合成了毒液Kazal丝氨酸蛋白酶抑制剂,利用原核表达纯化的蛋白制备了兔多克隆抗体。定量PCR检测发现毒液Kazal丝氨酸蛋白酶抑制剂基因在毒腺中特异性表达,且在羽化后第2天表达量最高；VWestern blotting检测发现毒液Kazal丝氨酸蛋白酶抑制剂只存在于毒液中,从羽化到羽化后第6天毒液中都存在。体外证实了融合表达的和化学合成的毒液Kazal丝氨酸蛋白酶抑制剂对寄主菜粉蝶和柑橘凤蝶血淋巴PO活性无影响,对菜粉蝶和柑橘凤蝶血淋巴PPO激活有抑制作用。
5.蝶蛹金小蜂毒液普通气味结合蛋白的特性分析
克隆了蝶蛹金小蜂毒液普通气味结合蛋白的基因,全长517bp,开放阅读框为396bp编码332个氨基酸。原核表达毒液普通气味结合蛋白,纯化后制备兔多克隆抗体。定量PCR结果表明蝶蛹金小蜂毒液普通气味结合蛋白基因在毒腺中特异性高表达,在羽化后第二天表达量最高,后表达量逐渐降低；Western blotting检测发现毒液普通气味结合蛋白存在于毒液和毒腺中,在羽化0-6天毒液中毒液普通气味结合蛋白量比较稳定。另外,交配对蝶蛹金小蜂毒液普通气味结合蛋白基因的表达没有影响；而喂食和寄生则能够促进蝶蛹金小蜂毒液普通气味结合蛋白基因的表达。利用毒液普通气味结合蛋白抗体免疫组织化学检测发现蝶蛹金小蜂毒液普通气味结合蛋白均匀的分布在毒腺内。
本研究通过结合转录组和蛋白质组技术,鉴定了蝶蛹金小蜂毒液的组分,获得了60个毒液蛋白,并选取了毒液钙网蛋白、Pacifastin丝氨酸蛋白抑制剂、Kazal丝氨酸蛋白酶抑制剂和毒液普通气味结合蛋白进行了表达模式及功能研究。本研究增加了对寄生蜂毒液组分的认识,扩展了寄生蜂毒液的作用模式,为下一步拓展利用寄生蜂毒液蛋白用于害虫控制具有指导意义。
Parasitoids are natural enemies of numerous insects that as important biological control agent to achieve pest status in agriculture system. Its deposit their eggs within the host insect along with virulence parasitic factors such as venom, polydnaviruse (PDV), virus-like particle (VLP), and teratocyte to interfere with host's immune response or to disrupt host's development to ensure their offspring successful development of in the hemocoel. The parasitic factors such as venom would be potentially valuable resources on developing new environmentally safe insect immune-suppression agents or transgenetic crops with modern biotechnologies. So, searching the parasitoid virulence parasitic factors and determining the functions of parasitic factors would help the next development. Up to date, only some parasitoids venom protein has been documented and only little venom protein has been studied. In this study, we chose Pteromalus puparum (Hymenoptera:Pteromalidae), a predominate pupal endoparasitoid of Pieris rapae (Lepidoptera:Pieridae) as object, combined venom gland transcriptome and venom proteomic method to analysis the constituents of Pteromalus puparum venom. And we selected some venom protein to expression in bacterial, and prepared rabbit polyclonal antibodies, studied their expression pattern and verified their biological function on its host.
1. The constituents of Pteromalus puparum venom
We sequenced the transcriptome of P. puparum venom gland and other tissue firstly, and analysed the transcriptome data. Then we used shotgun proteomic method to analysis P. puparum venom protein, the obtained data blasted with the database of P. puparum transcriptome and Metazoa database. And we obtained60venom protein from P. puparum venom, divided them into5types, including enzymes, proteinase inhibitors, recognition or binding proteins, unknown proteins and other.
2. Functional analysis of calreticulin from Pteromalus puparum venom and its relationship with calreticulin of Pieris rapae
We found a Coiled-coil domain in C terminal, and expressed the wild-type and the Coiled-coil domain deleted mutant P. puparum CRT (PpCRT) in bacterial and prepared the polyclonal antibody against PpCRT. RT-PCR results showed PpCRT gene expressed highest in venom gland, and the highest expression level in venom gland at after emergency2days. Western blotting analyses showed that PpCRT not only existed in venom but also in all tissues, and PpCRT lasted in venom after emergency. Recombinant His-tag wild-type and mutant PpCRT could unequally bind to P. puparum egg surface. PpCRT entered Pieris rapae hemocytes, and the Coiled-coil domain was important for entering hemocytes. Recombinant PpCRT inhibited host P. rapae hemocyte spreading and cellular encapsulation in vitro, and the Coiled-coil domain of PpCRT affected PpCRT exerting function. Injection PpCRT into P. rapae pupa, real time PCR results showed PpCRT inhibited P. rapae encapsulation-related genes (CRT, scavenger receptor) expression. PrCRT mRNA in hemocytes was significantly induced after injection of yeast or beads, but did not change noticeably after injection of Escherichia coli or Micrococcus lysodeikticus. Recombinant PrCRT enhanced cellular encapsulation by P. rapae hemocytes in vitro, and the N-domain of PrCRT was required for encapsulation. RNAi of PrCRT by dsRNA injection impaired the ability of hemocytes to encapsulate beads. After parasitization by P. puparum, PrCRT mRNA and protein levels in P. rapae pupal hemocytes were significantly suppressed compared to non-parasitized control.
3. Isolation and functional analysis of Pacifastin type serine protease inhibitor from Pteromalus puparum venom
We isolated the Pteromalus puparum venom by RP-HPLC, and obtained a single venom component. After MS/MS and N sequence, we identified the No.2peak as P. puparum venom Pacifastin type serine protease inhibitor (PpPI). We cloned the full sequence of PpPI, expressed in bacterial and purified, prepared the rabbit polyclonal antibody. RT-PCR results indicated PpPI gene special expression in venom gland and the highest expression level in venom gland at after emergency2days. Western blotting results showed PpPI special existed in venom protein and the highest level in venom at after emergency3days. We tested the recombinant or chemical synthesis PpPI effect on host or non-host hemolymph PO activity or PPO activation. Results showed PpPI had no effect on hemolymph PO activity, and inhibited the hemolymph PPO activation of host Pieris rapae and Papilio xuthus. PpPI also had no effect on hemolymph PO activity, inhibited the hemolymph PPO activation of non-host Bombyx mori. PpPI had no effect on hemolymph PO activity and PPO activation of non-host Chilo supperssalis. Interesting, PpPI enhanced the hemolymph PPO activation of non-host Manduca sexta. Through serine proteinase assay, PpPI inhibited trypsin activity, but had no effect on chymotrypsin and ptoteinase K activity.
4. Functional analysis of Kazal type serine proteinase inhibitor from Pteromalus puparum venom
We cloned the full sequence of P. puparum venom Kazal type serine proteinase inhibitor (PpKazal), expressed in bacterial and purified, prepared the rabbit polyclonal antibody. RT-PCR results showed PpKazal gene special expression in venom gland and the highest expression level in venom gland at after emergency2days. Western blotting results showed PpKazal special existed in venom protein, and lasted in venom through adult period. PpKazal had no effect on hemolymph PO activity, and inhibited the hemolymph PPO activation of host Pieris rapae and Papilio xuthus.
5. Characterization of general odorant binding protein from Pteromalus puparum venom
We cloned the full sequence of P. puparum venom general odorant binding protein (PpGOBP), expressed in bacterial and prepared the rabbit polyclonal antibody. PpGOBP gene special expression in venom gland and the highest expression level in venom gland at after emergency2days. PpGOBP existed in venom gland and venom, and its expression stable in venom at adult stage. Compared with non-mated, PpGOBP gene expression in venom gland after mated had no significant difference. After feeding honey or parasitized, PpGOBP gene expression in venom gland was siginificant promoted. Using PpGOBP polyclonal antibody, we detected PpGOBP located in venom gland.
Combined transcriptome and ptoteomic methods, we idenfified the constituents of Pteromalus puparum venom and obtained60venom proteins. We selected calreticulin, Pacifastin type serine protease inhibitor, Kazal type serine proteinase inhibitor and general odorant binding protein to study their expression pattern and biological functions. This study made a great contribution to gain insights into understanding parasitoid venom components and made the foundation for exploiting venom protein for agricultal insect control.
引文
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