草地暝围食膜蛋白的分离鉴定
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摘要
草地螟(Loxostege sticticalis L.)是我国华北、东北和西北地区农牧业生产的重要害虫,建国后,已三次暴发成灾,给农牧业生产造成了巨大的损失。由于草地螟迁飞、滞育和周期性暴发的特点,其基础研究相对比较薄弱。围食膜(PM)作为昆虫体内一道重要的物理屏障,逐渐成为害虫防治的新靶标,同时也是国内外研究的热点。本文即在构建草地螟中肠cDNA文库的基础上,利用围食膜蛋白的多克隆抗体免疫筛选来获得编码相关基因,并进行了初步的分离与鉴定。主要研究结果如下:
以草地螟中肠为材料,分别采用RNeasy Mini Kit、Oligotex mRNA kit、ZAP-cDNA? synthesis kit和ZAP-cDNA? Gigapack? III Gold Cloning Kit提取草地螟幼虫中肠总RNA,分离纯化mRNA并构建了草地螟中肠cDNA表达文库。测得文库的原始滴度为3.73×106pfu/ml,蓝白斑测定重组率达99.7%,插入片段平均长度约为1.7kb。
利用甜菜夜蛾围食膜多克隆抗体对草地螟中肠cDNA文库进行免疫筛选,共获得282个阳性克隆,并对其中六个蛋白进行了分离鉴定。其中LstiCBP基因在cDNA文库筛选时5′端缺少部分氨基酸序列,设计适宜的引物结合RACE技术成功克隆了基因全长,其基因序列开放阅读框长为2,403bp,GenBank登录号为FJ408730,编码了801个氨基酸,其中15个氨基酸组成信号肽,蛋白前体分子量为86.2kDa,含8个几丁质蛋白结合功能域CBD,利用pET30载体成功进行原核表达,在围食膜、中肠组织、血淋巴、体壁以及蜕皮等组织均可检测到LstiCBP的存在,但以中肠中的含量最高,M2R能够影响围食膜中LstiCBP的含量;Lsti99和Lsti201在国内外尚属首次发现,在GenBank中不能检索到相似序列,但是在草地螟的头部、围食膜、中肠组织、血淋巴以及体壁等组织检测到了此类蛋白的存在。其中Lsti99基因GenBank登录号为FJ798745,开放阅读框长为1 245 bp,编码415个氨基酸,蛋白前体分子量为47.1 kDa,存在17个氨基酸的信号肽,通过pET30载体在大肠杆菌中成功表达了Lsti99蛋白,尽管此蛋白含有his-tag,但是并不能用镍柱进行纯化;通过GatewayTM技术将Lsti99序列重组到V5-His6标记杆状病毒,得到具有独立转染活性的重组杆状病毒,转染Sf9细胞后成功进行了蛋白的表达。Lsti201基因GenBank登录号为FJ798746,开放阅读框长2 145 bp,编码715个氨基酸,蛋白前体分子量为80.5 kDa,存在17个氨基酸的信号肽,含有59个明显的结构功能域,其中包括1个苏氨酸富集区,通过pET30进行了蛋白的表达;LstiSLC25蛋白为典型的运载蛋白,其基因开放阅读框(ORF)长897 bp,编码299个氨基酸;其蛋白质分子量为32.1 kDa,等电点为9.46,实现了蛋白在原核细胞的表达。羧肽酶为中肠重要消化酶,LstiCPA基因GenBank登录号为EU924506,全序列为1 380 bp,编码434个氨基酸,蛋白前体分子量为49.1 kDa,由信号肽、前肽和酶体三部分组成,具有羧肽酶的典型特征,原核表达后检测酶活力为0.0005 U/mL;羧酸酯酶是中肠主要的解毒酶,LstiCarE基因GenBank登录号为EU339106,开放阅读框长1875 bp,编码625个氨基酸,蛋白前体分子量为69.0kDa,具有羧酸酯酶的典型特征,蛋白在大肠杆菌中获得表达。
围食膜含有至少19种蛋白质,分子量在94kDa以下,幼虫取食添加光增白剂(M2R)的食物可影响其围食膜中蛋白的种类和含量;正常的围食膜表面光滑致密、无孔洞和缝隙,幼虫取食光增白剂后其围食膜产生了孔洞,随光增白剂浓度的增加和取食时间的延长,则加重对其围食膜结构的破坏;食物添加光增白剂后能够显著缩短Bt对草地螟幼虫的杀虫时间,降低了Bt的使用浓度。
通过草地螟中肠围食膜蛋白的分离鉴定,为明确草地螟围食膜的靶标蛋白的生理功能提供了研究基础,通过探讨PM与病原微生物相互作用的机理,寻找生物防治新靶标,为开发新型高效生物杀虫剂提供理论依据。
The meadow moth,Loxostege sticticalis L.,is an important agricultural pest in the north (North China; the Northeast; the Northwest) of China. It has broken out three times since 1949,which lead to enormous loss on agricultural and animal husbandry. Because of the properties of periodicity,the fundamental research on Loxostege sticticalis L. is unsubstantial. As the first crucial barrier for insects, peritrophic membrane(PM)has gradually been the hotpoint of the latest research of pest control. Based on the constructed cDNA library of the meadow moth midgut, we used PM proteins polyclonal antibody to isolate and characterize the related coding genes. Main achievements summarized as follows:
RNeasy Mini Kit、Oligotex mRNA kit、ZAP-cDNA? synthesis kit and ZAP-cDNA? Gigapack? III Gold Cloning Kit were used to extract total RNA from larval midgut, then purify mRNA and construct cDNA library. Original library titer was 3.73×106pfu/ml, the recombinant rates of the blue-white spot screening were up to 99.7%, the average inserted gene fragment length was about 1.7kb.
The 282 positive colonies were obtained by PM proteins polyclonal antibody of Spodoptera exigua immunoscreening from meadow moth midgut cDNA library. Six proteins were isolated and identified. The appropriate primers were employed to clone the full length of LstiCBP gene using RACE technique because of 5’-terminus incompleteness. GenBank accession No. of LstiCBP gene was FJ408730, contained an open reading frame(ORF) 2,403bp and encoded 801 amino acids, 15 of which composed the signal peptide. The precursor of LstiCBP protein was 86.2kDa and contained 8 chitin binding domains (CBDs). The successful prokaryotic expression was performed when LstiCBP recombined into pET30 vector. The Western blotting analysis demonstrated that LstiCBP existed in PM, midgut tissue, hemolymph, integument and ecdysis, especially rich in midgut. The amount of LstiCBP in PM was affected by M2R. Lsti99 and Lsti201 were still unknow proteins until now while the proteins were detected in head, PM, midgut tissue, hemolymph and integument of larvae. GenBank accession No. of gene Lsti99 was FJ798745, contained an ORF of 1,245bp and encodied 415 amino acids. The molecular weight of the precursor, which contained a 17-amino-acid signal peptide, was 47.1kDa. The Lsti99 successfully expressed in Escherichia coli after being recombined with pET30 vector, but couldn’t be purified by Ni-NTA. The gene of Lsti99 was inserted to V5-His6 tagged baculovirus by GatewayTM, then Lsti99 protein was successfully expressed after recombined baculovirus transfecting the cell Sf9. The accession number in GenBank for gene Lsti201 was FJ798746. The cDNA was 2,145bp, and the longest open reading frame coded for 715 amino acids. The predicted precursor protein contained a signal peptide of 17 residues and 59 functional domains with a particular domain of Thr-rich area. The expressed protein was shown as 80.5kDa in E.coli.by SDS-PAGE analysis. The gene LstiSLC25, whose ORF was 897bp, encoded 299 amino acids. LstiSLC25 protein was a typical solute carrier with molecular weight 32.1kDa and realized the protein expression in prokaryotic cell. Carboxypeptidase was an important digestive enzyme in insect midgut, which had three compositions: signal peptide, propeptide and the enzyme. LstiCPA was 1 380 bp in full-length (GenBank accession no. EU924506), and the ORF encoded 434 amino acids, with the predicted MW 49.1kD and pI 9.56 respectively. The activity for Carboxypeptidase was 0.0005 when it expressed in E.coli.. Carboxylesterase was an important detoxificative enzyme of insect midgut. The GenBank accession No. of gene LstiCarE was EU339106. The open reading frame of LstiCarE was 1875bp and encoded 625 amino acids with the precursor’s molecular weight 69.0kDa. It possessed the typical characters of carboxylesterase and had been successfully expressed in E.coli.
There were at least 19 proteins in PM and most of them were lower than 94kDa. Feeding the larva with M2R could affect the types and the contents of the PM protein. The normal PM surface was smooth without pores and slits while the treated PM had pores and slits. The higher concentration and longer treatment, the more damage could cause to PM. Once M2R were added to Bt, the treated larva not only showed obviously shorter time of Bt toxicity to Loxostege sticticalis, but also reduced Bt using concentration.
By isolating and identifying the PM proteins of meadow moth, we could get a clear idea of the physiological function toward the target site of PM. Through investigating the interaction mechanism between PM and pathogenic microorganism, we could hunt for novel target site and explore high efficient biological pesticide.
以草地螟中肠为材料,分别采用RNeasy Mini Kit、Oligotex mRNA kit、ZAP-cDNA? synthesis kit和ZAP-cDNA? Gigapack? III Gold Cloning Kit提取草地螟幼虫中肠总RNA,分离纯化mRNA并构建了草地螟中肠cDNA表达文库。测得文库的原始滴度为3.73×106pfu/ml,蓝白斑测定重组率达99.7%,插入片段平均长度约为1.7kb。
利用甜菜夜蛾围食膜多克隆抗体对草地螟中肠cDNA文库进行免疫筛选,共获得282个阳性克隆,并对其中六个蛋白进行了分离鉴定。其中LstiCBP基因在cDNA文库筛选时5′端缺少部分氨基酸序列,设计适宜的引物结合RACE技术成功克隆了基因全长,其基因序列开放阅读框长为2,403bp,GenBank登录号为FJ408730,编码了801个氨基酸,其中15个氨基酸组成信号肽,蛋白前体分子量为86.2kDa,含8个几丁质蛋白结合功能域CBD,利用pET30载体成功进行原核表达,在围食膜、中肠组织、血淋巴、体壁以及蜕皮等组织均可检测到LstiCBP的存在,但以中肠中的含量最高,M2R能够影响围食膜中LstiCBP的含量;Lsti99和Lsti201在国内外尚属首次发现,在GenBank中不能检索到相似序列,但是在草地螟的头部、围食膜、中肠组织、血淋巴以及体壁等组织检测到了此类蛋白的存在。其中Lsti99基因GenBank登录号为FJ798745,开放阅读框长为1 245 bp,编码415个氨基酸,蛋白前体分子量为47.1 kDa,存在17个氨基酸的信号肽,通过pET30载体在大肠杆菌中成功表达了Lsti99蛋白,尽管此蛋白含有his-tag,但是并不能用镍柱进行纯化;通过GatewayTM技术将Lsti99序列重组到V5-His6标记杆状病毒,得到具有独立转染活性的重组杆状病毒,转染Sf9细胞后成功进行了蛋白的表达。Lsti201基因GenBank登录号为FJ798746,开放阅读框长2 145 bp,编码715个氨基酸,蛋白前体分子量为80.5 kDa,存在17个氨基酸的信号肽,含有59个明显的结构功能域,其中包括1个苏氨酸富集区,通过pET30进行了蛋白的表达;LstiSLC25蛋白为典型的运载蛋白,其基因开放阅读框(ORF)长897 bp,编码299个氨基酸;其蛋白质分子量为32.1 kDa,等电点为9.46,实现了蛋白在原核细胞的表达。羧肽酶为中肠重要消化酶,LstiCPA基因GenBank登录号为EU924506,全序列为1 380 bp,编码434个氨基酸,蛋白前体分子量为49.1 kDa,由信号肽、前肽和酶体三部分组成,具有羧肽酶的典型特征,原核表达后检测酶活力为0.0005 U/mL;羧酸酯酶是中肠主要的解毒酶,LstiCarE基因GenBank登录号为EU339106,开放阅读框长1875 bp,编码625个氨基酸,蛋白前体分子量为69.0kDa,具有羧酸酯酶的典型特征,蛋白在大肠杆菌中获得表达。
围食膜含有至少19种蛋白质,分子量在94kDa以下,幼虫取食添加光增白剂(M2R)的食物可影响其围食膜中蛋白的种类和含量;正常的围食膜表面光滑致密、无孔洞和缝隙,幼虫取食光增白剂后其围食膜产生了孔洞,随光增白剂浓度的增加和取食时间的延长,则加重对其围食膜结构的破坏;食物添加光增白剂后能够显著缩短Bt对草地螟幼虫的杀虫时间,降低了Bt的使用浓度。
通过草地螟中肠围食膜蛋白的分离鉴定,为明确草地螟围食膜的靶标蛋白的生理功能提供了研究基础,通过探讨PM与病原微生物相互作用的机理,寻找生物防治新靶标,为开发新型高效生物杀虫剂提供理论依据。
The meadow moth,Loxostege sticticalis L.,is an important agricultural pest in the north (North China; the Northeast; the Northwest) of China. It has broken out three times since 1949,which lead to enormous loss on agricultural and animal husbandry. Because of the properties of periodicity,the fundamental research on Loxostege sticticalis L. is unsubstantial. As the first crucial barrier for insects, peritrophic membrane(PM)has gradually been the hotpoint of the latest research of pest control. Based on the constructed cDNA library of the meadow moth midgut, we used PM proteins polyclonal antibody to isolate and characterize the related coding genes. Main achievements summarized as follows:
RNeasy Mini Kit、Oligotex mRNA kit、ZAP-cDNA? synthesis kit and ZAP-cDNA? Gigapack? III Gold Cloning Kit were used to extract total RNA from larval midgut, then purify mRNA and construct cDNA library. Original library titer was 3.73×106pfu/ml, the recombinant rates of the blue-white spot screening were up to 99.7%, the average inserted gene fragment length was about 1.7kb.
The 282 positive colonies were obtained by PM proteins polyclonal antibody of Spodoptera exigua immunoscreening from meadow moth midgut cDNA library. Six proteins were isolated and identified. The appropriate primers were employed to clone the full length of LstiCBP gene using RACE technique because of 5’-terminus incompleteness. GenBank accession No. of LstiCBP gene was FJ408730, contained an open reading frame(ORF) 2,403bp and encoded 801 amino acids, 15 of which composed the signal peptide. The precursor of LstiCBP protein was 86.2kDa and contained 8 chitin binding domains (CBDs). The successful prokaryotic expression was performed when LstiCBP recombined into pET30 vector. The Western blotting analysis demonstrated that LstiCBP existed in PM, midgut tissue, hemolymph, integument and ecdysis, especially rich in midgut. The amount of LstiCBP in PM was affected by M2R. Lsti99 and Lsti201 were still unknow proteins until now while the proteins were detected in head, PM, midgut tissue, hemolymph and integument of larvae. GenBank accession No. of gene Lsti99 was FJ798745, contained an ORF of 1,245bp and encodied 415 amino acids. The molecular weight of the precursor, which contained a 17-amino-acid signal peptide, was 47.1kDa. The Lsti99 successfully expressed in Escherichia coli after being recombined with pET30 vector, but couldn’t be purified by Ni-NTA. The gene of Lsti99 was inserted to V5-His6 tagged baculovirus by GatewayTM, then Lsti99 protein was successfully expressed after recombined baculovirus transfecting the cell Sf9. The accession number in GenBank for gene Lsti201 was FJ798746. The cDNA was 2,145bp, and the longest open reading frame coded for 715 amino acids. The predicted precursor protein contained a signal peptide of 17 residues and 59 functional domains with a particular domain of Thr-rich area. The expressed protein was shown as 80.5kDa in E.coli.by SDS-PAGE analysis. The gene LstiSLC25, whose ORF was 897bp, encoded 299 amino acids. LstiSLC25 protein was a typical solute carrier with molecular weight 32.1kDa and realized the protein expression in prokaryotic cell. Carboxypeptidase was an important digestive enzyme in insect midgut, which had three compositions: signal peptide, propeptide and the enzyme. LstiCPA was 1 380 bp in full-length (GenBank accession no. EU924506), and the ORF encoded 434 amino acids, with the predicted MW 49.1kD and pI 9.56 respectively. The activity for Carboxypeptidase was 0.0005 when it expressed in E.coli.. Carboxylesterase was an important detoxificative enzyme of insect midgut. The GenBank accession No. of gene LstiCarE was EU339106. The open reading frame of LstiCarE was 1875bp and encoded 625 amino acids with the precursor’s molecular weight 69.0kDa. It possessed the typical characters of carboxylesterase and had been successfully expressed in E.coli.
There were at least 19 proteins in PM and most of them were lower than 94kDa. Feeding the larva with M2R could affect the types and the contents of the PM protein. The normal PM surface was smooth without pores and slits while the treated PM had pores and slits. The higher concentration and longer treatment, the more damage could cause to PM. Once M2R were added to Bt, the treated larva not only showed obviously shorter time of Bt toxicity to Loxostege sticticalis, but also reduced Bt using concentration.
By isolating and identifying the PM proteins of meadow moth, we could get a clear idea of the physiological function toward the target site of PM. Through investigating the interaction mechanism between PM and pathogenic microorganism, we could hunt for novel target site and explore high efficient biological pesticide.
引文
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