【作者】 刘小民；

【导师】 郭巍；

【作者基本信息】 河北农业大学 ， 农业昆虫与害虫防治， 2011， 博士

【摘要】 华北大黑鳃金龟(Holotrichia oblita)为鞘翅目鳃金龟科昆虫,是国内外公认的难防治的土栖性害虫。围食膜(peritrophic membrane, PM)是昆虫体内保护其中肠的第一道天然屏障,在昆虫中肠的消化过程及保护昆虫免受微生物和寄生虫侵害等方面发挥着重要作用。昆虫中肠围食膜蛋白的分离鉴定能够为深入研究害虫的生防机制,寻找生物防治新靶标,以及进一步明确围食膜与病原微生物之间相互作用的分子机理等提供前提和基础。本研究在构建华北大黑鳃金龟中肠cDNA表达文库的基础上,对其进行免疫筛选,并对筛选得到的围食膜靶标蛋白进行了分离与鉴定,所得的主要结果如下:1.提取华北大黑鳃金龟幼虫中肠总RNA,分离得到mRNA,利用Uni-ZAP XR载体,成功构建高质量的华北大黑鳃金龟幼虫中肠cDNA表达文库。经鉴定,该cDNA文库滴度为5.18×10~6pfu/mL,重组率为98.8%,扩增后文库滴度为2.36×109pfu/mL,插入cDNA片段的长度平均为1.85kb。其后,以棉铃虫(Helicoverpa armigera)围食膜蛋白血清为抗体,对该文库进行免疫筛选。经过筛选,共得到阳性克隆254个。其中包括4个全长基因HoCBP2、HoCBP76、HoSCP、HoChi,以及1个基因片段HoIIM。2. HoCBP2基因及HoCBP76基因,编码华北大黑鳃金龟幼虫围食膜几丁质结合蛋白。其中HoCBP2基因全长2226bp,GenBank登录号为JF681185,开放阅读框长1947bp,编码649个氨基酸。HoCBP2蛋白N-端具有19个氨基酸的前导信号序列,预期蛋白分子量70.2kDa,等电点3.52。Blast比对结果显示,其序列与鞘翅目昆虫赤拟谷盗(Tribolium castaneum)围食膜蛋白PMP14(GenBank登录号GU12810~6)相似性最高,为35%。结构域分析表明HoCBP2蛋白包含8个具有peritrophin-A结构特征的几丁质蛋白结合功能域(CBD, chitin binding domain)。HoCBP76基因全长2019bp,GenBank登录号为JF681186,开放阅读框长1725 bp,编码575个氨基酸。HoCBP76蛋白N-端具有19个氨基酸的前导信号序列,预期蛋白分子量62.3kDa。等电点3.51。Blast比对结果表明,其序列与Tribolium castaneum围食膜蛋白PMP14相似性最高,为34%。结构域分析表明HoCBP76蛋白包含7个CBD。成功构建原核表达载体pET21b-HoCBP2和pET21b-HoCBP76,分别表达了约120 kDa及110 kDa的目的蛋白。利用Bac-to-Bac昆虫杆状病毒表达系统成功构建了重组表达载体Bac-HoCBP2及Bac-HoCBP76,转染BTI-Tn-5B1-4细胞,实现了其在昆虫细胞中的成功表达。Western blot检测表明,重组后的HoCBP2和HoCBP76蛋白表达的蛋白分子量约为120kDa和110kDa。几丁质结合活性试验结果表明,HoCBP2和HoCBP76重组蛋白均具有几丁质结合活性,重组蛋白经PBS及1M NaCl处理后,不能从几丁质/蛋白复合物中解离,而用强变性剂6M尿素及1% Calcoflour处理后,重组蛋白从几丁质/蛋白复合物中大量释放。成功分离得到HoCBP2及HoCBP76的特异抗体,Western blot检测发现HoCBP2和HoCBP76在华北大黑鳃金龟幼虫中肠前、中,后部均匀分布,围食膜、中肠、粪便及卵中均存在CBP蛋白,而在其体壁、消化液、脂肪体、蜕及马氏管中未见阳性信号。3. HoChi基因,编码华北大黑鳃金龟幼虫几丁质酶,该基因全长1636bp,GenBank登录号为HM596340,开放阅读框长1458bp,编码486个氨基酸,预期蛋白分子量为51.8kDa,等电点为5.58。其5’端和3’端各有一个长27bp和145bp的非翻译区,在polyA末端上游39bp处有一个多聚腺甘酸终止信号序列AAGAAA。该蛋白具有典型的几丁质酶特性,即一个信号肽、一个几丁质酶活性位点、一个C端苏氨酸富集区和一个几丁质结合域,属于几丁质酶18家族。其氨基酸序列与赤拟谷盗几丁质酶蛋白(GenBank登录号NM001044629)具有较高的相似性。将该基因与pET28b载体重组后,经IPTG诱导,Western blot鉴定,该蛋白在大肠杆菌中得到表达。利用Bac-to-Bac昆虫杆状病毒表达系统成功构建重组表达载体pFastBac-HoChi,转染BTI-Tn-5B1-4细胞后,实现了其在昆虫细胞中的成功表达。4. HoSCP基因,编码华北大黑鳃金龟幼虫丝氨酸羧肽酶,GenBank登录号为JF681184。该基因全长1830bp,开放阅读框长1371 bp,编码457个氨基酸。推导的蛋白质分子量为51.2kDa,等电点为6.12。其5’端具有78bp的非翻译区及起始密码子ATG,3’端具有381bp的非翻译区,并且含有终止密码子TAA,在polyA末端上游14bp处有一个多聚腺甘酸终止信号序列AATAAA。N-末端具有15个氨基酸的前导信号序列,表明其是分泌型蛋白。序列分析表明,HoSCP含有一个保守的S10肽酶结构域、一个丝氨酸活性位点序列IAGESYAG、一个组氨酸活性位点序列FAFLkVYGAGHMVPMDQP以及两个N-联糖基化位点,并且含有丝氨酸羧肽酶保守催化三联体位点Ser-185,Asp-361,His-418。将该基因与pET28b载体重组后,经IPTG诱导,Western blot鉴定,该蛋白在大肠杆菌中得到表达。利用Bac-to-Bac昆虫杆状病毒表达系统成功构建重组表达载体pFastBac-HoSCP,转染BTI-Tn-5B1-4细胞后,实现了其在昆虫细胞中的成功表达。5. HoIIM基因,编码华北大黑鳃金龟肠幼虫围食膜粘蛋白,长1879bp,最长读码框长1707bp,编码569个氨基酸,GenBank登录号为JF681187。结构域分析表明,HoIIM蛋白5个具有peritrophin-A结构特征的几丁质结合功能域、5个粘蛋白结构域。其粘蛋白结构域富含苏氨酸(Thr,T)、脯氨酸(Pro,P)和丝氨酸(Ser,S),分别占29.3%、15.5%及3.91%,并含有大量重复序列,其中PTTTPTT共重复15次,PTSTTTPTTITT及STTTT分别重复5次,TTPTTP重复4次。对华北大黑鳃金龟幼虫围食膜蛋白组分的银染分析表明,其围食膜蛋白种类较多。其中分子量大小大约为160kDa和80kDa的两条蛋白条带,与经PAS方法检测到的呈明显红色的两种糖蛋白大小相符。从粉纹夜蛾颗粒体病毒中分离得到增效蛋白Enhancin,离体处理华北大黑鳃金龟幼虫围食膜后,Western blot检测发现有一条分子量较大的围食膜蛋白条带明显消失,而相应的一些小分子量的围食膜蛋白条带有所增加。更多还原

【Abstract】 Holotrichia oblita is one of the most destructive agricultural and landscape pests in the world, and belongs to the family Scarabaeidae (Coleoptera, Scarabaeidae). Peritrophic membrane is a physical barrier, protecting the midgut epithelium from abrasive food particles, digestive enzymes, and pathogens infectious peros. Identifying and characterizing of the peritrophic membrane proteins can lay a solid foundation for its using as biological control targets and the understanding of the interaction between peritrophic membrane and pathogenic microorganism. We have constucted a cDNA expression library of the Holotrichia oblita midgut, identified and chacterized several peritrophic membrane target proteins. Main achievements summarized as follows:1. Midgut RNA and mRNA were isolated from the Holotrichia oblita larvae using the RNeasy total RNA isolation kit and the Oligotex mRNA isolation kit, and a cDNA library was constructed from Holotrichia oblita midgut mRNA using the ZAP-cDNA Gigapack Cloning Kit.The cDNA was unidirectionally ligated into the Uni-ZAP XR vector. The library had a complexity of 5.18×10~6 plaques, of which over 98.8% were recombinants. Titer of the amplified library was 2.36×109pfu/mL, and the average length of the inserted fragments was about 1.85kb by PCR analysis. About 254 positive clones were obtained by screening the library with an aitiserum specific to the PM protein of Helicoverpa armigera, including HoCBP2, HoCBP76, HoSCP, HoChi and HoIIM.2. HoCBP2 and HoCBP 76 encoded the chitin binding protein. The cDNA HoCBP2 (GeneBank Accession No. JF681185) was 2226 bp in length, containing an ORF of 1947 bp. The deduced protein sequence showed that HoCBP2 was synthesized as a preprotein of 649 amino acid residues with the predicted molecular weight of 70.2 kDa, pI of 3.52 and a 19-amino acid signal peptide predicted by the software SignaIP. The cDNA HoCBP76 (GeneBank Accession No. GU12810~6) was 2019 bp in length, containing an ORF of 1725 bp, The deduced protein sequence showed that HoCBP76 was synthesized as a preprotein of 575 amino acid residues with the predicted molecular weight of 62.3 kDa, pI of 3.51 and a 19-amino acid signal peptidel. Search of the GenBank database using blastp programs showed that HoCBP2 and HoCBP76 had sequence similarity with PMP14 (GeneBank Accession No. GU12810~6) from Tribolium castaneum (Coleoptera: Tenebrionidae), the identity were 35% and 34% respectively. Deduced amino acid sequence analysis indicated that HoCBP2 and HoCBP76 contained eight and seven tandem putative chitin binding domains which belonged to the peritrophin-A domains, respectively. The HoCBP2 and HoCBP76 were recombined into pET21b vector, transformed into E.coli., and were successfully expressed after IPTG induction.Recombinant HoCBP2 and HoCBP76 were successfully expressed in insect cells (Tn-5B1-4) as secreted proteins using recombinant baculoviruses. Western blot analysis showed that the apparent molecular weight for recombinant HoCBP2 and HoCBP76 were about 120 and 110kDa. Subsequent chitin binding assays demonstrated that both of the recombinant HoCBP2 and HoCBP76 had chitin binding a?nity. The HoCBP2 and HoCBP76 tightly bound to chitin and did not dissociate from the chitin following treatment with PBS and 1 M NaCl. However, they were solubilized from the bound chitin by 6M Urea or by 1% Calco?uor. Immunolocation analysis using the antibodies reacting to HoCBP2 and HoCBP76 showed that the abundance of HoCBP2 and HoCBP76 in the anterior, middle and posterior regions of the midgut was similar, HoCBP2 and HoCBP76 was mainly present in the peritrophic membrane, midgut and ovum, some weak postitive staining was detected in the extract from the fecal pellet, integument, digesive fluid, fat body, exuviae and Malpighian tubules extracts did not show any positive reaction.3. HoChi encoded the chitinase protein. The cDNA was 1636bp in length (GenBank Accession No. HM596340), containing an ORF of 1458bp, and flanked by a 5’untranslated region of 27bp and a 3’untranslated region of 145bp. The deduced protein sequence showed that HoChi was synthesized as a preprotein of 486 amino acid residues with the predicted molecular weight of 51.8 kDa, pI of 5.58. A potential polyadenylation signal sequence AAGAAA was located at 39bp upstream of the polyA tail. It shared the typical character of chitinase protein, including a signal peptide, a chitinase active site, a C-terminal threonine-rich region and a chitin-binding domain, belonging to the chitinases family 18. The deduced amino acid sequence showed a high identity to reported chitinases from other insect, especially the Coleopteran insect Tribolium castaneum (GenBank Accession No. NM001044629). The HoChi was recombined into pET28b vector, and transformed into E.coli. Western blot analysis demonstrated that HoChi protein was successfully expressed after IPTG induction, and recombinant HoChi was successfully expressed in insect cells (Tn-5B1-4) using recombinant baculoviruses.4. HoSCP encoded the serine carboxypeptidases protein. The cDNA was 1830 bp in length (GenBank Accession No. JF681184), containing an ORF of 1830 bp, and flanked by a 5’untranslated region of 78bp and a 3’untranslated region of 381bp. The deduced protein sequence showed that HoSCP was synthesized as a preprotein of 457 amino acid residues with the predicted molecular weight of 51.2 kDa, pI of 6.12 and a 15-amino acid signal peptide. A potential polyadenylation signal sequence AATAAA was located at 14bp upstream of the polyA tail. Deduced amino acid sequence analysis showed that HoSCP contained an eonserved peptidase S10 domain, the serine active site IAGESYAG, the histidine active site AFLkVYGAGHMVPMDQP, and two potential sites for N-glycosylation. The sequence also indicated the presence of a catalytic triad (Ser-185,Asp-361,His-418). The HoSCP was recombined into pET28b vector, and transformed into E.coli. Western blot analysis demonstrated that HoSCP protein was successfully expressed after IPTG induction, and recombinant HoSCP was successfully expressed in insect cells (Tn-5B1-4) using recombinant baculoviruses.5. HoIIM encoded the insect intestianl mucin protein. The cDNA was 1879 bp in length (GenBank Accession No. JF JF681187), Deduced amino acid sequence analysis indicated that the HoIIM protein contained five tandem putative chitin binding domains which belonged to the peritrophin-A domains, and five mucin domains which riched in threonine, proline and serine, and also contained several repeating units, PTTTPTT, STTTT PTSTTTPTTITT and TTPTTP, which repeated 15, 5, 5 and 4 times, respectively. The periodic acid-Schiff (PAS) stained SDS-PAGE analysis showed that the two red glycoprotein bands accorded with the 160kDa and 80kDa protein bands of the PM proteins from the Holotrichia oblita larvae by the silver-stained SDS-PAGE analysis. Western blot analysis showed that the bigger molecular weight PM protein band disappeared after the treatment of the PM from the Holotrichia oblita larvae with the enhancin isolated from the Trichoplusia ni granulosis virus (TnGV), and some smaller molecular weight PM proteins increased correspondingly.更多还原