【作者】 周洪旭；

【导师】 孙绪艮；

【作者基本信息】 山东农业大学 ， 农业昆虫与害虫防治， 2009， 博士

【摘要】 围食膜(peritrophic matrix,PM)是大多数昆虫中肠内壁的一层厚薄均匀的长管状薄膜,成圆桶状把肠腔和中肠壁细胞分开,自中肠前端一直延伸至后肠。它主要由几丁质和蛋白质组成,是昆虫中肠天然的防御体系,具有保护中肠上皮细胞、阻止有害物质侵入和帮助消化等多种功能,是害虫生物防治的潜在靶标。鳃金龟是我国重要的地下害虫,其幼虫(蛴螬)在土壤中生活,取食植物的地下部分,并把周围的土壤颗粒、病原微生物等物质一同带入消化道,因此围食膜对蛴螬的生长发育具有重要的作用。本文采用生化和分子生物学技术等手段,对华北大黑鳃金龟(Holotrichia oblita)和暗黑鳃金龟(Holotrichia parallela)围食膜的形态结构和化学组成进行了研究,构建了华北大黑鳃金龟中肠cDNA表达文库,分离鉴定新的PM靶标蛋白,明确其生理功能,并研究生物防治促进因子对围食膜的作用与影响,寻找生物防治新靶标,为发展新的高效生物杀虫剂和鳃金龟的生物防治提供理论依据。1.通过光学显微镜和电子显微镜观察发现,鳃金龟幼虫围食膜位于中肠两端的两个“液囊环”(sac-circles)之间,是一层厚薄均匀的长管状薄膜,表面平滑致密,无孔无缝,具有一定弹性,但与鳞翅目昆虫围食膜相比,韧性较差,解剖时其围食膜易破碎。应用Bradford法和苯酚-硫酸法,测定华北大黑鳃金龟围食膜蛋白含量为36.38%,糖含量为8.84%;暗黑鳃金龟围食膜蛋白含量为38.26%,糖含量为9.08%。2.SDS-PAGE分析发现华北大黑鳃金龟PM蛋白至少有28条多肽,暗黑鳃金龟比较清楚的PM蛋白带有15条,而鳞翅目昆虫粉纹夜蛾、甜菜夜蛾、棉铃虫和粘虫PM蛋白分别有19、17、16和9条,与鳞翅目昆虫围食膜相比,华北大黑鳃金龟围食膜蛋白种类相对较多。肠粘蛋白(insect intestinal mucin, IIM)是昆虫围食膜中的重要蛋白,而应用TnPM-IIM特异性抗血清对华北大黑鳃金龟和暗黑鳃金龟围食膜蛋白做Western blot检测,未发现两种鳃金龟围食膜中有类似于鳞翅目昆虫的粘蛋白。3.应用QIAGEN公司的RNeasy Mini Kit、Oligotex mRNA Mini Kit以及Stratagene公司cDNA Synthesis Kit,成功构建了华北大黑鳃金龟3龄幼虫中肠cDNA表达文库,原始文库滴度为1.9×10~6 pfu/mL,扩增后滴度为1.4×10~9pfu/ml,文库重组率为99.97%,插入片段在0.8-2.3kb之间,平均长度为1.6kb。4.应用Western blot检测发现棉铃虫围食膜蛋白多克隆抗体、粉纹夜蛾围食膜蛋白多克隆抗体与华北大黑鳃金龟围食膜蛋白之间有交叉反应,并用这两种抗体免疫筛选华北大黑鳃金龟中肠cDNA文库,得到122个阳性克隆,测序及序列比对后得到Ho-Peritrophin1、Ho-Peritrophin2、Ho-Peritrophin3、Ho-Peritrophin4四个华北大黑鳃金龟围食膜蛋白基因,Genbank登录号分别是FJ393548、FJ393549、FJ393550和FJ573145,最长读码框(ORF)分别编码729、477、528和324个氨基酸,含有9、6、5和4个几丁质结合功能域(CBD),不含粘蛋白功能域,含有1-4个N-连糖基化位点,N端不含有信号肽。Ho-Peritrophin1、Ho-Peritrophin2、Ho-Peritrophin4的胰蛋白酶和胰凝乳蛋白酶的酶切位点大部分位于CBD内部,受到保护,不会被胰蛋白酶和胰凝乳蛋白酶降解。而Ho-Peritrophin3的第5个CBD下游区域富含大量的胰蛋白酶和胰凝乳蛋白酶的酶切位点。通过构建重组表达质粒,Ho-Peritrophin1、Ho-Peritrophin2和Ho-Peritrophin3在大肠杆菌中分别表达出78、51和57KD的目的蛋白,几丁质结合实验表明Ho-Peritrophin1和Ho-Peritrophin3具有几丁质结合活性。5.从华北大黑鳃金龟中肠cDNA表达文库中筛选得到了HoSP1和HoSP2两种华北大黑鳃金龟丝氨酸蛋白酶全长基因,在Genbank登录号分别为FJ573146和FJ573147,开放阅读框(ORF)长783和786bp,编码260和261个氨基酸,均含有3对半胱氨酸残基,与CzSP3相似性最高,分别为52.47%和51.52%,催化活性中心位于His80、Asp125、Ser215,His80、Asp125、Ser216。通过构建重组表达质粒,HoSP1和HoSP2在大肠杆菌中表达出26.7kDa和27.1kDa的蛋白。筛选得到了华北大黑鳃金龟羧酸酯酶基因HoCL,在Genbank登录号为FJ573148,开放阅读框(ORF)长1599bp,编码532个氨基酸,含有3个半胱氨酸残基,具有Ser207,Asp333和His422的催化活性中心,推导的蛋白质分子量为59.5kDa,属于B类羧酸酯酶。6.以棉铃虫颗粒体病毒增效蛋白和荧光增白剂FB28为先导物,测定了其对华北大黑鳃金龟和暗黑鳃金龟围食膜的作用和影响,发现棉铃虫颗粒体病毒增效蛋白在不同浓度和不同时间下均不能降解两种鳃金龟的围食膜;采用液滴法喂食华北大黑鳃金龟和暗黑鳃金龟10uL 1%的FB28,2.5h时,PM前端破损,扫描电镜观察中后段围食膜,发现围食膜上出现一些分布均匀的空洞;喂食至6h时,PM完全崩解,喂食至12h,PM基本恢复。可见,荧光增白剂FB28能够破坏鳃金龟围食膜,但破坏作用是短暂的、可修复的。为进一步研究荧光增白剂与病原微生物联合使用防治鳃金龟提供了理论依据。更多还原

【Abstract】 Peritrophic matrix (PM) is an invertebrate unique structure that lines the digestive tract, playing important roles in facilitating food digestion and providing protection to the gut epithelium. The important physiological functions of the PM suggest that PM can be a significant structural target for insect control. In this paper, the structure and chemical composition of PMs of both Holotrichia oblita and H. parallela were studied by using the biochemistry and molecular biology; the expression library of midgut cDNA of Holotrichia oblita was constructed to screen and separate new target protein of PMs. The effects of biocontrol-promoting factors on the PMs were also studied.1. By light microscopy and scanning electron microscope, the studies showed that PMs of H. oblita and H. parallela, situated between two sac-circles, were tubelike matrixes with even thickness and there were no holes and crevices on the smooth and compact surface. Compared to lepidopteran insects, grub PMs had poor tenacity and were destructible when dissected. By Bradford and phenol-sulfuric acid method, the protein contents of H. oblita and H. parallela PMs were 36.38% and 38.26%, and their carbohydrate contents were 8.84% and 9.08% respectively.2. SDS-PAGE analysis revealed that H. oblita PM contained at least 28 clear polypeptides and H. parallela PM 15 polypeptides; and in PMs of lepdopteran insects, Trichoplusia ni, Spodoptera exigua, Helicoverpa armigera and Mythimna separata, 19, 17, 16 and 9 clear polypeptides were found respectively. Compared with PMs of four lepidopteran larvae, H. oblita contained more polypeptides. Mucin was an important kind of PM proteins. Western blot analyses did not reveal a positive interaction between the protein of grub PMs and IIM-specific polyclonal antiserum from T. ni, and no mucin similar to that of lepidopteran insects were found in the two grubs. 3. The cDNA expression library from H. oblita larvae midgut was constructed successfully. The titer of primary and amplified library was 1.9×10~6 pfu/mL and 1.4×10~9pfu/ml respectively. The recombination rate was 99.97%, and inserted fragments ranged between 0.8-2.3kb with the average length of 1.6kb.4. By Western blot, the PM proteins of H. oblita produced positive interactions with two kinds of polyclonal antiserums from total PM proteins of H. armigera and T. ni on the nitrocellulose membrane. 122 positive clones were acquired from the midgut cDNA library of H. oblita by immunoscreening with the two kinds of antiserums, from which PM protein genes of Ho-Peritrophin1, Ho-Peritrophin2, Ho-Peritrophin3 and Ho-Peritrophin4 were found by sequence determination and multiple sequence alignment. Their GenBank accession numbers were FJ393548, FJ393549, FJ393550 and FJ573145, and their longest open reading frames (ORFs) coded for 729, 477, 528 and 324 amino acids respectively. The four genes contained 9, 6, 5 and 4 chitin binding domains (CBDs), 1 to 4 N-glycosylation sites and no mucin domains. Signal peptides were not found at N end of the protein. The trypsin and chymotrypsin cleavage sites were mostly located within the CBDs and protected by them, thus these PM proteins could not be degraded by the trypsin and chymotrypsin. Otherwise, it contained multiple cleavage sites of trypsin and chymotrypsin at the downstream of fifth CBDs in Ho-Peritrophin3. By construction of recombination plasmids, Ho-Peritrophin1, Ho-Peritrophin2 and Ho-Peritrophin3 were expressed 78, 51 and 57KD PM proteins in the Escherichia coli respectively. The chitin binding experiment showed that Ho-Peritrophin1 and Ho-Peritrophin3 had the chitin binding activities.5. Two whole length genes of serine protease of H. oblita, HoSP1 and HoSP2, were screened from the midgut cDNA library of H. oblita. GenBank accession number were FJ573146 and FJ573147 respectively, and the longest ORFs were 783bp and 786bp coding for 260 and 261 amino acids which contained three pairs of cysteine residues respectively. The similarities of HoSP1 and HoSP2 to CzSP3 were found to be highest, up to 52.47% and 51.52% respectively. The catalytic sites of HoSP1 were His80, Asp125, Ser215 and those of HoSP2 were His80, Asp125 and Ser216. By construction of recombination plasmids, HoSP1 and HoSP2 were expressed 26.7 and 27.1KD proteins in the Escherichia coli. Carboxylesterase gene of H. oblita, HoCL, was screened, and its GenBank accession number was FJ573148. The longest ORF was 1599bp coding for 532 amino acids that contained three cysteine residues. HoCL, belonging to B type carboxylesterase, had the catalytic sites of Ser207, Asp333 and His422, and its predicted preotein molecular weight was 59.5kDa.6. The effects of Helicoverpa armigera granulosis virus enhancin (Ha-En) and Calcoflour FB28 on the PMs of H. oblita and H. parallela were determined. The study showed that the two grub PMs were not degraded at different time and concentrations by Ha-En. After the two grubs were fed with 10uL 1% FB28, their PMs were observed at different time points. Observation revealed that the fore part of PMs was destroyed and by scanning electron microscope some evenly distributed holes appeared on the mid-and-hind gut PMs in 2.5 hours, the PMs were cracked completely 6 hours later and then restored to good state 12 hours later. Therefore, Calcoflour FB28 was able to destroy grub PMs temporarily which could be repaired by PM’s regeneration. This provided the theoretical basis to control Melolonthid larvae with Calcoflour and pathogens.更多还原