Peritrophic Matrixes (PMs) From Two Spices Of Melolonthid Larvae And Identification Of The Structural Proteins

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.