Isolation, Cloning And Verification Of Cuticle-degrading Protease Genes From Metarhizium Anisopliae And Locust

The migratory locust is a widespread agriculture pest in China with a comparatively broad geographic distribution that includes both temperate and tropical climatic zones. It can cause signicant damage to pastures and crops and is a major threat for food safety, social stability and economic development. So we should strengthen on the research and pest control of agriculture pest.For a long time, the use of chemical insecticides for the control of insect pests was proven very effective at increasing agriculture and forestry productivities. However, the abuse of poisonous chemical substances directly led to three main environmental problems (residue, resistance and resurgence). To reduce environmental pollution, the usage of chemical pesticide should be reduced. Our immediate concern is to develop efficient, stable and safe bio-pesticides. Microbial pesticides have many advantages such as: safety, no-residue, hard to appear resistances and so on, and therefore are applied extensively. Entomopathogenic fungi are a big group of entomopathogenic microorganisms (bacteria, fungi and virus). Unlike entomopathogenic bacteria and viruses that invade insects though the alimentary canal, fungi can actively penetrate the host cuticle. Metarhizium anisopliae is an effective fungus for locust control. However, like other enfomopathogenic fungi, Metarhizium anisopliae needs a long time to kill pest insects, which impedes its wide application. To the invading fungus, insect cuticle represents the first barrier to penetration. In common with other entomopathogenic fungi the initial stage of infection involves penetration of the insect cuticle, so the success rate and speed of cuticle-penetration have a great effect on time of pest killing. Mechanical pressure plays a role in this process but of major importance is enzymatic degradation of cuticle within the immediate vicinity of infection hyphae. Cuticle-degrading proteases play a great role in the cuticle-degrading activity. Thus, this kind of proteases has been used as virus factors for developing high effective fungi pesticide by genetic engineering.Moreover, we found that besides entomopathogenic fungi, insects could also generate proteases to degrade their own cuticle. During the molting process, degradation and recycling of the old cuticle are brought about by enzymes present in the molting fluid filling the space between the old cuticle and the new cuticle. So, this study took entomopathogenic fungi Metarhizium anisopliae and Locusta migratoria as research targets, isolated and cloned cuticle-degrading protease genes and then verified their functions. This research could provide virus genes and basic work for developing high effective fungi pesticide by genetic engineering, which would speed the process of industrialization of fungi pesticides and promote their core competitive capability. Furthermore, cuticle-degrading proteases in the molting fluid of insects play an important role in molting. Silencing these proteases caused molt defects and finally led to death in locust. So this study is very important for the research on ecdysis of locust and for the development of novel, high effective and safe pesticides by taking these kinds of proteases as screening target. The main results are as follows:(1) PR1A gene was cloned from Metarhizium anisopliae strain CQMa102 using primers designed according conserved region of other Metarhizium anisopliae PR1A genes. The DNA, cDNA and and its deduced protein sequences were deposited in GenBank (accession numbers AY839935, EF627449 and ABR20899, respectively). The DNA sequences were 1571 bp. The 1319-bp cDNA sequence contained an 1173-bp single open reading frame encoded a protein of 390 amino acids and a 146-bp 3'untranslated region. Analysis of the amino acid sequences by computer using the NCBI database and BLAST revealed that PR1A belonged to subtilisin-like serine protease family. Amino acid sequences of PR1A from CQMa102 with other 16 subtilisin-like proteases from filamentous fungi, yeast, bacteria, cyanobacteria, vertebrate and invertebrate were used for the multiple sequence alignment, homology tree and phylogenetic tree. The results showed that PR1A had significant homology and close relationship with subtilisin-like proteases from filamentous fungi, and had the closest relationship to the protein from Metarhizium anisopliae var. acridum strain ARSEF 324 with 99% identity. Furthermore, the gene was cloned into and secretively expressed in Pichia pastoris KM71 under the control of the AOX1 promoter. Compared with controls, the product of PR1A recombinant (KM71/pPIC9k-PR1A) added a new band at about 26 kDa by SDS-PAGE and Weatern blot, and was good accordance with the nature peotein from CQMa102. The recombinant protein showed significant specific protease activity, total protease activity and cuticle-degrading activity. From these results, we could conclude that we cloned PR1A gene from Metarhizium anisopliae strain CQMa102 and its encoded protein had cuticle-degrading function.(2) Trypsin-like proteases and serine proteases from insects were used for multi-sequence alignment. Two pairs of degenerate primers were designed according to the conserved region. For RT-PCR cloning experiments, mRNA was prepared from locusts at the time when the molting fluid protease activity increased sharply. We cloned a novel molting fluid protease gene from Locusta migratoria manilensis,designated as Lm-TSP. The cDNA and its deduced protein sequences were deposited in GenBank (accession numbers EF081255 and ABN13876, respectively). The 1587-bp cDNA sequence contained a 735-bp single open reading frame encoded a protein of 244 amino acids and an 852-bp 3'untranslated region. Analysis of the amino acid sequences by computer using the NCBI database and BLAST revealed that Lm-TSP belonged to trypsin-like serine protease family. Amino acid sequences of Lm-TSP with other 14 trypsin-like serine proteases from insects, arachnid, mammals and bacteria were used for the multiple sequence alignment, homology tree and phylogenetic tree. The results showed that Lm-TSP had significant homology and close relationship with trypsin-like serine proteases from insects, and had the closest relationship to the protein from Tribolium castaneum with 82% identity. Furthermore, RNA interference (RNAi) studies showed that the silencing of Lm-TSP caused dramatic reduction in specific protease activity and cuticle degrading activity of the molting fluid, and caused cuticle degrading defects as a consequence, which led to molt defect from the fifth instar larvae (L5) to adult and molt defect from the fourth instar larvae (L4) to L5 and finally led to death. The results described herein suggested that Lm-TSP plays a critical role in controlling L. migratoria manilensis ecdysis(3) A relative mature larvae RNAi system of Locusta migratoria was established.