Cloning and Expression of Gallerimycin from Spodoptera Litura
|Keywords||Antifungal Peptide gallerimycin RNAi Spodoptera litura RT-qPCR|
Gallerimycin genes are lepidopteran insects in an anti-fungal peptides, which can be a variety of bacteria, fungi and even metalloproteinase inducer, was synthesized in the fat body, and then secreted into the hemolymph works. Gallerimycin mainly filamentous fungi have significant activity and no resistance to the bacteria and yeast play an important role in insect humoral immunity. The Spodoptera litura Lepidoptera vegetables pests, larvae not only mixed diet, the mature larva formation gluttony, has a variety of vegetables hazards. Against the larvae of Spodoptera litura chemical method is still the use of chemical pesticides. Chemical pesticides as the major hazards of pesticides pollute the environment; easy pest resistance; easy to harm human health residues on vegetables. With the improvement of people's awareness of environmental protection, as well as the improvement of the life quality requirements, the hazards of chemical pesticides appear gradually attracted attention. Therefore the Leydig Nomura Jundeng entomogenous fungi and biocontrol applications get more and more attention. This study than protein sequences in GenBank gallerimycin identify gallerimycin conserved sequence of amino acids, design degenerate primers PCR amplified conserved sequence; to gallerimycin conserved region of the gene sequence as a basis, specific primers were designed using 3 'RACE and 5'RACE amplified the Spodoptera litura aging the larvae gallerimycin gene length cDNA sequence; on the basis obtain gallerimycin gene full-length cDNA, gene function by expression profiling and RNA interference gallerimycin. Spodoptera litura aging the larvae gallerimycin gene function studies help to clarify the molecular biology of the of Spodoptera litura gallerimycin gene in the humoral immune mechanisms, from the molecular level has important guiding significance for the research and development of new bio-pesticides. 1. Of Spodoptera litura gallerimycin gene cloning to Leydig Nomura infect bacteria CQNr01, Spodoptera litura mature larva of cDNA as a template, conserved sequences designed simple according gallerimycin and primer, a 86 bp gene fragment obtained by PCR. By NCBI BLAST biological information analysis, the results showed that the gene sequence of the fragment and Spodoptera frugiperda (Spdoptera frugiperda) with 91% similarity. Design gene-specific primers based on the sequence in the the Spodoptera litura gallerimycin gene conserved region, amplified with 3'RACE and 5'RACE get gallerimycin gene full-length cDNA sequence, GenBank accession number HM747937. The Spodoptera litura gallerimycin cloned cDNA sequence length 380bp, open reading frame 225bp, which encodes 75 amino acids, respectively, long 22bp and 133bp of the 5 'end and 3' end of the non-coding region. 8.1KDa of the gene encoding the protein isoelectric point of 8.79, having a signal peptide composed of 20 amino acid residues. Phylogenetic analysis and amino acid sequence comparison showed that the product of the gene encoding belongs gallerimycin. An the structure having gallerimycin the common features: having six cysteine ??residues form three disulfide bonds. 2. Of Spodoptera litura gallerimycin gene expression profiling mature larvae of Spodoptera litura Leydig Nomura bacteria infection in different periods, gallerimycin gene expression measured by RT-qPCR. The results showed that the gene expression gallerimycin a linear relationship between time and infestation. gallerimycin expression amount of over infection time gradually increased to 24 hours to reach the maximum, and then gradually reduce. RNA interference analysis of 3.gallerimycin genes according gallerimycin gene coding region primer design interference by Ambion MEGAscript? High Yield Transcription Kit kit to get there T7 promoter gallerimycin the dsRNA. Using a micro syringe dsRNA injection into the abdominal cavity of the mature larvae of Spodoptera litura. By real-time PCR analysis, interfere with efficiency of 73%, found that to Spodoptera litura gallerimycin gene interference mature larva antifungal decreased ability biometric. This study shows that gallerimycin genes and the Spodoptera litura antifungal ability to have a direct relationship and play an important role.