Cloning And Expression Of Gallerimycin From Spodoptera Litura

Gallerimycin is a gene coding for an antifungal peptide of Lepidoptera. Gallerimycin is synthesised in fat body and then secreted into the haemolymph after infection with microbes and kill the microbes rapidly. This antifungal peptide plays an important part in the immunity of Lepidoptera .It against the entomopathogenic fungus Metarhizium anisopliae, but not against yeast, gram-negative and gram-positive bacteria.Spodoptera litura is a kind of vegetable insect pests,the larvae of it eat some kinds of vegetables and the old larvae eat a lot ,so Spodoptera litura is harmful to many kinds of vegetables. The main method of control Spodoptera litura is still using chemical pesticides,but chemical pesticides pollute the environment , easy to make pests resist to it ,easy to remain in vegetables and harmful to humanbeing.With the development of environmental awareness of people, the requirement increase of life quality , people begin to pay attention to the harm of chemical pesticides. Therefore, the fungi pesticides just like Beauveria and Metarhizium will get more attention of people.A pair of degenerate primers of gallerimycin were designed by oligonucleotide high conservative sequence and we got the conservative sequence. Specificity primers for 3' RACE and 5' RACE were designed according to the conservation sequence. 3' RACE and 5' RACE were carried out using SMARTTM RACE cDNA Amplification Kit (Clontech). Through the real-time PCR detect the expression of gallerimycin and the RNAi of gallerimycin to characterize the function of gallerimycin. Study the function of gallerimycin will help to clarify the immunity mechanism of Spodoptera litura and this will be instructive to the study of new biological insecticides.1. Cloning of gallerimycin cDNA from Spodoptera lituraThe full-length cDNA sequence of gallerimycin was cloned from immuned Spodoptera litura. The GenBank accession number is HM747937. The full length of the cDNA was 380 bp, with a single open reading frame encoding 75 amino acid residues. Behind the stop codon, a polyadenylation site is detectable. The calculated molecular weight of the deduced protein was 8.1 kDa and the pI was 8.79 (http://expasy.org/tools/ protparam.html). The maturation protein contains a signal peptide which is coded by the front 20 amino acids. The primary structure of it has the common point of gallerimycin:with three disulfide linkages composed with six cysteine residues. 2. Induction of expression of the gallerimycin gene in the larval fat bodyIn naive larvae, the expression level of this gene was very low, but it can be strongly induced in larval fat bodies by Nomuraea rileyi strain CQNr01 after 24 h. This was demonstrated by quantitative real-time PCR (RT-qPCR). The induction of gallerimycin could already be observed at 8 hours post-infection and continued to increase slowly from 8 h–16 h after infection, after which it suddenly made an obvious increase and reached a maximum at 24 h. A remarkable decrease was observed starting at 48 h.3.The RNAi of gallerimycinThe interference dsDNA sequence (196 bp) was cloned from fat body total cDNA using the primer with T7 promoter. RNAs were synthesised in vitro using the MEGAscript? High Yield Transcription Kit (Ambion). In this experiment, knockdown of gallerimycin led to the quantity of gallerimycin in the fat body of the experimental group being much lower than in the control group injected with gfp ds-RNA. The ratio was 46%-73.3%.Knockdown of gallerimcyin also led to faster death of the larvae. These insects were more susceptible than pre-infected controls given a control gfp ds-RNA.These results show that gallerimycin is associated with the ability of Spodoptera litura to defend itself against fungal infection.