Abstract [Objectives]This study was conducted to synthesize sea anemone peptide Ap-GI and investigate its insecticidal activity. [Methods]The linear peptide Ap-GI was synthesized by solid phase peptide synthesis （SPPS）， and the linear peptide was subjected to the two-step oxidative folding， mass spectrometry identification and high performance liquid chromatography （HPLC） purification. Then， the MTT method and insect injection method were used to study its insecticidal activity. [Results]The synthesized sea anemone peptide had a purity of 95%. The test results of the MTT method showed that the peptide Ap-GI had the activity of inhibiting the growth of insect cells sf9 with the median effective dose of 0.7 nM; and the test results of the injection method on yellow mealworms showed that the peptide Ap-GI had high insecticidal activity， and the median lethal dose was 16.9 nM. [Conclusions]The sea anemone peptide Ap-GI from Aiptasia pallida has a good inhibitory effect on the growth of insect cells and high-efficiency insecticidal activity， which can lay a foundation for the development of new， safe and efficient peptide biological insecticides.
　　Key words Aiptasia pallida; Sea anemone peptide; Solid phase peptide synthesis; Oxidative folding; Insecticidal activity
　　In recent years， with the abuse of chemical pesticides， the problem of agricultural pollution has become more and more serious， which directly threatens agricultural ecological safety and sustainable development of agriculture， and then brings food safety problems to human health[1]. How to solve the agricultural environmental crisis and ecological security issues is imminent， so the demand for new， efficient and safe biological pesticides is very urgent[2-3]. At present， animals that prey on insects can usually secrete peptide toxins， which can poison and capture prey through specific ion channels or receptors of insects， and most of which have little toxicity or no toxic and side effects to mammals[4-5]. It has been reported in the literatures that sea anemones， snails， scorpions， spiders and predatory mites can secrete peptide toxins[6-9]. Therefore， the search for insecticidal active peptides from sea anemones and other marine toxic biological resources has become one of the hotspots in the research of efficient and safe insecticides.
　　Sea anemones， also known as Haijuhua， is a primitive marine metazoan belonging to Anthozoa of Cnidaria. More than 1 100 species of anemone has been recorded worldwide， belonging to about 400 genera in 50 families. China's sea anemone species account for about 1/10 of those in the world， and they are widely distributed in temperate tropical and tropical waters[10-11]. The tentacles of sea anemones have stinging cells that can secrete venom to prey on fish， shellfish， copepods， crustaceans and worms. The venom is rich in various peptide toxins and is an important marine drug resource[12-15]. Anthopleura xanthogrammica （Berkly） is used wholly as a traditional Chinese medicine used in coastal areas. It is salty and neutral in nature and flavor， and attributes to the liver， spleen and large intestine meridians. Chinese Materia Medica and Chinese Medicinal Animal History record that sea anemones have astringency-inducing， moisturizing and insecticidal effects， and are used in Chinese medicine for treating prolapse， hemorrhoids， and diarrhea[10]. Qingdao Handbook of Chinese Herbal Medicine records the treatment of enterobiasis： one piece of sea anemone is stuffed into the anus， once a night， for one week. Modern pharmacological studies have also shown that sea anemone peptide toxins have insecticidal， anti-tumor， antihypertensive， antibacterial， analgesic and neural inhibition effects[16-18]. 　　In this study， sea anemone peptide toxin Ap-GI was discovered by high-throughput transcriptomics from Aiptasia pallida. Its sequence is CIGCYYQVGNECKLDKFC， with 4 cysteines forming 2 disulfide bonds. The linear peptide Ap-GI was synthesized by the solid phase peptide synthesis （SPPS）， and the oxidized peptide Ap-GI with two disulfide bonds was obtained by the disulfide bond directional oxidation method （disulfide bonding mode： C1-C3， C2-C4 ）， and then purified by high performance liquid chromatography for mass spectrometry identification. The synthesized peptide Ap-GI was tested for its inhibitory effect on insect cells by the MTT method， and for its insecticidal effect on yellow mealworms by the insect injection method. The tests proved that the sea anemone peptide had high-efficiency insecticidal activity， which lays a foundation for the development of new， efficient and safe biological insecticides.
　　Materials and Methods
　　Experimental materials
　　Chromatographic grade trifluoroacetic acid （TFA） and chromatographic grade acetonitrile （Acetonitrile， ACN）， purchased from Thermo Fisher Scientific; Vydac analytical C18 column （5 μm， 4.6 mm×250 mm） and preparative C18 column （10 μm， 22 mm×250 mm）， purchased from Shanghai Chenqiao Biotechnology Co.， Ltd.; conventional molecular biology reagents such as plasmid extraction kits， purchased from Tiangen Biotech （Beijing） Co.， Ltd.
　　Experimental instruments
　　CEM automatic microwave peptide synthesizer （LibertyBlue， USA）; reversed-phase high performance liquid chromatograph （Agilent， USA）; liquid chromatograph-triple quadrupole tandem mass spectrometer （Shimadzu， Japan）; desktop freeze dryer （Biosafer ， China）; microplate reader （MR-96A， Shenzhen Mindray）.
　　Experimental methods
　　Linear peptide synthesis
　　Peptide Ap-GI （CIGCYYQVGNECKLDKFC） was synthesized by SPPS referring specifically to literatures[6]. Cysteines Cys 1 and Cys 3 were protected by the protecting group S-acetamidomethyl （Acm）. The resin peptide was cut with a cutting fluid （TFA∶TIPS∶DODT∶H2O=92.5%∶2.5%∶2.5%∶2.5%） at 42 ℃ for 30 min， then filtered and precipitated using 5 times volume of diethyl ether pre-cooled with dry ice， and centrifuged to recover the crude peptide product. Then the linear peptide was purified by preparative HPLC using Vydac C18 column. The HPLC was performed with mobile phase A （H2O， 0.1% TFA） and mobile phase B （ACN， 0.1% TFA） at a flow rate of 15 ml/min through 60 min of linear gradient elution， by which phase B was changed from 0% to 60%， and the detection was carried out at the wavelength of 214 nm. The purity of the linear peptide was more than 90%， and it was lyophilized and stored after being identified by mass spectrometry to be correct. 　　Two-step oxidative folding
　　The linear peptide was oxidized by the two-step oxidation method referring specifically to literatures[6]. In the first step of oxidation， the purified linear peptide was dissolved in 50% methanol solution at 10 mg/ml， then diluted to 1 mg/ml with acetic acid， and gradually added with methanol iodine solution （10 mg/ml） dropwise with stirring until the yellow color did not disappear when the first disulfide bond （Cys2 and Cys4） was formed. A small amount of the solution was taken out for mass spectrometry identification. In the second step of oxidation， a hydrochloric acid solution （50 mM HCl/50% methanol） was first added to remove the protective group Acm， followed by the addition of an iodine solution with a volume 10 times of the amount of the peptide and stirring for 1 h. Ascorbic acid was added until the color of the solution disappeared， and mass spectrometry was performed to identify the formation of the second disulfide bond （Cys1 and Cys3）.
　　MTT methods
　　The MTT test was carried out using insect cells sf9 referring to literatures[6]. Specifically， about 103 insect cells sf9 were inoculated into a 96-well plate. After 4 h of culture， the peptide Ap-GI with concentrations of 0.1， 0.25， 0.5， 0.75 and 1.0 nM were added in three replicates， with the blank experimental group as a negative control.
　　Insect injection method
　　Yellow mealworms weighing about 180 mg were selected for insect injection， the specific steps of which referred to literatures[6]. Specifically， the peptide Ap-GI was dissolved to 2.5， 5， 10， 15 and 20 nM with 0.7% NaCl， and 5 μl of the peptide Ap-GI was injected into the abdomens of yellow mealworms， with yellow mealworms free of liquid injection as a blank control and yellow mealworms injected with 5 μl of 0.7% NaCl solution as a negative control group.
　　Data processing
　　The data was all statistically processed by the software GraphPad Prism6. The differences between the control group and the experimental groups were analyzed by t test， in which * meant a significant difference （P<0.05）， and ** meant an extremely significant difference （P<0.01）.
　　Results
　　Peptide synthesis and oxidative folding
　　The theoretical molecular weight of the linear peptide Ap-GI was 2 086.458 Da. After cutting of the resin peptide， the linear peptide Ap-GI was subjected to two-step oxidative folding using an iodine solution， by which two disulfide bonds were respectively formed. The molecular weights were identified by mass spectrometry to 694.80 and 1 041.8， respectively， and the two bonds existed in the form of [M+H]2H+ and [M+H]3H+ （Fig. 1）， respectively. Therefore， mass spectrometry identified the molecular weight of the oxidized peptide Ap-GI to be 2 082.60， which was about 4 different from the molecular weight of the linear peptide， proving that the 4 H were successfully removed to form 2 disulfide bonds. 　　Separation and purification of the oxidized peptide
　　The oxidized peptide Ap-GI was separated and purified by preparative HPLC， and then analyzed by analytical HPLC after purification. The result is shown in Fig. 2. The elution time of the oxidized peptide Ap-GI was 9.605 min， and the purity was calculated based on the peak area to be 96.793%.
　　MTT method
　　The inhibitory effect of the sea anemone peptide Ap-GI on insect cells sf9 was tested by the MTT method. The test results （Fig. 3） showed the experimental groups had significant differences from the control group （0.7% NaCl solution）. The sea anemone peptide Ap-GI had a dose effect on insect cells， and the median effective dose was calculated to be 0.7 nM.
　　Insect injection method
　　The experimental results are shown in Fig. 4. The death rate of yellow mealworms in the blank control and the negative control group were both 0， indicating that it is feasible to evaluate the insecticidal effect of peptide Ap-GI by injection. The death rate of yellow mealworms increased with the increase of the dose of the sea anemone peptide Ap-GI， and was significantly different from the control group. The high-dose 20 nM peptide Ap-GI showed a death rate up to 56%， and the median lethal dose calculated by the software was 16.9 nM.
　　Discussion
　　In recent decades， the research on the activity of marine peptides has focused on major diseases such as analgesic， anti-cancer and anti-epileptic. For example， the conotoxin MVIIA has been approved by the FDA as an intrathecal analgesic for advanced cancer[19-22]. The peptide toxins secreted by sea anemones can be used to prey on shrimps and other arthropods， and it is speculated that their venom targets insects specifically with a poisoning effect[23]. In the early stage， our research group obtained the peptide Ap-GI gene sequence with 4 cysteines forming 2 disulfide bonds from A. pallida by the transcriptome sequencing technology.
　　In this study， the sea anemone peptide Ap-GI was synthesized by the SPPS method and the two-step oxidative folding method. The test results of the MTT method showed that the peptide Ap-GI had the activity of inhibiting the growth of insect cells sf9 with the median effective dose of 0.7 nM; and the test results of the injection method on yellow mealworms showed that the peptide Ap-GI had high insecticidal activity， and the median lethal dose was 16.9 nM. Therefore， the sea anemone peptide Ap-GI can act on the specific target of insects and has a highly effective insecticidal ability. 　　In recent years， the abuse of chemical pesticides has led to pesticide residues polluting the environment and pest resistance. Meanwhile， the natural enemies of pests have also been poisoned， thereby destroying the ecological balance. Insect virus insecticides have the advantages of being safe against natural enemies of pests， being friendly to environment and producing no drug tolerance， so they are widely used[2]. However， insect virus insecticides have significant shortcomings such as narrow insecticidal spectrum and slow insecticidal speed. With the development of molecular biology and genetic engineering， the use of exogenous insecticidal genes to modify insect viruses will become an effective way to overcome their shortcomings[24]. Therefore， the next step is to use the sea anemone peptide Ap-GI gene as a synergistic gene to construct a recombinant baculovirus， which can effectively solve the shortcomings of chemical pesticides and wild baculovirus insecticides， and lay a foundation for the development of new， efficient and safe recombinant insecticides.
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