The nucleotide sequence reported in this paper has been submitted to GenBank with accession number: EU874846.
The impacts of classical insect hormones on the expression profiles of a new digestive trypsin-like protease (TLP) from the cotton bollworm, Helicoverpa armigera
Article first published online: 20 APR 2009
© 2009 The Authors. Journal compilation © 2009 The Royal Entomological Society
Insect Molecular Biology
Volume 18, Issue 4, pages 443–452, August 2009
How to Cite
Sui, Y.-P., Wang, J.-X. and Zhao, X.-F. (2009), The impacts of classical insect hormones on the expression profiles of a new digestive trypsin-like protease (TLP) from the cotton bollworm, Helicoverpa armigera. Insect Molecular Biology, 18: 443–452. doi: 10.1111/j.1365-2583.2009.00884.x
- Issue published online: 6 JUL 2009
- Article first published online: 20 APR 2009
- Helicoverpa armigera;
- trypsin-like protease;
- expression patterns;
Trypsin proteinases perform important roles in the protein digestion of an insect midgut. A 1042 bp full-length cDNA was cloned from Helicoverpa armigera. The gene encoded a 32 kDa protein, with a predicted isoelectric point of 5.7. The amino acid sequence of the protein had a trypsin-like serine protease domain, and the gene was named Ha-TLP. The expression of the gene was tissue-specific and the transcript of Ha-TLP existed only in the midgut and was not found in the head-thorax, integument, fat body and haemocytes from 5th instar larvae, with similar expression levels between those in feeding larvae and in molting larvae. In the midgut, the gene transcription level declined from 6th instar 72 h after the larvae entered the wandering stage, and disappeared from 6th instar at 96 h until the pupal stage. By immunohistochemistry, Ha-TLP was detected in the cytoplasm of the midgut epithelial cells of the 6th instar feeding stage worms. The expression of Ha-TLP could be up-regulated by a juvenile hormone (JH) analog methoprene and down-regulated by 20-hydroxyecdysone (20E). These facts indicate that Ha-TLP was involved in food digestion during larval growth and probably up-regulated by JH and suppressed by extra 20E in vivo.
Proteases are present in insects primarily as major digestive enzymes (Terra & Ferreira, 1994). Proteinases play important roles in protein digestion and absorption (Zhu et al., 2003), and much research has focused on midgut enzymes. In most species, the major endopeptidases (or proteinases) are trypsin and chymotrypsin (Chapman, 1998). Molting and metamorphosis of larvae are important physiological stages in insect lifecycles. They are controlled by two hormones: ecdysone (20-hydroxyecdysone, 20E) and juvenile hormone (JH) (Hiruma & Riddiford, 2001). Methoprene, an analog of JH, is often used to imitate the influence of JH in insects (Wu et al., 2006; Nishiura et al., 2007). In Schistocerca gregaria, it has been reported that the expression level of a Ser-protease-related protein increased after JH treatment and decreased after treatment with 20E (Chiou et al., 1998).
Trypsin is a member of the serine proteinase superfamily, which have a common catalytic triad including specific residues: serine, histidine and aspartic acid (Klein et al., 1996). Trypsin-like protease is a type of secretory endopeptidase (Kraut, 1977), and is highly specific to the positively charged side chains of lysine and arginine (Brown & Wold, 1973).
A trypsin-like serine protease is expressed at a high level in the larval gut in Drosophila melanogaster, suggesting its role in food digestion (Ahrens & Mahoney, 1998). In Lygus lineolaris, the main proteinases that exist in the gut and salivary glands are serine proteinases, especially trypsins (Zhu et al., 2003). Moreover, trypsins are only expressed in larval stages suggesting that proteinase activity occurs in protein digestion after food ingestion (Zhu et al., 2005). In hematophagous Diptera, such as Phlebotomus papatasi, Anopheles gambiae and Aedes aegypti, blood meal digestion is catabolysed mainly by trypsin and chymotrypsin serine proteases, produced by the midgut epithelial cells (Ramalho-Ortigão et al., 2003). In Lepeophtheirus salmonis, LsTryp1, a trypsin-like serine protease, functions as a digestive enzyme via secretion into the lumen of the salmon louse intestine (Kvamme et al., 2004). It has been intimated that trypsins from lepidopterans and dipterans come from the same ancestor; however, in subsequent gene duplications, additional copies have be found to be retained by some species (Wang et al., 1995). In Helicoverpa armigera, the main digestive enzymes are extracellular trypsin and chymotrypsin type serine proteinases, of which several cDNAs have been reported and some have been purified (Mazumdar-Leighton et al., 2000; Chougule et al., 2005; Telang et al., 2005).
As an important digestive enzyme, trypsin is not only one of the major components of digestive juices, but is also involved in the subsequent activation of other enzymes. As an example, in Manduca sexta, it has been reported that the activated trypsin could convert chymotrypsinogen into mature peptides (Taylor & Lee, 1997). In lepidopteran insects like Sesamia nonagrioides, chymotrypsins are associated with Cry1Ab activation, however the primary enzymes involved in Cry1Ab activation were trypsins. Although the purified trypsins were not involved in toxin degradation, trypsins and other proteases were implicated in the first step of protoxin processing and trypsins played the most important role (Díaz-Mendoza et al., 2007).
The cotton bollworm, Hercoverpa armigera, is a polyphagous pest, universally plaguing cotton, wheat and other arable crops (Huang et al., 2002); therefore, it is important to explore the functional genes involved in its physiology.
In our current study, a trypsin-like proteinase (Ha-TLP) cDNA, which deduced 299 amino acids, was obtained from H. armigera. The sequence of the protein had very low similarity with the trypsins previously being reported in the cotton bollworm. Semi-quantitative reverse trancription PCR (RT-PCR) was used to characterize the expression profiles of tissue-specificity and developmental stages. Localization in the midgut and activity of endogenous Ha-TLP were demonstrated. Hormonal influences on this gene, including 20E and methoprene, were also investigated.
Gene Cloning of Ha-TLP
Through random sequencing of the sequential expression from the midgut of H. armigera we obtained a fragment DNA (345 bp). Based on this fragment, the 5′ and 3′ ends of the cDNA were obtained through the SMART technique as described in Experimental procedures, below. By overlapping three fragments, a 1042 bp full-length cDNA was obtained, including a 31 bp 5′ untranslated region (UTR), a 900 bp open reading frame (ORF), and a 111 bp 3′ UTR. The 3′ UTR contained a 17 bp polyadenylation (A)+ tail that was 14 bp from AATAAA, the eukaryotic consensus polyadenylation signal. The ORF encoded a 299 amino acid protein with a 20 amino acid signal peptide. The calculated molecular weight of the protein was 32 kDa, with a predicted isoelectric point of 5.7 and a 29.3 kDa mature peptide (Ile 28-Thr 299) which was predicted by ProP 1.0 Server (http://www.cbs.dtu.dk/services/ProP/). The protein included one predicted N-glycosylation site, three putative protein kinase C phosphorylation sites, three presumed N-myristoylation sites, and four disulfide bonds (Cys 60-Cys 76, Cys 165-Cys 236, Cys 196- Cys 215, Cys 226-Cys 250), which were found by analysis with ExPASy (http://www.au.expasy.org/prosite/) (Fig. 1). The result of SMART analysis (http://www.smart.embl-heidelberg.de/) showed that the protein was probably inactive due to the substitution of two required catalytic sites: His 75 replaced by Ser 75; and Ser 230 replaced by Ile 230.
Identification of Ha-TLP
By analysis with ExPASy software (http://www.au.expasy.org/prosite/), Ha-TLP was found to contain a trypsin-like serine protease domain (Ile 28-Asn 274). Ha-TLP had the highest similarity with the azurocidin-like precursor protein from Trichoplusia ni (33% identity). Moreover, the Ha-TLP showed 30% identity to the serine protease from Lutzomyia longipalpis, 29% identity to the trypsin-like protease from Nilaparvata lugens and 28% identity to the trypsin from A. aegypti (Fig. 2).
Phylogenetic analysis utilizing MEGA 3.1 showed that the trypsin-like serine proteases from various animals could be divided into two main groups: Group 1 included two subgroups: 1) trypsin from the malaria mosquito (A. gambiae), trypsin from the fruit fly (D. melanogaster), pretrypsinogen II from the European honey bee (Apis mellifera), trypsinogen from the parasitoid wasp (Nasonia vitripennis), trypsinogen from the red jungle fowl (Gallus gallus), and trypsin-like protease from the brown plant hopper (N. lugens); and 2) trypsin from the yellow fever mosquito (A. aegypti) and serine protease from the sand fly (L. longipalpis). Group 2 contains azurocidin-like precursor protein from the cabbage looper (T. ni) and trypsin-like protease from the cotton bollworm (H. armigera) (Fig. 3).
Expression profiles of Ha-TLP during larval development
To understand which tissues Ha-TLP were distributed into, total RNA from different tissues at both 5th instar feeding larvae and molting larvae were analyzed with RT-PCR. The target band was only detected in the midgut, however, no signal was observed in the other tissues including the head-thorax, the integument, the fat body and the haemocytes. The result of T-test statistical analysis (P > 0.05) indicated that the expression levels of Ha-TLP transcript in the midgut between the 5th instar feeding and molting stages had no significant difference (Fig. 4A).
To investigate how Ha-TLP were expressed in the midgut at different developmental stages, total RNA isolated from the midgut from the 5th instar 12 h larvae (5th 12 h) to 1 d pupae was analyzed with RT-PCR. The results showed that the expression of the gene could be detected in the midgut from 5th 12 h larvae to 6th 48 h larvae, decreased from 6th 72 h worms, and were undetectable in the midgut from 6th 96 h larvae to 1 d pupae. It seemed that the expression level of 5th 36 h larvae was slightly higher than those of 5th 24 h and 5th 42 h according to the statistical analysis (P < 0.05). However the total expression levels of Ha-TLP from between the whole 5th instar larval stage and 6th instar feeding stage were very stable (Fig. 4B).
Immunoblotting and immunohistochemistry
To prepare the antibody, Ha-TLP-pGEX-4T-1 was expressed in Escherichia coli. A 58 kDa protein band was induced to express. It mainly existed in the precipitate after sonication in the form of inclusion body. After denaturalization and refolding, the inclusion body which was cut from the gel was electrophoresed in a dialysis tube and was subsequently injected into a rabbit (Fig. 5A). Through western blot, a 29 kDa band of mature peptide was detected in the midguts from several important physiological periods of the larvae, including the 5th instar feeding stage, molting stage and the 6th instar feeding stage (Fig. 5B). To determine the distribution of Ha-TLP into the cell, immunohistochemistry was demonstrated in the midguts from the 6th instar feeding stage larvae. The results showed that the fluorescence signals were observed mainly in the cytoplasm of midgut epithelial cells (Fig. 6).
Activity of endogenous Ha-TLP
To detect the activity of Ha-TLP, endogenous peptide was purified by its antibody and examined using western blot. A 29 kDa band was observed in the eluted product, which was consistent with the band detected in the midgut (Fig. 7A). The proteolytic activity of eluted protein was determined utilizing in situ hydrolysis and digestion to bovine serum albumin (BSA). Via digesting the gelatin, a single hydrolyzate band of the purified protein could be noticed on the gelatin-gel (Fig. 7B). Moreover, after incubated with BSA for 5 min, the eluted endogenous Ha-TLP began to hydrolyze the substrate BSA (Fig. 7C). Furthermore, antimicrobial experiments showed that no inhibition zone was observed from the all four bacteria and one fungus examined (data not shown).
Hormonal effects on Ha-TLP
The 6th instar 0 h larvae (6th 0 h, with white head capsule) were injected with 20E to examine the effect of ecdysone on Ha-TLP expression. The results revealed that the gene began to decline evidently compared to the control after the injection of 3 to 12 h (P < 0.05). From 18 h to 36 h after injection, however, the expression level of Ha-TLP recovered as compared to the control levels (Fig. 8A).
In order to identify the influence of methoprene on the expression of Ha-TLP, 6th instar 24 h larvae (6th instar feeding larvae) were injected with methoprene. In comparison to the control, the level of the gene started to ascend at 3 h after injection which continuously increased through the 6 h to 18 h after the challenge, and reached a high level at 24 h after injection (Fig. 8B).
We identified a 1042 bp full-length trypsin-like protease gene from H. armigera which encoded 299 amino acids, containing a trypsin-like serine protease domain. BLAST results suggested that Ha-TLP had high homology with azurocidin-like precursor protein from T. ni (33% identity), serine protease from L. longipalpis (30%), and trypsin-like protease from N. lugens (29%). Phylogenetic tree analysis showed that the Ha-TLP and azurocidin-like precursor protein from T. ni belonged to an independent group, suggesting that the two proteins were much closer in the evolution. The evolution of Ha-TLP was far from the other trypsin-like serine proteases, implying that they probably had different ancestors and Ha-TLP could acquire unique properties.
The expression of the Ha-TLP transcript was tissue-specific. The gene was detected only in the midgut, and not in the other tissues, such as the head-thorax, the integument, the fat body and the haemocytes. This observation was similar to the azurocidin-like precursor protein from T. ni. In T. ni, azurocidin-like precursor protein was found in the midgut, but not in the fat body and the haemocytes (Kang et al., 2002). In the H. armigera midgut, Ha-TLP was detectably expressed from the 5th instar feeding stage to the 6th instar feeding stage on both the transcription level by RT-PCR analysis and the translation level by western blot analysis. A subsequent decrease of Ha-TLP expression was noted from the wandering stage. These results implied that the main function of the protein was diet digestion since worms stopped ingesting food after the 6th instar 48 h. By immunohistochemistry in the midgut of the 6th instar feeding stage larva, Ha-TLP was detected in the cytoplasm of midgut epithelial cells which was in accordance with the trypsins reported in some hematophagous Diptera (Ramalho-Ortigão et al., 2003), suggesting that after the food ingested by worms went into the lumen of the midgut, Ha-TLP could then be synthesized in the cytoplasm and subsequently secreted into the digestive tract to be utilized to hydrolyze proteins into free amino acids for maintaining physiological functions. Activity experiments showed that endogenous Ha-TLP possessed the capability of hydrolyzing the protease substrates gelatin and BSA, implying the probable proteolytic activity of the Ha-TLP in vivo although some of required catalytic sites (His 75 and Ser 230) were lacked by theoretical analysis. This suggested that Ha-TLP could be synthesized as an inactive proenzyme and then could be cleaved during proteolysis to generate its active form. Furthermore, the gene was undetectable from the 6th instar 96 h to the pupal stage, interestingly implying that Ha-TLP was a larval-specific gene and was turned off before the worms entered its pupal stage, which was similar with the phenomenon observed in Hessian fly (Zhu et al., 2005).
Previous studies showed that H. armigera had a similar developmental schedule with M. sexta (Wang et al., 2007). According to the studies in M. sexta, JH titer in cotton bollworm was highest at the point of 0 h larva and then declined to basic lower level, while 20E titer was highest at the near point of head capsule slippage (HCS) and decreased into the lowest level at the point of 0 h larva (Riddiford et al., 2003). In Apanteles congregatus, larval ecdysis, emergence, and metamorphosis could be suppressed by methoprene which functioned as juvenile hormone (Beckage & Riddiford, 1982). After injection with 20E and methoprene, the expression of Ha-TLP transcript was suppressed by 20E and enhanced by methoprene, suggesting that Ha-TLP in vivo could be down-regulated by ecdysone and up-regulated by JH, which was in accordance with the trypsin from A. aegypti. In the midgut of A. aegypti mosquito after adult emergence, the early trypsin (AaET) could be increased by JH stimulation, and the transcription of a chymotrypsin-like protease gene (JHA15) could also be significantly activated by JH (Bian et al., 2008; Noriega & Wells, 1999).
In some herbivorous insect pests a novel, inhibition-resistant suite of midgut trypsin-like serine proteinases was produced to adapt to the proteinase inhibitors from their diets. It was also reported that the midgut serine proteinases could resist Bt δ-endotoxins (Mazumdar-Leighton et al., 2000). Ha-TLP was obviously an important digestive protease but no antimicrobial activity, however, it was interesting that the expression level of Ha-TLP during 5th instar molting stage did not decrease in response to the high titer of 20E, but on the contrary maintained its high level. It suggested that except for the role as a midgut digestive proteinase during the feeding period, Ha-TLP possibly played another role during the 5th instar larval molting stage, which required further future investigation.
Molecular cloning of Ha-TLP gene
Total RNA (5 µg), isolated from the midguts of three 5th instar larvae with Unizol reagent (Biostar, Shanghai, China), was used for reverse transcription of cDNA, according to SMART cDNA (Clontech, Palo Alto, CA, USA) method with primer oligo-anchorR (5′-GACCACGCGTATCGATGTCGACT16(A/C/G)-3′) and SmartF (5′-TACGGCTGCGAGAAGACGACAGAAGGG-3′). The product was utilized as the template of RT-PCR.
A sequential expression tag of Ha-TLP was obtained by a random sequence of the midgut cDNA. Based on the trypsin-like protease gene fragment, a specific primer TrypF1 (5′-ACATCAGAGTTCACCCCTC-3′) was designed to amplify the 3′ end of the gene with 3′anchor R primer (5′-GACCACGCGTATCGATGTCGAC- 3′) under the following conditions: one cycle (94 °C, 2 min); 35 cycles (94 °C, 30 s; 55 °C, 45 s; 72 °C, 45 s); one cycle (72 °C, 10 min). The 5′end of the gene was obtained using gene specific reverse primer TrypR1 (5′-TCGACTTCCCCAACCCGATAG-3′) and 5′ PCR primer (5′-TACGGCTGCGAGAAGACGACAGAA-3′) followed by the PCR conditions described above.
Bioinformatics analysis and phylogenetic tree analysis
DNA-protein translation and prediction of deduced protein were achieved with software from ExPASy (http://www.au.expasy.org/). Similarity analysis was performed by BLASTX (http://www.ncbi.nlm.nih.gov). The signal sequence and motifs were predicted by SMART (http://www.smart.embl-heidelberg.de/). Alignments were made with the ClustalW and GENEDOC computer software (http://www.nrbsc.org/downloads/gd322700.exe). After the sequences were aligned, a phylogenetic tree was produced by the neighbor-joining method in MEGA 3.1 (http://www.megasoftware.net/). Via the bootstrap method, statistical analysis was done using 1000 repetitions (Gorman et al., 2008; Kumar et al., 2004). The prediction of the mature peptide was performed with ProP 1.0 Server (http://www.cbs.dtu.dk/services/ProP/).
Unizol reagent (Biostar) was used to isolate total RNAs from various tissues including head-thorax, integument, midgut, fat body and haemocytes from 5th instar feeding and molting larvae, and the midguts at different development stages. Five µg of the total RNA was reverse transcribed into first strand cDNA (FirstStrand cDNA Synthesis Kit, MBI Fermentas, St. Leon-Rot, Germany), for using as the PCR template. Before the RT-PCR analysis the PCR templates were amplified for different number of cycles (18 to 30, sample every three cycles) to find out an appropriate condition to make sure that Ha-TLP and β-actin products were analyzed in the amplification phase of PCR. Then the 26 cycles was chosen to do the subsequent analysis. The gene specific primers TrypF2 (5′-TCGGAGTATCTCAAACATCAGAGTTCACCC-3′) and TrypR3 (5′-AGTCTTGGGTCTTGACAGCAATGCCCGTAA-3′) were utilized to do RT-PCR (416 bp) under the following conditions: one cycle (94 °C, 2 min); 26 cycles (94 °C, 30 s; 55 °C, 45 s; 72 °C, 45 s); one cycle (72 °C, 10 min). A pair of primers, actinF (5′-CCTGGTATTGCTGACCGTTGC-3′) and actinR (5′-CTGTTGGAAGGTGGAGAGGGAA-3′), were used to amplify a 150 bp fragment of β-actin as a quantitative control (Fabrick et al., 2003). The experiment was repeated three times using three independent samples. RT-PCR products were separated on 2% agarose gels, stained by ethidium bromide and photographed over UV light with Quantity One software (Bio-Rad, Hercules, CA). Then the intensities of absorbance of the bands were read from the pictures using Quantity One software. The intensity ratios of Ha-TLP to β-actin were calculated according to the above data. The averages of the ratios from three independent experiments were calculated and were utilized to construct the histograms (Du et al., 2007).
Recombinant expression of Ha-TLP
A 900 bp ORF, encoding the trypsin-like proteinase from H. armigera was inserted into expression vector pGEX-4T-1 and transformed into competent E. coli BL21 host cells. The target protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG, 0.5 mM) in Luria-Bertani/Ampicillin (100 µg/ml) medium, and expressed as inclusion bodies. The inclusion bodies were washed twice with buffer A (50 mM Tris-HCl, pH 8.0; 5 mM EDTA), subsequently washed twice with buffer B (50 mM Tris-HCl, pH 8.0; 5 mM EDTA; 2 M urea) and then dissolved in buffer C (0.1 M Tris-HCl, pH 8.0; 10 mM DTT; 8 M urea). The proteins were refolded in dialysis buffer (0.1 M Tris-HCl, pH 8.0; 5 mM EDTA; 5 mM cysteine) for 16 h at 4 °C (Kuhelj et al., 1995). After refolding, the samples were loaded into the gel with the SDS-PAGE and the target protein was cut from the gel. The cut samples were electrophoresed for 16 h at 4 °C in a dialysis tube. After this process, the samples in the tube were refolded in deionized water for 16 h at 4 °C and the resultant solution was injected into a rabbit.
Preparation of antiserum against Ha-TLP protein
About 200 µg of purified recombinant Ha-TLP protein in 1 ml TBS (10 mM This-HCl, pH 7.5; 150 mM NaCl) was mixed with 1 ml complete Freund's adjuvant and then injected hypodermically into the back of a rabbit. Three weeks later, the rabbit was injected with the recombinant protein (200 µg) mixed with incomplete Freund's adjuvant. After two further weeks, 500 µg protein was injected into the rabbit without Freund's adjuvant. The antiserum titer was determined by double immunodiffusion according to the method by Ouchterlony & Nilsson (1978) using 1% agar in 0.02 M Tris-HCl (pH 8.0), containing 0.15 M NaCl and 0.01% sodium azide.
After SDS-PAGE, the proteins were transferred onto a nitrocellulose membrane electrically and the bands were detected as described by Sambrook et al. (1989). The membrane was treated as the following procedures, at room temperature, and with shaking: blocked in blocking solution (2% nonfat dry milk in TBS) for 1 h, incubated in antiserum against Ha-TLP (1:100 in blocking solution) for 1 h, washed in TBST (0.1% Tween-20 in TBS) for 3 × 15 min, incubated in peroxidase-conjugated goat-anti-rabbit IgG (1:10 000 in blocking solution) for 1 h, followed by 3 × 15 min washes in TBST and 1 × 15 min wash in TBS. The target protein was then visualized by allowing peroxidase to react with peroxidase staining reaction mixture (1 ml 4-chloro-1-naphtholin methanol (6 mg/ml); 9 ml TBS; 6 µl H2O2) in the dark for five to 30 min (Zhao et al., 2005).
Midguts were removed and embedded into Frozen Section Medium (Richard-Allan Scientific, Kalamazoo, MI) and were immediately frozen in liquid nitrogen. Using MICROM HM550 cryostat microtome (Richard-Allan Scientific), 7 µm cryosections were cut and placed onto glass slides and then dried at room temperature overnight. After 24 h, the sections were fixed with cold acetone (−20 °C) for 10 min and were dried at room temperature for 1 h. The sections were washed three times for 5 min with phosphate buffered saline (PBS, 140 mM NaCl, 10 mM sodium phosphate, pH 7.4) after drying and were blocked with blocking buffer (2% bovine serum albumin in PBS) for 1 h at room temperature. The samples were subsequently incubated with anti-Ha-TLP (diluted 1:100 in blocking buffer) overnight at 4 °C. The sections were washed 6 × 10 min with PBS and incubated with goat anti-rabbit-ALEXA 488 (Molecular Probes, Eugene, OR, USA) which was diluted to 1:1000 with 2% BSA in PBS at room temperature for 1 h. After washing with PBS for 6 × 10 min, the sections were stained with 4′-6-Diamidino-2-phenylindole dihydrochloride (DAPI, 1 µg/ml in PBS, Adobe Systems, San Jose, CA, USA) for 10 min and again washed with PBS for 3 × 10 min. After the samples were mounted with glycerol in PBS (1:1), fluorescence of the section was detected with an Olympus BX51 fluorescence microscope (Olympus Optical Co., Tokyo, Japan). Negative controls were treated with the same procedure except that the pre-immune rabbit serum replaced the anti-Ha-TLP as the primary antibody.
Purification of endogenous Ha-TLP
Protein A Sepharose CL-4B medium (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was suspended in distilled water by gentle swirling and equilibrated in binding buffer (7.8 mM NaH2PO4, 12.2 mM Na2HPO4, pH 7.0). To purify antibody against Ha-TLP, the antiserum was added to Protein A Sepharose medium, washed three times by binding buffer, and eluted by elution buffer (93 mM citric acid, 7 mM sodium citrate, pH 3.0). Then the purified antibody was dialyzed in coupling buffer (0.1 M NaHCO3 containing 0.5 M NaCl, pH 8.3) for 20 h at 4 °C. The purified anti-Ha-TLP (0.3 mg) was then coupled with 0.3 ml of CNBr-activated Sepharose 4B (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) in coupling buffer. The mixture was rotated end-over-end in a stoppered vessel overnight at 4 °C. The next day the resin was washed five times with coupling buffer and blocked in 0.1 M Tris-HCl (pH 8.0) for 2 h at room temperature. Then the resin was washed with four cycles of alternating pH. Each cycle consisted of a wash with acetic acid buffer (0.1 M sodium acetate and 0.5 M NaCl, pH 4.0) followed by a wash with 0.1 M Tris-HCl, pH 8.0 containing 0.5 M NaCl. After that the resin was equilibrated with PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl and 2.7 mM KCl, pH 7.4). One milligram of midgut homogenate (in PBS) from 5th instar feeding larvae was then added to the medium and shaked in a stoppered vessel for 1 h at 4 °C. After washing six times with PBS, the endogenous Ha-TLP was eluted with acetic acid buffer. GeneQuant pro RNA/DNA Calculator Spectrophotometer (Amersham Biosciences Corp., Piscataway, NJ) was used to measure the concentration of the protein. The eluted product was detected by western blot.
Activity of endogenous enzyme
To detect the proteolytic activity of purified Ha-TLP, in situ hydrolysis (gelatin-SDS-PAGE) was performed in 50 mM Tris-HCl (pH 8.0) using the methods described previously (Zhao et al., 2005). Besides, the proteolytic activity of purified Ha-TLP (1.5 µg) to 40 µg of BSA (Takara Shuzo Co. Ltd., Kyoto, Japan) was also tested in 50 mM Tris-HCl buffer (pH 8.0) at 37 °C for different durations. Cylinder-Plate Method was used to detect the antimicrobial activity of Ha-TLP in order to do the inhibition zone assay (Tian et al., 2008). Two Gram-negative bacteria (E. coli and Klebsiella oxytoca), two Gram-positive bacteria (Staphylococcus aureus and Bacillus thringiensis) and one fungus (Candida Albicans) were examined. Purified Ha-TLP (20 µg) was added into an Oxford Cup which stood onto a plate containing 5 ml PB medium (LB medium without yeast extract), with the solvent (no Ha-TLP) in another cup as a negative control. And the mixture of ampicillin (10 µg) and kanamycin (10 µg) was added into a cup as a positive control. Then the plates with the Oxford Cups were placed for 16 h at 37 °C.
Hormonal regulation of Ha-TLP gene
20-hydroxyecdysone (20E; Sigma, St, Louis, MO) and methoprene (Dr. Ehrenstorfer, Augsburg, Germany) were dissolved in dimethyl sulphoxide (DMSO) at the storage concentration of 10 mg/ml. The storage solution of 20E and methoprene were respectively diluted into 0.1 mg/ml with phosphate buffered saline (PBS, 10 mM Na2HPO4; 1.8 mM KH2PO4; 140 mM NaCl and 2.7 mM KCl, pH 7.4) prior to injecting the worms. The 6th instar 0 h larvae were injected with 20E (500 ng/larva), while the methoprene was injected into 6th instar 24 h larvae (500 ng/larva). All untreated controls were only injected by equivalent amounts of carrier. The total RNA of midgut was extracted from the injected worms at different developmental periods. The differences between the control and the challenged were compared by RT-PCR analysis. The experiment was repeated three times using three independent samples.
This work was supported by grants from the National Natural Science Foundation of China (No: 30670265, 30710103901) and the National Basic Research Program of China (2006CB102001).
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