Molecular Identification of Sunn Pest some Vital Genes and Analysis Effect of Different hosts on Polygalacturonase Expression  

Amiri Azam 1 , Bandani Ali Reza 1 , Allahyari Morteza 2
1. Plant Protection Department, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.
2. Fars Research Center for Agriculture and Natural Resources, Iran
Author    Correspondence author
Molecular Entomology, 2015, Vol. 6, No. 4   doi: 10.5376/me.2015.06.0004
Received: 11 May, 2015    Accepted: 15 Jun., 2015    Published: 21 Jul., 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Amiri Azam, Bandani Ali Reza and Allahyari Morteza, 2015, Molecular Identification of Sunn Pest some Vital Genes and Analysis Effect of Different hosts on Polygalacturonase Expression, Molecular Entomology, Vol.6, No.4 1-9 (doi: 10.5376/me.2015.06.0004)

Abstract

Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae) is a serious pest of cereals which causes severe quantitative and qualitative damage to crops. Since there is less molecular data about this important pest, Sunn pest molecular gene identification can be targets for developing new tools in pest control. This study described for the first time in the world, molecular identification of Sunn pest V-ATPase subunit E, cytochrome P450 and polygalacturonase transcripts, which might serve as possible targets for integrated pest management. Also the tissue specific relative gene expression levels of polygalacturonase was determined under different host kernels (wheat, barley, rye and triticale) feeding conditions. Result showed that host plant quality can significantly influence the Sunn pest polygalacturonase expression and wheat fed larvae had higher mRNA abundant.

Keywords
V-ATPase; cytochrome P450; polygalacturonase; Eurygaster; gene expression

1 Introduction
E. integriceps, commonly known as Sunn pest, belongs to the order Hemiptera and family Scutelleridae. Sunn pest is a serious pest of cereals causes severe quantitative and qualitative (destruction of gluten protein) damage to crops (sometime up to 100%) by feeding on leaves, stems and grains (Konarev et al., 2011; Amiri and Bandani, 2013; Dolgikh et al., 2014; Yandamuri et al., 2014).

Pesticides as a major approach of insect pest control worldwide are associated with significant hazards to the environment and human health. As a result, there is a need to develop more benign and ecologically safe alternative methods for pest control. Since there is less molecular data about this important pest, Sunn pest molecular gene identification can be targets for developing new tools in integrated pest management.

Between different genes, this study focused on Sunn pest V-ATPase, cytochrome P450, and polygalacturonase gene identification because of their importance.

Vacuolar-type ATPases (V-ATPase) are multisubunit enzymes, which are composed of V0 (membrane bound subunits) and V1 (peripherally associated subunits) complex. The V1 complex that catalyzes the hydrolysis of ATP includes eight different subunits (A, B, C, D, E, F, G and H) and V0 that forms the proton conducting pore contains subunits with the a, c, c',c″, and d isoforms (Jefferies and Michael, 2007; Ma et al., 2011).

V-ATPases are multisubunit and multifunctional enzymes in eukaryotic cells that are ubiquitously found in the membranes of intracellular compartments, such as lysosomes, vacuoles, secretary granules, the trans-Golgi network and coated vesicles or are also found in the plasma membranes of cells.

V-ATPases are involved in the pH homeostasis and membrane secondary active transport processes energization in cell plasma membranes; while they pumps H+ (coupled with ATP hydrolysis) from the cytoplasm into the lumen of the organelles likelysosomes, secretory vesicles, and endosomes to acidify them in every eukaryotic cell or even can acidify whole insect’s midgut. Acidification is required for multiple cellular processes and modifying fluid and electrolyte transport (Jefferies et al., 2008; Wieczorek et al., 2009; Yang et al., 2009; Baumann and Walz, 2012; Holliday, 2014; Lu et al., 2014; Sahara et al., 2014).

Cytochrome P450 was another gene selected in this research. Cytochrome P450 monooxygenases are present in almost all organisms such as bacteria, fungi, plants and animals (Feyereisen, 2006). They are superfamily of heme-containing ubiquitous enzymes that play vital roles in herbivorous insect biology and physiology. Cytochrome P450s are important enzymes because their fundamental physiological functions in the hormone, pheromones, fatty acids, and steroids regulation such as juvenile hormone (JH) or 20- hydroxyecdysone (Feyereisen, 2012), biosynthesis or inactivation of endogenous or xenobiotic compounds (pesticides and plant phytochemicals) by catalyzing oxidation reactions (Feyereisen, 2005; Nielsen and Moller, 2005; Scott, 2008; Mizutani and Ohta, 2010; Schuler, 2011), insect growth, development, reproduction and evolutionary adaptation to environments (Hu et al., 2014; Chen et al., 2015; Yu et al., 2015). Insect P450s can induce insecticide resistance in pest population by catalysis of many reactions (Hardwick, 2008; Zhou et al., 2010).

According diverse functions, there are multiple P450 forms. More than 1000 cytochrome P450 genes have been reported in insects and new sequencing technologies facilitate rapidly increasing of this number (Nelson, 2009; Ai et al., 2011).

Plant innate immunity is based on an ancient system of molecules that defend the host against infection. Pectin is a complex carbohydrate and one of major component of plant physiological barrier against pathogens and insects. Polygalacturonases (PGs; EC 3.2.1.15) are produced by bacteria, fungi, nematodes and insects (Jaubert et al., 2002; De Lorenzo et al., 2003; Allen and Mertens, 2008; Prabhu et al., 2012). Insect PGs are hydrolytic enzymes degrade the α(1-4) linkages between the D-galacturonic acid residues of homogalacturonan, the main component of pectin and can play important role in plant-pest interactions (Kalunke et al., 2015). Fungi and bacteria polygalact-uronase have been studied extensively, but there are less PGs reports from several species of insects belonging to different taxa (Garcia-Maceira et al., 2001; Gotesson et al., 2002; D’Ovidio et al., 2004; Shackel et al., 2005; Frati et al., 2006; Allen and Mertens, 2008; Prabhu et al., 2012).

Therefore, the objectives of this research were to: 1) Molecular identify V-ATPase, cytochrome P450, and polygalacturonase genes in the Sunn pest genome for the first time; and 2) to study alteration in expression of salivary glands and gut polygalacturonase in response to different host plants.

2 Results
2.1 Identification and Homologous alignment
By searching in database, the E. integriceps V-ATPase subunit E and P450 protein homologues were found in various insects. The other protein or homologue sequences used in this study were downloaded from GenBank database. Protein alignment of V-ATPase subunit E and P450 demonstrated a high homology to that of Riptortus pedestris (Hemiptera: Alydidae) (97% identity) and Dendroctonus rhizophagus (Coleoptera: Curculionidae) (81% identity) respectively (Figure 1 and 2).

 
Figure 1 The alignment of amino acid sequences of V-ATPase subunit E from E. integriceps with the sequences of other insect V-ATPase subunit E: Plutella xylostella (Lepidoptera: Plutellidae), Culex quinquefasciatus (Diptera: Culicidae), Bombyx mori (Lepidoptera: Bombycidae), Danaus plexippus (Lepidoptera: Nymphalidae), Acyrthosiphon pisum (Hemiptera: Aphididae), Zootermopsis nevadensis (Isoptera: Termopsidae), Riptortus pedestris (Hemiptera: Alydidae), Cerapachys biroi (Hymenoptera: Formicidae), Papilio xuthus (Lepidoptera: Papilionidae), Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), Maconellicoccus hirsutus (Hemiptera: Coccoidea), Coptotermes formosanus (Isoptera: Rhinotermitidae), Nilaparvata lugens (Hemiptera: Delphacidae), Anopheles darling (Hemiptera: Culicidae)


 
Figure 2 The alignment of amino acid sequences of cytochrome P450 from E. integriceps with the sequences of other insect cytochrome P450:  Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), Dendroctonus ponderosae (Coleoptera: Curculionidae), Tribolium castaneum (Coleoptera: Tenebrionidae), Aedes aegypti (Diptera: Culicidae) 


A 238 bp partial cDNA encoded a protein was denoted as E. integriceps V-ATPase subunit E, based on the BLASTx analysis indicating homology with V-ATPase subunit E Protein from Riptortus pedestris (Hemiptera: Alydidae) (97% identity), Culex quinquefasciatus (Diptera: Culicidae), Zootermopsis nevadensis (Isoptera: Termopsidae) (95%), Bombyx mori (Lepidoptera: Bombycidae) (94%) and Danaus plexippus (Lepidoptera: Nymphalidae) (94%). This cDNA sequence has been deposited in GenBank under accession number KR045860. The next 154 bp partial cDNA encoded a protein that it’s BLASTx analysis revealed homology with cytochrome P450 from Dendroctonus rhizophagus (Coleoptera: Curculionidae) (81% identity), D. valens (Coleoptera: Curculionidae) (79%), Aedes albopictus (Diptera: Culicidae) (43%), and Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) (43%). Therefore, it was referred to the protein as E. integriceps cytochrome P450. This cDNA sequence has been deposited in GenBank under accession number KR045862. High level of conservation of the amino acid sequence among different insect V-ATPase subunit E proteins and also cytochrome P450 proteins indicates that these proteins are highly conserved during the evolution of insects.

Also the last partial cDNA encoded a 251 bp sequence that homology search revealed 100% identity with Lygus lineolaris (Hemiptera: Miridae) polygalacturonase PG1 mRNA, so was denoted as E. integriceps polygal- acturonase (PG). The PG sequence was deposited to GenBank with accession number KP295959.

2.2 Phylogenetic analysis
To understand the relationship between insect V-ATPase subunit E and cytochrome P450 and Sunn pest corresponding genes, phylogenetic analysis were performed using cDNA sequences available in GenBank. Neighbor-joining trees were constructed using amino acid sequences and a poisson-corrected distance with bootstrap test of 1000 replications. Phylogenetic analysis of proteins from insect species in the different orders such as Hemiptera, Diptera, Hymenoptera, Coleoptera, and Lepidoptera showed that V-ATPase subunit E and cytochrome P450 from the Sunn pest E. integriceps was closely related to R. pedestris (Hemiptera: Alydidae) and A. darlingi (Diptera: Culicidae) (Figure 3 and 4). The molecular evolution of V-ATPase genes showed a high level of conservation and a correlation with the taxonomy of the selected species.

 
Figure 3 Phylogenetic tree of amino acid sequences of V-ATPase subunit E of Sunn pest and other insects. Neighbour- joining method with genetic distance calculated. The numbers above the branches indicate the percentages of times that the species are grouped together in the bootstrap trees. The scale bar indicates the number of substitutions per site for a unit branch length 


 
Figure 4 Phylogenetic tree of amino acid sequences of cytochrome P450 of Sunn pest and other insects. Neighbour- joining method with genetic distance calculated. The numbers above the branches indicate the percentages of times that the species are grouped together in the bootstrap trees. The scale bar indicates the number of substitutions per site for a unit branch length 


2.3 Sunn pest response to different host plants
This experiment was done to investigate the effects of different hosts on the transcript level of PG gene. Wheat is the preferred host for the Sunn pest. However, the insect can also survive on barley, rye and triticale. To gain insight on the impact of different host types on the expression of PG gene, the expression patterns of the gene in the Sunn pest larvae feeding on wheat, barley, rye and triticale kernels were examined. To determine tissue and host dependent expression of PG, total RNA samples from the salivary glands and midgut were analyzed by real-time quantitative PCR. The results showed that this gene was differentially expressed in Sunn pest tissues and in response to different host plants kernels. In other words, the expression of PG gene was affected by host genus. This polygalacturonase was expressed only in the gut and there wasn’t any detectable PG transcript in the salivary glands (Figure 5A). For the Sunn pest larvae fed with barley, rye and triticale, gut PG mRNA expression decreased significantly comparing with that of the wheat fed larvae. Results showed that barley, rye and triticale caused 39%, 100% and 100% reduction in PG expression respectively (p<0.05) (Figure 5B). In conclusion, all these results indicated that the application of all-plant-based diet could decrease gene expression when compared with wheat.

 
Figure 5 Relative expression of polygalacturonase in fifth instar larvae of E. integriceps. A: in salivary glands and gut of wheat fed Sunn pest. B: in gut of insects feeding on either barley (B), rye (R) or triticale (T) in comparison with transcript levels in insects feeding on wheat (W) kernels.
Ct values were first normalized to the endogenous control gene 18S ribosomal RNA gene, followed by normalization to the control without treatment using the 2-∆∆Ct method. Each kinetic point was performed in triplicate on 5 pooled larvae. Asterisks indicate significant difference (P<0.05) according to the iteration test (Rest 2009 Software). The values represent averages with vertical bars indicating SE 


3 Discussion
E. integriceps causes serious economic losses to cereals especially to wheat and barley in wide areas of the world almost every year. Despite the importance of this key pest, almost no functional genomics data exist for the Sunn pest. In this study, for the first time cDNAs encoding V-ATPase subunit E, cytochrome P450 and polygalacturonase were isolated and identified from the Sunn pest, E. integriceps.

V-ATPases are multifunctional and important genes ofdifferent insect species and have potential for pest management (Baumann and Walz, 2012; Holliday, 2014; Lu et al., 2014; Sahara et al., 2014). Midgut lumen acidification by V-ATPases was reported in different insects such as Bombyx mori (Wieczorek et al., 2000; Lu et al., 2013), Aedes aegypti (Weng et al., 2003) and the American visceral leishmaniasis vector Lutzomyia longipalpis (Ramalho-Ortigão et al., 2007).

Other than acidifying, in insects, V-ATPases function as midgut, salivary glands and labial glands epithelia cation transporter (Wieczorek et al., 2009; Thakur et al., 2014). Also Huang et al. (2006) reported V-ATPases seem to associate with gregarine infection of the mosquito Aedes albopictus. As well as Lu et al. (2013) observed V-ATPases play important role in the defense response against Bombyx mori Nucleopolyhe-drovirus.

In insects, cytochrome P450s enzymes are very important, large and diverse group of enzymes that play vital roles in many different physiological processes (Hu et al., 2014; Chen et al., 2015; Yu et al., 2015). Therefore, identification of Sunn pest P450 could be useful in pest management strategies. To date, however, no P450 gene from E. integriceps has been reported. Our research is the first report of this vital gene in the Sunn pest.

There are many convincing evidence for P450s in several insect species. For example identification of Cytochrome P450 Genes have been reported in Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) (Chen et al., 2015); diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)(Hu et al., 2014; Yu et al., 2015); the mosquito, Aedes aegypti (Diptera: Culicidae) (Elgarj and Wajidi, 2013); Locusta migratoria (Orthoptera: Acrididae)(Guo et al., 2012), Asian corn borer, Ostrinia furnacalis (Lepidoptera: Crambidae) (Zhang et al., 2011), and the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae) (Xu et al., 2009).

Molecular characterization of polygalacturonases from agricultural pests will be helpful in formulating and selecting pest resistant crops. Phytophagous insect’s salivary glands PGs are considered as a main cause of plant damage (Boyd et al., 2002; Frati et al., 2006).

Following finding Sunn pest polygalacturonase gene (PG), we investigated whether and how different host plants affect the transcription levels of this gene. Host plant quality can significantly influence gene expression and the insect growth (Oppert et al. 2010; Vogelweith et al., 2011; Wang et al., 2013; Spit et al., 2014; Wang et al., 2015). Likewise in this study results clearly indicated that host plants quality can affect gene expression. So transcript level of PG was most abundant in the wheat fed Sunn pest larvae in compare to other hosts. Such results were observed in other insects. Phytophagous insects may exhibit differential expression patterns of various genes in response to plant defense (Bown et al., 1997).

Frati et al. (2006) reported PG activity in the salivary glands and the gut of different species of mirid bugs [Lygus rugulipennis Popp., L. pratensis (L.), Orthops kalmi (L.), Adelphocoris lineolatus (Goeze) and Closterotomus norwegicus (Gmelin)]. They also examined effect of different plant source polygalacturonase- inhibiting proteins on inhibition of mirid PGs. PGs of all the mirid bugs were inhibited by PGIPs from Phaseolus vulgaris.

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall leucine-rich repeat glycoproteins that upregulated and counteract insect and patogen polygalacturonases (Frati et al., 2006; Janni et al., 2008; Prabhu et al., 2012; Tamburino et al., 2012; Kalunke et al., 2015). They can bind to the plant cell and play an important role in plant defense with inhibiting the pectin-depolymerizing activity of PGs. Sevaral studies reported plant cell wall PGIPs have been inhibited the PGs of insects and pathogens (Al-Obaidi et al., 2010; Prabhu et al., 2012; Bashi et al., 2013; Ferrari et al., 2013; Kalunke et al., 2015).

No plant species or mutants totally lacking PGIP activity has been characterized so far (Kalunke et al., 2015). Di Giovanni et al., (2008) genome analysis showed PGIPs exist as a single gene in diploid wheat species. However, other plant species have more PGIPs, such as two in Arabidopsis thaliana (Ferrari et al., 2003), and sixteen in Brassica napus (Hegedus et al., 2008). Al-Obaidi et al., (2010) reprted PGIP from barley.

We observed PG expression in the Sunn pest feed on rye and triticale was zero. Allen and Mertens (2008) reported isolation of three unique cDNAs encoding putative polygalacturonase from the tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae). They studied differentially expression of Lygus PG genes in all feeding stages and observed a zero expression reading in at least one life stage of this hemipteran too. Also, Walker and Allen (2010) reported cotton-fed bugs had consistently lower PG transcript levels than those reared on artificial diet.

To conclude, we have for the first time identified three different genes encoding V-ATPase, cytochrome P450, and polygalacturonase from an important agriculture pest E. integriceps and analyzed polygalacturonase expression in different tissues of the Sunn pest reared on various hosts. Variation in host plant, strongly affects the level of polygalacturonase expression.

4 Materials and Methods
4.1 Insect rearing
Sunn pest adults were collected from wheat field during spring 2014. The insects were maintained on soaked wheat kernels and water in the laboratory as described by Allahyari et al. (2010). To determine the effect of different host species on gene expression, 100 eggs for each replicate (three replicates were considered for each treatment) in each treatment were reared separately on various hosts including wheat, barley, rye and triticale kernels. The insects were reared to fifth-instar when used for sample collection. In order to establish the developmental stage and to synchronize the treated fifth instar larvae, the molt was observed every day and the newly emerged larvae were collected every 24 h and placed on fresh soaked kernels and one day after ecdysis, fifth instar larvae were collected for gene expression experiment.

4.2 Tissue sampling
To obtain selected tissue samples (midgut and salivary glands), E. integriceps fifth instar larvae were dissected in sterile water under a stereoscopic microscope. Each tissue (midgut and salivary glands) was collected and pooled from individual insects and were transferred into a microtube containing 0.5 mL TRI reagent (~ 30 mg wet tissue) (Sigma, Aldrich).

4.3 RNA extraction and cDNA cloning
Samples were processed for total RNA extraction by TRI reagent using instruction provided by the manufacturer. To remove potential DNA contamination, total RNA samples were treated with DNase I. First strand cDNA was prepared with RNA (DNA free) and the oligo-dT primer using AccuPower® RocketScript™ Cycle RT PreMix cDNA synthesis kit (Bioneer, Korea). All cDNA concentrations were determined using a Nanodrop 1000 Thermo Scientific, diluted (ten-fold) and used for the next PCR reactions consisting of preheating 95°C for four min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 30 s, and a final extension step of 72°C for seven min. Each 25µL PCR reaction contained 1µL of cDNA template, 2.5 µL of 10x PCR buffer, 1.5 µL of MgCl2 (25 mmol/µL), 2µL of dNTP (2 mmol/µL each), 1.5 µL of forward and 1.5 µL of reverse primers (10 pmol/µL) (E primers for V-ATPase, P450 primers for cytochrome P450, PG primers for polygalacturonase, Table 1), 0.2 µL of Taq polymerase (5 U/µL), and 14.8 µL of ddH2O. E and P450 primers were designed from two highly conserved regions of other insect genes and PG primers were obtained from the tarnished plant bug,Lygus lineolaris (Walker and Allen, 2010) (Table 1). Six microlitter of the PCR product was analyzed on a 1% TAE-ethidium bromide gel and the target band was isolated from the gel and recovered and purified by using the AccuPrep Gel Extraction Kit (Bioneer, Korea), cloned into the pTG19-T PCR cloning vector (Vivantis) and transformed into Escherichia Coli DH5α competent cells. Colonies with inserts were screened under standard ampicillin conditions. Recombinant plasmids were sequenced from both directions and hereby, gene sequences were confirmed by sequencing. The sequencing reaction was performed commercially.


Table 1 The primer sequences were used in the study


4.4 Sequences and phylogenetic analysis
The sequences of the V-ATPase, cytochrome P450, and polygalacturonase cDNA were confirmed by a homology search of other corresponding gene sequences known within the Blast program available on the GenBank database of National Center for Biotechnology Information (NCBI) website (http:// www.ncbi.nlm.nih.gov/BLAST/). The amino acid sequences of corresponding gene homologs from different organisms were retrieved from GenBank database. Sequences were aligned by using ClustalX, and phylogenetic trees were constructed by the neighbor-joining method with a Poisson correction model (1000 bootstrap replications to check for repeatability of the results) using the MEGA 5.0 software.

4.5 Qualitative gene expression assays
Real time PCR was employed to determine the expression of polygalacturonase in various tissues of E. integriceps in response to different hosts. The reactions were performed with Eva green master mix on a Rotor-Gene® Q system (QIA-Gene). Gene-specific primers (re-PG and 18S, Table 1) were designed using Primer Blast. PCR amplification using forward and reverse primers resulted in an approximately 100 bp fragment within the coding region of each gene. E. integriceps specific 18S ribosomal RNA gene (18S) was used as internal control. The 18S primers were designed according to Sunn pest 18S ribosomal RNA sequence in GenBank with accession number KP890857 by using primer blast online software (authors unpublished data). Prior to PCR, cDNA preparations from developmental stages were quantified. For every sample, three technical replicates were performed. Each reaction was performed with 5 μl of cDNA, 0.5 μM of forward and reverse primer (re-PG and 18S for separate reactions) and 4 μl of Eva green master mix in 20 μl total volumes. The PCR amplifications were performed with the following cycling conditions: one cycle at 95°C (15 min), followed by 40 cycles of denaturation at 95°C (15 seconds), annealing at 62°C for 20 sec and extension at 72°C for 20 sec. At the end of the program a melting curve for each primer (65-94°C read every 1°C) was acquired to ensure that only single products were generated.

The data on gene expression in different tissues of E. integriceps were normalized by subtracting cycle threshold (Ct) values from the corresponding 18S Ct values. The relative expression level of genes in different tissues was determined by comparative Ct method (2-∆∆Ct) (Pfaffl, 2001).

4.6 Statistical Analysis
The data were given as means ± SE. Statistical significance was established as P<0.05. The significance of differences in the gene expression was determined by t-test using the Rest 2009 Software (Pfaffl et al., 2002). The PG expression levels were normalized relative to those of the control gene (18S).

5 Author contributions

Azam is a student and carried out the experiments and drafted the manuscript. Ali is Azam’s supervisor and participated in the design of the study, helped in statistical analysis and correction of the manuscript. Ali also paid from his grant for the project expenditure. Morteza is Azam’s advisor. All authors read and approved the final manuscript.

6 Acknowledgements

This research was founded by a grant (No. 86025.11) from the Iran National Science Foundation (INSF).

References
Ai J.W., Zhu Y., Duan J., Yu Q.Y., Zhang G.J., Wan F., and Xiang Z.H., 2011, Genomewide analysis of cytochrome P450 monooxygenase genes in the silkworm, Bombyx mori, Gene, 42-50
http://dx.doi.org/10.1016/j.gene.2011.03.002

Allahyari M., Bandani A.R., and Habibi-Rezaei M., 2010, Subcellular fractionation of midgut cells of the sunn pest Eurygaster integriceps (Hemiptera: Scutelleridae): enzyme markers of microvillar and perimicrovillar membranes, J Insect Physiol, 56(7):710-717
http://dx.doi.org/10.1016/j.jinsphys.2009.12.010

Allen M.L., and Mertens J.A., 2008, Molecular cloning and expression of three polygalacturonase cDNAs from the tarnished plant bug, Lygus lineolaris, J Insect Sci,8:1-14
http://dx.doi.org/10.1673/031.008.2701

Al-Obaidi J.R., Mohd -Yusuf  Y., Chin-Chong T., Mhd-Noh N., and Othman R.Y., 2010, Identification of a partial oil palm polygalacturon-aseinhibiting protein (EgPGIP) gene and its expression during basal stem rot infection caused by Ganoderma boninense, African Journal of Biotechnology, 9(46):7788-7797.

Amiri A., and Bandani A.R., 2013, Comparison of energy reserves in prediapause and diapausing adult Sun pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), J. Agr. Sci. Tech, 15:435-444.

Bashi Z.D., Rimmer S.R., Khachatourians G.G., and Hegedus D.D., 2013, Brassica napus polygalacturonase inhibitor proteins inhibit Sclerotinia sclerotiorum polygalacturonase enzymatic and necrotizing activities and delay symptoms in transgenic plants, Can. J. Microbiol, 59(2):79-86
http://dx.doi.org/10.1139/cjm-2012-0352

Baumann O., and Walz B., 2012, The blowfly salivary gland - a model system for analyzing the regulation of plasma membrane V-ATPase, J Insect Physiol. 58(4):450-8
http://dx.doi.org/10.1016/j.jinsphys.2011.11.015

Bown D.P., Wilkinson H.S., and Gatehouse J.A., 1997, Differentially regulated inhibitor sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families, Insect Biochem. Mol. Biol, 27:625-638
http://dx.doi.org/10.1016/S0965-1748(97)00043-X

Boyd D.W. J.r., Cohen A.C., and Alverson D.R., 2002, Digestive enzymes and stylet morphology of Deraeocoris nebulosus (hemiptera: Miridae), a predacious plant bug, Ann. Entomol. Soc. Am. 95:395-401
http://dx.doi.org/10.1603/0013-8746(2002)095[0395:DEASMO]2.0.CO;2

Chen J., Li C., and Yang Z., 2015, Identification and Expression of Two Novel Cytochrome P450 Genes, CYP6CV1 and CYP9A38, in Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), J. Insect Sci, 20:15(1).

De Lorenzo G., D’Ovidio R., and Cervone F., 2003, The role of polygalacturonase-inhibiting proteins (PGIP) in defence against pathogenic fungi, Ann. Rev. Phytopathol, 39: 313-335
http://dx.doi.org/10.1146/annurev.phyto.39.1.313

Di Giovanni M., Cenci A., Janni M., and D’Ovidio R., 2008, A LTR copia retrotransposon and Mutator transposons interrupt pgip genes in cultivated and wild wheats, Theor. Appl. Genet, 116: 859-867
http://dx.doi.org/10.1007/s00122-008-0719-1

Dolgikh V.V., Senderskii I.V., and Konarev A.V., 2014, Production and properties of recombinant glutenin-hydrolyzing proteinases from Eurygaster integriceps Put, Prikl. Biokhim. Mikrobiol, 50(5):466–74
http://dx.doi.org/10.7868/S0555109914040205 

D’Ovidio R., Raiola A., Capodicasa C., Devoto A., Pontiggia D., Roberti S., Galletti R., Conti E., O’Sullivan D., and De Lorenzo G., 2004, Characterization of the complex locus of Phaseolus vulgaris encoding polygalacturonase-inhibiting proteins (PGIPs) reveals subfunctionalization for defense against fungi and insects, Plant Physiol,135: 2424-2435
http://dx.doi.org/10.1104/pp.104.044644

Elgarj F.N.A, and Wajidi M.F.F., 2013, Molecular Cloning and Characterization a Novel Gene Encoding CYP4H28v2 from the Mosquito, Aedes aegypti, International Journal of Chemical, Environmental & Biological Sciences (IJCEBS), 1(2): 240-243

Ferrari S., Vairo D., Ausubel F.M., Cervone F., and De Lorenzo G., 2003, Tandemly duplicated Arabidopsis genes that encode polygalacturonase- inhibiting proteins are regulated coordinately by different signal transduction pathways in response to fungal infection, Plant Cell, 15(1): 93-106
http://dx.doi.org/10.1105/tpc.005165

Ferrari S., Savatin D.V., Sicilia F., Gramegna G., Cervone F., and De Lorenzo G., 2013, Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth and development, Front. Plant Sci, 4:49
http://dx.doi.org/10.3389/fpls.2013.00049

Feyereisen R., 2005, Insect cytochrome P450, In: Gilbert L.I., Iatrou K., and Gill S.S., (eds.), Comprehensive Molecular Insect Science, Elsevier Press, Oxford, United Kingdom, vol. 4, pp. 1-77
http://dx.doi.org/10.1016/B0-44-451924-6/00049-1

Feyereisen R., 2006, Evolution of insect P450, Biochemical Society Transactions, 34:1252-1255
http://dx.doi.org/10.1042/BST0341252

Feyereisen R., 2012, Insect CYP genes and P450 enzymes, In: Gilbert L., (ed) InInsect Molecular Biology and Biochemistry, London, Elsevier, pp.236-316
http://dx.doi.org/10.1016/b978-0-12-384747-8.10008-x

Frati F., Galletti R., DeLorenzo G., Salerno G., and Conti E., 2006, Activity of endo-polygalacturonases in mirid bugs (Heteroptera: Miridae) and their inhibition by plant cell wall proteins (PGIPs), European Journal of Entomology, 103:515-522
http://dx.doi.org/10.14411/eje.2006.067

Garcia-Maceira F., Di Pietro A., Huertas-Gonzalez M.D., Ruiz-Roldan M.C., and Roncero M.I.G., 2001, Molecular Characterization of an endo- polygalacturonase from Fusarium oxysporum expressed during early stages of infection, Appl. Environ. Microb., 67(5): 2191-2196
http://dx.doi.org/10.1128/AEM.67.5.2191-2196.2001

Götesson A., Marshall J.S., Jones D.A., and Hardham A.R., 2002, Characterization and evolutionary analysis of a large polygalacturonase gene family in the oomycete plant pathogen Phytophthora cinnamomi, Mol. Plant-Microbe. In., 15(9): 907-921.

Guo Y., Zhang J., Yu R., Zhu K.Y., Guo Y., and Ma E., 2012, Identification of two new cytochrome P450 genes and RNA interference to evaluate their roles in detoxification of commonly used insecticides in Locusta migratoria, Chemosphere, 87(7):709-17
http://dx.doi.org/10.1016/j.chemosphere.2011.12.061

Hardwick J.P., 2008, Cytochrome P450 omega hydroxylase (CYP4) function in fatty acid metabolism and metabolic diseases, Biochem. Pharmacol., 75(12):2263-75
http://dx.doi.org/10.1016/j.bcp.2008.03.004

Hegedus D.D., Li R., Buchwaldt L., Parkin I., Whitwill S., and Coutu C., et al., 2008, Brassica napus possesses an expanded set of polygala- cturonase inhibitor protein genes that are differentially regulated in response to Sclerotinia sclerotiorum infection, wounding and defense hormone treatment, Planta, 228: 241-253
http://dx.doi.org/10.1007/s00425-008-0733-1

Holliday L.S., 2014, Vacuolar H+-ATPase: An essential multitasking enzyme in physiology and Pathophysiology, New J. Sci., 1-21
http://dx.doi.org/10.1155/2014/675430

Hu Z., Lin Q., Chen H., Li Z., Yin F., and Feng X., 2014, Identification of a novel cytochrome P450 gene, CYP321E1 from the diamondback moth, Plutella xylostella (L.) and RNA interference to evaluate its role in chlorantraniliprole resistance, Bull. Entomol. Res., 104(6):716-23
http://dx.doi.org/10.1017/S0007485314000510

Huang C.G., Tsai K.H., Wu W.J., and Chen W.J., 2006, Intestinal expression of H+ V-ATPase in the mosquito Aedes albopictus is tightly associated with gregarine infection, J. Eukaryot Microbiol., 53(2):127-35
http://dx.doi.org/10.1111/j.1550-7408.2005.00086.x

Janni M., Sella L., Favaron F., Blechl A.E., De Lorenzo G., and D'Ovidio R., 2008, The expression of a bean pgip in transgenic wheat confers increased resistance to the fungal pathogen Bipolaris sorokiniana, Mol. Plant-Microbe Interactions, 21: 171-177
http://dx.doi.org/10.1094/MPMI-21-2-0171

Jaubert S., Laffaire J.B., Abad P., and Rosso M.N., 2002, A polygalac- turonase of animal origin isolated from the root-knot nematode Meloidogyne incognita, FEBS Lett, 522:109-112
http://dx.doi.org/10.1016/S0014-5793(02)02906-X

Jefferies K.C., and Michael F., 2007, Subunit H of the vacuolar (H+) ATPase inhibits ATP hydrolysis by the Free V1 domain by interaction with the rotary subunit F, J. Biol. Chem., 283:4512-4519
http://dx.doi.org/10.1074/jbc.M707144200

Jefferies K.C., Cipriano D.J., and Forgac M., 2008, Function, structure and regulation of the vacuolar H+-ATPases, Archives of Biochemistry and Biophysics, 476:33-42
http://dx.doi.org/10.1016/j.abb.2008.03.025

Kalunke R.M., Tundo S., Benedetti M., Cervone F., De Lorenzo G., and D'Ovidio R., 2015, An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens, Front Plant Sci., 20:6:146.

Konarev A.V., Beaudoin F., Marsh J., Vilkova N.A., Nefedova L.I., Sivri D., Köksel H., Shewry P.R., and Lovegrove A., 2011, Characterization of a glutenin-specific serine proteinase of Sun bug Eurygaster integriceps Put., J. Agric. Food Chem., 59(6):2462-2470
http://dx.doi.org/10.1021/jf103867g

Lu P., Xia H., Gao L., Pan Y., and Wang Y. et al., 2013, V-ATPase Is Involved in Silkworm Defense Response against Bombyx mori Nucleopolyhedrovirus, PLoS ONE, 8(6): e64962
http://dx.doi.org/10.1371/journal.pone.0064962

Lu L., Qi Z., and Wu W., 2014, Cloning, expression and purification of subunit H of vacuolar H=-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae), Int. J. Mol. Sci., 15(9):15443-55
http://dx.doi.org/10.3390/ijms150915443

Ma B., Xiang Y., and An L., 2011, Structural bases of physiological functions and roles of the vacuolar H(+)-ATPase, Cell Signal, 23(8):1244-56
http://dx.doi.org/10.1016/j.cellsig.2011.03.003

Mizutani M., and Ohta D., 2010, Diversification of P450 genes during land plant evolution. Annu. Rev. Plant Biol., 61:291-315
http://dx.doi.org/10.1146/annurev-arplant-042809-112305

Nelson D.R., 2009, The cytochrome P450 homepage, Human Genomics, 4:59-65.

Nielsen K.A., and Moller B.L., 2005, Cytochrome P450s in Plants, In: Ortiz de Montellano P.R., (ed.), Cytochrome P450: Structure, Mechanism, and Biochemistry, 3rd ed. Kluwer Academic/Plenum Publishers, New York, pp. 553-583
http://dx.doi.org/10.1007/0-387-27447-2_12

Oppert B., Elpidina E.N., Toutges M., and Mazumdar-Leighton S., 2010, Microarray analysis reveals strategies of Tribolium castaneum larvae to compensate for cysteine and serine protease inhibitors, Comp. Biochem. Physiol. D, 5(4):280-287
http://dx.doi.org/10.1016/j.cbd.2010.08.001

Pfaffl M.W., 2001, A new mathematical model for relative quantification in realtime RT-PCR, Nucleic Acids Res., 29:e45
http://dx.doi.org/10.1093/nar/29.9.e45

Pfaffl M.W., Horgan G.W., and Dempfle L., 2002, Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR, Nucleic Acids Res., 30:e36
http://dx.doi.org/10.1093/nar/30.9.e36

Prabhu S.A., Kini K.R. and Shetty H.S., 2012, Partial purification and characterization of polygalacturonase-inhibitor proteins from pearl millet, African Journal of Biotechnology, 11(27): 7000-7008.

Ramalho-Ortigão J.M., Pitaluga A.N., Telleria E.L., Marques C., Souza A.A., and Traub-Cseko Y.M., 2007, Cloning and characterization of a V-ATPase subunit C from the American visceral leishmaniasis vector Lutzomyia longipalpis modulated during development and blood ingestion, Mem. Inst. Oswaldo. Cruz., 102(4):509-15
http://dx.doi.org/10.1590/S0074-02762007000400013

Sahara Y., Horie S., Fukami H., Goto-Matsumoto N., and Nakanishi-Matsui M., 2014, Functional roles of V-ATPase in the salivary gland, Journal of Oral Biosciences, doi:10.1016/j.job.2014.11.002.
http://dx.doi.org/10.1016/j.job.2014.11.002

Shackel K.A., de la Paz Celorio-Mancera M., Ahmadi H., Greve L.C., Teuber L.R., Backus E.A., and Labavitch J.M., 2005, Micro-injection of Lygus salivary gland proteins to simulate feeding damage in alfalfa and cotton flowers, Archives of Insect Biochemistry and Physiology,  58:69-83
http://dx.doi.org/10.1002/arch.20033

Schuler, M.A., 2011, P450s in plant–insect interactions, Biochim. Biophys. Acta.,1814:36-45
http://dx.doi.org/10.1016/j.bbapap.2010.09.012

Scott J.G., 2008, Insect cytochrome P450s: thinking beyond detoxification. In: Liu N., (ed.), Recent Advances in Insect Physiology, Toxicology and Molecular Biology, Research Signpost, Kerala, India, pp.117-124.

Spit J., Zels S., Dillen S., Holtof M., Wynant N., and Vanden Broeck J., 2014, Effects of different dietary conditions on the expression of trypsin- and chymotrypsin-like protease genes in the digestive system of the migratory locust, Locusta migratoria, Insect Biochem. Mol. Biol., 48:100-109
http://dx.doi.org/10.1016/j.ibmb.2014.03.002

Tamburino R., Chambery A., Parente A., and Di Maro A., 2012, A novel polygalacturonase-inhibiting protein (PGIP) from Lathyrus sativus L. seeds, Protein Pept. Lett.,19(8):820-5
http://dx.doi.org/10.2174/092986612801619561

Thakur N., Upadhyay S.K., Verma P.C., Chandrashekar K., and Tuli R. et al., 2014, Enhanced Whitefly Resistance in Transgenic Tobacco Plants Expressing Double Stranded RNA of v-ATPase A Gene, PLoS ONE, 9(3): e87235
http://dx.doi.org/10.1371/journal.pone.0087235

Vogelweith F., Thiery D., Quaglietti B., Moret Y., and Moreau J., 2011, Host plant variation plastically impacts different traits of the immune system of a phytophagous insect, Funct. Ecol., 25:1241-1247
http://dx.doi.org/10.1111/j.1365-2435.2011.01911.x

Walker W.B., and Allen M.L., 2010, Expression and RNA interference of salivary polygalacturonase genes in the tarnished plant bug, Lygus lineolaris, J. Insect Sci.,10:173
http://dx.doi.org/10.1673/031.010.14133

Wang Z.Z., Shi M., Ye X.Q., Chen M.Y., and Chen X.X., 2013, Identification, characterization and expression of a defensin-like antifungal peptide from the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), Insect Mol. Biol. 22(3):297-305
http://dx.doi.org/10.1111/imb.12021

Wang R.L., Li J., Staehelin C., Xin X.W., Su Y.J., and Zeng R.S., 2015, Expression Analysis of Two P450 Monooxygenase Genes of the Tobacco Cutworm Moth (Spodoptera litura) at Different Developmental Stages and in Response to Plant Allelochemicals, J. Chem. Ecol., 41(1):111-119
http://dx.doi.org/10.1007/s10886-014-0540-z

Wieczorek H., Grber G., Harvey W.R., Huss M., and Merzendorfer H. et al., 2000, Structure and regulation of insect plasma membrane H (+) V-ATPase, J. Exp. Biol., 203:127-135.

Wieczorek H., Beyenbach K.W., Huss M., and Vitavska O., 2009, Vacuolar-type proton pumps in insect epithelia, J. Exp. Biol., 212:1611-1619
http://dx.doi.org/10.1242/jeb.030007

Xu Y., Wang J., Jiang H., Dou W., Tang P., and An F., 2009, Identification, Characterization, and Expression of P450 Gene Encoding CYP6BQ13v2 from the Red Flour Beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), Agricultural Sciences in China, 8(10):1210-1218
http://dx.doi.org/10.1016/S1671-2927(08)60331-4

Yandamuri R.C., Gautam R., Darkoh C., Dareddy V., El-Bouhssini M., and Clack B.A., 2014, Cloning, Expression, Sequence Analysis and Homologyodeling of the Prolyl Endoprotease from Eurygaster integriceps Puton, Insects, 5:762-782
http://dx.doi.org/10.3390/insects5040762

Yang H., Chen H., Chen K., Yao Q., Zhao G., Wu C., Lv P., and Wang L., 2009, Characterization and localization of the vacuolar-type ATPase in the midgut cells of silkworm (Bombyx mori), Z Naturforsch C., 64(11-12):899-905
http://dx.doi.org/10.1515/znc-2009-11-1223

Yu L., Tang W., He W., Ma X., Vasseur L., Baxter S.W., Yang G., Huang S., Song F., and You M., 2015, Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.), Sci. Rep., 10:5:8952.

Zhang Y-L., Kulye M., Yang F-S., Xiao L., Zhang Y-T., Zeng H-M., Wang J-H., and Liu Z-X., 2011, Identification, characterization, and expression of a novel P450 gene encoding CYP6AE25 from the Asian corn borer, Ostrinia furnacalis, Journal of Insect Science, 11:37
http://dx.doi.org/10.1111/j.1744-7917.2004.tb00178.x
http://dx.doi.org/10.1673/031.011.0137

Zhou X.J., Ma C.X., Li M., Sheng C.F., Liu H.X. and Qiu X.H., 2010, CYP9A12 and CYP9A17 in the cotton bollworm Helicoverp armigera: sequence similarity, expression profile and xenobiotic response, Pest Management Science, 66:65-73
http://dx.doi.org/10.1002/ps.1832

Molecular Entomology
• Volume 6
View Options
. PDF(805KB)
. HTML
Associated material
. Readers' comments
Other articles by authors
pornliz suckporn sex videos bbw mom xxx big fucking arabin porn videos teen gril sex video riding hard cock woman hard vagina . Amiri Azam
. Bandani Ali Reza
. Allahyari Morteza
Related articles
. V-ATPase
. cytochrome P450
. polygalacturonase
. Eurygaster
. gene expression
Tools
. Email to a friend
. Post a comment