Author running head: Y. Sun et al. Title running head: Expression analysis of Alhsc70 Correspondence: Li-Xin Bai, Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China. Tel: +86 025 84390392; fax: +86 025 84391175; email: [email protected]
ORIGINAL ARTICLE Identification of heat shock cognate protein 70 gene (Alhsc70) of Apolygus lucorum and its expression in response to different temperature and pesticide stresses Yang Sun1,2, Jing Zhao1, Yang Sheng1, Ying-Fang Xiao3, Yong-Jun Zhang2, Li-Xin Bai1, Yongan Tan1, Liu-Bin Xiao1 and Guang-Chun Xu1 1
Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014,
China; 2Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 3Entomology and Nematology, Mid-Florida Research and Education Center, University of Florida, FL 32703, USA
This is an Accepted Article that has been peer-reviewed and approved for publication in the Insect Science but has yet to undergo copy-editing and proof correction. Please cite this article as doi: 10.1111/1744-7917.12193. This article is protected by copyright. All rights reserved.
1
Abstract Heat shock cognate protein 70 (Hsc70) is a very important stress-resistance protein of insects against environmental stresses. We employed fluorescent real-time quantitative PCR and western-blot techniques to analyze the transcriptional and translational expression profiles of AlHSC70 under extreme temperature (4°C and 40°C) or 4 pesticide stresses in Apolygus lucorum. The results showed that the expression of AlHSC70 were significantly induced by cyhalothrin or extremely high temperature (40°C) in both transcriptional and translational levels (P < 0.05), while the transcriptional and translational level of AlHSC70 decreased significantly in treatments of chlorpyrifos or extreme cold temperature (4°C) (P < 0.05). Moreover, after A. Lucorum treated by imidacloprid or emamectin benzoate, the expression of AlHSC70 were only up-regulated significantly in transcriptional level (P < 0.05), though obviously up-regulated in translational level of AlHSC70 were also showed. Therefore, this study confirmed that the Alhsc70 played important roles in response to both temperature and pesticide stresses, especially for cyhalothrin or extremely high temperature (40°C). In addition, the significant polynomial regression correlations between temperature and the Alhsc70 expression level were showed in all the nymph and adult stages (P < 0.01), indicating temperature was an important factor to affect the relative expression of Alhsc70. Key words A. lucorum; expression files; extreme temperature; hsc70; pesticide
Introduction Heat shock proteins (HSPs) belonging to a protein superfamily are rapidly synthesized in response to a series of environmental stressors (Feder & Hofmann, 1999; Sonoda & Tsumuki, This article is protected by copyright. All rights reserved.
2
2007). Among these HSPs, the HSP70 family represents one of the most highly conserved proteins (Mayer & Bukau, 2005), which also has several functions in Eukaryotic organisms including translocation, folding newly synthesized proteins, degradation of unstable, and misfolded proteins, as well as prevention and dissolution of protein complexes (Daugarrd et al., 2007). Genes encoding HSP70 traditionally are divided into two groups. The first group is the most abundantly induced proteins, which can be induced quickly under a variety of stress conditions, while returned to a normal expression level under non-stressful conditions. However, the second group is not stress-inducible and is generally referred to as being constitutively expressed or as Heat Shock Cognates protein 70 (HSC70) (Denlinger et al., 2001; Qin et al., 2003; Bahar et al., 2013). As an important member of HSP70 family, HSC70 played important roles in homeostasis and immunity by functioning in protein synthesis, folding, transporting and degradation (Hartl & Hayer-Hartl, 2002; Lee et al., 2009; Su et al., 2010; Stankowski et al., 2011). Some reports indicated that hsc70 in organism was presented under most conditions and may or may not be influenced by heat, cold, heavy metal pollutant or endocrine disrupting pollutant stresses (Shim et al., 2006; Yoshimi et al., 2009; Gkouvitsas et al., 2009; Zhang & Denlinger, 2010; Morales et al., 2011). However, recent evidence indicated that the general pattern of hsc70 expression is also variable in response to different development stages (Mahroof et al., 2005) or stressors, such as temperature stress (Sonoda et al., 2006a,b; Wang et al., 2008; Wang et al., 2012), insecticides (Yoshimi et al., 2009), heavy metals (Yoshimi et al., 2009; Wang et al., 2012), and food restriction (Shim et al., 2006; Wang et al., 2012). Mirid bug, A. Lucorum, currently is one of key pests in Bt cotton fields in China (Lu et al., This article is protected by copyright. All rights reserved.
3
2010), and their feeding resulted in extremely plants losses both yield and quality (Lu et al., 2010; Lu & Wu, 2008). Due to the strong temperature adaptability of both nymphs and adults, A. lucorum has spread over to most cotton planting regions in China (Lu et al., 2007; Lu et al., 2010; Lu & Wu, 2008), and calendar-based insecticide sprays are the sole management option for A. lucorum (Lu et al., 2007). To better understand how A. lucorum regulated its hsp in response to thermal and pesticide stresses, Sun et al. (2014) early studied the expression of hsp90 in A. lucorum treated with four highly-active pesticides (cyhalothrin, imidacloprid, chlorpyrifos, and emamectin benzoate) and thermal stresses, indicating that Alhsp90 of A. lucorum may be an important gene involving in the resistance or tolerance to both temperature and pesticide stresses. However, exception for hsp90, whether hsc70 was involved in the response to pesticide and thermal stresses on A. lucorum was still unknown. Therefore, in this study, we firstly identified the full-length cDNA of A. lucorum hsc70 (denoted as Alhsc70) by RT-PCR and RACE, and then investigated the transcriptional and translation responses of Alhsc70 in A. lucorum in response to different temperatures, extreme temperatures (4°C and 40°C) or pesticides by means of qRT-PCR and western blot techniques.
Materials and methods Collection, culture, and treatment of A. lucorum under different temperatures A colony of A. lucorum was established from about 900 adults collected from the broad bean fields of Dafeng and Dongtai City, Jiangsu Province, China. The colony was reared on sauteed green beans in the light incubator at the temperatures of 25 ± 1°C, RH 70% ± 5% This article is protected by copyright. All rights reserved.
4
humidity, and photoperiod 12 : 12 (L : D). According to the study reported by Sun et al. (2014) the tested temperatures were setup at 18, 21, 24, 27, 31, and 33°C, with 24°C as the control. More than 400 A. lucorum eggs were reared at each aforementioned temperature. A. lucorum samples collected were separated into nymph and adult stages. The nymph stage was divided into 1st, 2nd, 3rd, 4th, or 5th instars, and the adult stages were categorized based on new emergence (1-day-old), pre-mating (5-day-old) and post-mating (11-day-old). Each treatment sampled five A. lucorum adults or nymphs with 5 replicates for a total of 25 adults or nymphs were sampled at each stage or temperature. More than 120 2-d old female or male adults were treated at extreme temperatures (4 and 40°C) in insect rearing boxes with sauteed green beans as host for 1 h. Same sampling number of 2-d old female or male adults reared at 24°C were set as controls. Five female or male adults were collected each time after treatment, and this collection was repeated 12 times for a total of 60 adults at each temperature. These treatments were replicated two times, one was for real-time PCR, and the other was for western blot.
Bioassay and pesticide treatment For the pesticide treatments, the topical treatment technique reported by FAO (1980), Shi et al. (2011) and Sun et al. (2014) were adopted for the toxicity bioassay of cyhalothrin, imidacloprid, chlorpyrifos, emamectin benzoate, and acetone alone. The detail information about aforementioned 4 pesticides, such as the purity and manufactures of these 4 pesticides, the LD50 and LD20 value of each aforementioned pesticide to A. lucorum adults were obtained This article is protected by copyright. All rights reserved.
5
in our previous study reported by Sun et al. (2014) and listed in supplementary Table 1. Sixty female or male adults were treated with each aforementioned pesticide (Induction dose = LD20, LD50), respectively. The same batches of female or male adults reared at 24°C were set as CK controls (Ct), and the same batches of Ct controls adults treated with acetone alone were set as Ac controls. For pesticide treatments and Ac controls, 2-day old adults were treated with acetone (0.25 μL/A. lucorum). Mortality was assessed after 48 h treatment. Surviving adults after pesticides treatments, Ct controls and Ac controls were collected and stored at −80°C. These testaments, which adults were treated with each aforementioned 4 pesticides (Induction dose = LD50), Ct controls and Ac controls, were also performed two times, one was for real-time PCR, and the other was for western blot.
Cloning and sequencing the full length cDNA of Alhsc70 In this study, A. lucorum hsc70 (Alhsc70) fragment from 1 bp to 1605 bp of this 2333 bp full-length gene was first obtained from adult cDNA library that had been reported by Sun et al. (2014). And then, the 5' and 3' ends of Alhsc70 were obtained by rapid amplification of cDNA ends (RACE) and SMARTer™ RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA). Two pairs of Gene-specific Primers (GSPs) for RACE were used to obtain the full-length
cDNA
of
Alhsc70.
Two
AACCTGGCCCTGGTGATGGAAGTGT-3', GTTGTCAAAGTCCTCACCGCCCAAGT-3') CCATCGCCTACGGCCTCGACAAG-3',
5'GSPs
(5'GSP1:
5'-
5'GSP2: and
two
3'GSPs,
5'(3'GSP1:5'3'GSP2:5'-
CACAGTCCCCGCCTACTTCAACGACT-3') were designed based on the partial sequence This article is protected by copyright. All rights reserved.
6
obtained by adult cDNA library using Primer 5.0 software.
Bioinformatics analysis of Alhsc70 A sequence similar search both in the nucleotide and amino acid levels were performed with the BLAST program at the National Center for Biotechnology Information (http://www. ncbi. Nlm. Nih. gov/BLAST/). The inferred amino acid sequence was analyzed with the Expert Protein Analysis System (http://www.expasy.org/). Multiple alignment of AlHSC70 was performed with GeneDoc 3.2 software. Therefore, a phylogenic tree was constructed by ClustalX 2.0 and MEGA 4.0 based on the obtained AlHSC70 sequences and other known insect HSC70 amino acid sequences. Bootstrap analysis was used with 1000 replicates to estimate the confidence of branches produced by the neighbor-joining method.
Relative quantitation of AlHSC70 mRNA expression by real-time quantitative RT-PCR after A. lucorum treated by different temperatures and pesticides Total RNA of A. lucorum samples treated with different temperatures or pesticides was extracted using the SV Total RNA Isolation System (Promega, Madison, WI, USA). And then, cDNA was synthesized using MMLV Reverse Transcriptase (Promega, Madison, WI, USA), treated with Ribonuclease H (TaKaRa, Tokyo, Japan), and quantified by spectrophotometry. The SYBR Premix Ex Taq Kit (TaKaRa, Tokyo, Japan) were used to conduct the qRT-PCR reactions, and the qRT-PCR products of this Kit were limited from 100 bp to 300 bp. Therefore, the 279 bp length of Alhsc70 (GenBank accession no. KC119044) product and 177 bp length of β-actin (GenBank accession no. JN616391) product were designed for qRT-PCR. This article is protected by copyright. All rights reserved.
7
The primers used for qRT-PCR of Alhsc70 were 5'-CGCCGCCAAGAACACCTTG-3' and 5'-AATCCGCCGGGGAAACCT-3'. The endogenous reference gene β-actin of A. lucorum was used for data normalization with primers were 5'-ACCTGTACGCCAACACCGT-3' and 5'-TGGAGAGAGAGGCGAGGAT-3'. All the primers for qRT-PCR were designed by Primer 5.0 software. Dissociation curve analysis and gel electrophoresis showed that only the target gene was synthesized. Real-time fluorescence data of reactions containing SYBR Green I were carried out using the Bio-rad iCycler real-time quantitative RT-PCR detection system, and values were determined using iCycler iQ real-time detection system software (version 3.0a; Bio-Rad). Real-time reactions of each treatment were replicated 5 times, and non-template control reactions were performed in triplicate for each primer pair. The qRT-PCR cycling parameters were 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, 58°C for 15 s and then 95°C for 19s, and finally a dissociation curve from 60 to 95°C with an increment of 0.5°C per 5 s. To estimate the real-time quantitative RT-PCR amplification efficiency of primers, a standard curve of five dilution series (1 × 102, 1 × 101, 1 × 100, 1 × 10−1, and 1 × 10−2 ng) was constructed from purified cDNA fragments obtained from previous qRT-PCRs using the same primer set. The efficiency of primers was calculated using the formula E = 10−1/SLOPE. The amplification efficiency values of Alhsc70 and β-actin were 2.162 ± 0.091 and 2.115 ± 0.046, respectively. The relative gene expression levels of Alhsc70 after A. lucorum samples treated with different temperatures and pesticides were represented by relative quantification (RQ) values calculated by the 2-ΔΔCt method (Livak & Schmittgen, 2001).
This article is protected by copyright. All rights reserved.
8
Western blot To identify AlHSC70 expression at protein level after extreme heat, extreme cold or different pesticide stresses, more than 30 of the 2-d female or male adults were selected to perform western blot for each sample according to our method (Sun et al., 2014). Total proteins were extracted using the Tissue Protein Extraction Reagent Kit (Nanjing Zoonbio Tech, Co., Ltd.). Total protein concentrations were determined using the bicinchoninic acid (BCA) method (Nanjing Zoonbio Tech, Co., Ltd.). Western blot analysis was performed according to Song et al. (2012) with small modification. Protein samples were electrophoresed on 10% SDS-polyacrlamide gel and electro-blotted to NC membrane (Bio-rad). Transfer was run at 100 mA for 3 h, using Tris/glycine buffer. Membranes were blocked with 5% nonfat powdered milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and incubated for 1 h at 37°C. The primary antibody against AlHSC70, which was produced from the AlHSC70 protein by Nanjing Zoonbio Tech, Co., Ltd., was used at 1:1000 dilutions and incubated for 1 h at 37°C. The AlHSC70 protein obtained by prokaryotic expressed from the cDNA of Alhsc70 ORF was also by Nanjing Zoonbio Tech, Co., Ltd. The membrane was washed three times in TBS-T, 5min each, and then incubated with HRP conjugated goat anti-rabbit IgG secondary antibody (Nanjing Zoonbio Tech, Co., Ltd.) diluted 1 : 5000 in blocking buffer for 1 h at 37°C. The membrane was washed three times with TBS-T, 5 min each, and then developed by using enhanced chemiluminescence advance kit (GE Healthcare). For each experiment, samples containing an equal amount of total protein were run on the same gel. Densitometric analysis of the immunoblots was performed using
a
desktop
scanner
(Hanwang
This article is protected by copyright. All rights reserved.
E60)
and
Image
J
free
software 9
(http://rsb.info.nih.gov/ij/index.html). Furthermore, the western blot for β-actin was also conducted with above method at the same time, and mouse anti-β-actin antibody using for western blot was purchased from Nanjing Zoonbio Tech, Co., Ltd. The optical density of immunoblots was determined by scanning densitometry and the result was presented in densitometric units. Translation was calculated by dividing the normalized AlHSC70 density by the normalized β-actin density (AlHSC70/β-actin ratio) (Song et al., 2012). The western blotting was replicated three times.
Statistical analyses All statistical analyses were executed using the software package SAS version 9.0. Statistical differences of Alhsc70 gene and protein expression in A. lucorum treated with different pesticide and temperature stresses were determined by the one-way analysis of variance, followed by the Tukey’s honestly significance difference (HSD) test (P < 0.05 and P < 0.01). The P-value, F-value, R-value, SE-value (Standard Error of regression function) and the shape of the regression figure were used to identify which mathematical model or function was the most suitable for revealing the regression correlation between relative expression of Alhsc70 and temperature.
Results Phylogenetic analyses By combining the SMART cDNA library of A. lucorum with RACE approaches, two fragments corresponding to the 5' and 3' ends of Alhsc70 cDNA were amplified. Finally, a This article is protected by copyright. All rights reserved.
10
nucleotide sequence of 2333 bp representing the complete cDNA sequence of Alhsc70 was obtained and deposited in GenBank (accession no. KC119044). The full-length cDNA of Alhsc70 was 2333 bp long, including a 5'-untranslated region (UTR) of 108 bp, a 3'-UTR of 254 bp, a canonical polyadenylation signal sequence AATAAA, a poly(A) tail, and an ORF of 1971 bp. The ORF encoded a polypeptide of 656 amino acids with a predicted molecular weight of 71.57 kDa and a theoretical isoelectric point of 5.38. In addition, Classical HSP70 protein signature motifs include IDLGTTYS (AA 10–17), IFDLGGGTFDVSIL (AA 200–213) and IVLVGGSTRIPKVQKL (AA 337–352). Three other typical motifs are also found in the Alhsc70: the first is a deduced ATP-GTP binding site, AEAYLGQK (AA 132–139);
the
second
is
a
putative
bipartite
nuclear
localization
signal
[(KRKYKKDLTT(Q)NKRALRRL (AA 249–266); KRALRRLRTA(S)CERAKRTL (AA 260–277)], which is needed for the selective translocation of HSP70 into the nucleus (Knowlton & Salfity, 1996); the third is a non-organellar consensus motif RARFEEL (AA 302–308). The sequence of the ATPase domain (AA 1–336) of HSP70 is highly conserved, while the C-terminal domain is less conserved than the ATPase domain (Figs. 1, 2) (Su et al., 2010). Furthermore, conserved motif EEVD was also found in the C-terminal of AlHSC70. In addition, 15 characteristic residues (IVLVGGSTRIPKVQKL, AA 337–352), which only presented in constitutive HSC70 amino acid sequence but not in inducible HSP70 were also found in AlHSC70 (Chen et al., 2006). Among the HSC70 proteins compared, the highest percentage identity of AlHSC70 was with HSP70 from Nilaparvata lugens (95% identity). The lowest percentage identity and similarity was Plutella xylostella HSC70 with HSC70 from Aedes aegypti (87% identity). This article is protected by copyright. All rights reserved.
11
BLAST results from GenBank indicated a high percentage identity of AlHSC70 compared with other insect HSC70s, and the lowest percentage identity and similarity was those of AlHSC70 with HSP70 from Anopheles gambiae (85% identity, accession no. XP_315042). A phylogenetic tree was constructed with CLUSTAL X 2.0 and MEGA 4 based on 29 HSC70s from eight different insect orders (Fig. 3). Results of the phylogenetic tree showed that 24 HSC70s
belonged
to
Hemiptera,
Lepidoptera,
Blattodea,
Anoplura,
Coleopteran,
Hymenopera, and Orthoptera insects located in a close branch, and the sequence similarity among these HSC70s was at least 85%. HSC70s belonged to Diptera insects located in the branch far from that of the other seven insect orders. However, the sequence similarity among these 29 HSC70s was at least 85%, which may indicate that these HSC70 genes were derived from the same ancestral gene.
Quantitative analysis of Alhsc70 expression when A. lucorum was reared at different temperatures The expression of Alhsc70 in A. lucorum nymphs and adults reared at ~24°C were lower than other temperatures (Fig. 4), which indicated that the expression of Alhsc70 in A. lucorum nymphs and adults can be up-regulated by both cold shock (18°C) and heat shock (30°C and 33°C) significantly (P < 0.01) (Fig. 4). Moreover, the polynomial correlation with mathematical function y = ax2 + bx + c was regarded as the most suitable regression correlation for indicating the function relationship between relative expression of Alhsc70 and temperature among all these functions of software package SAS version 9.0, because of the lower SE-value, P-value and higher F-value, R-value. After A. lucorum culturing from 18 This article is protected by copyright. All rights reserved.
12
to 33°C, significant polynomial regression correlations between the relative expressions of Alhsc70 with temperatures were found in all the A. lucorum nymph stages and adult stages (Figs. 5, 6) (P < 0.01).
Transcriptional quantitative analysis of Alhsc70 expression when A. lucorum adults treated with extreme temperatures (4 and 40 °C) or 4 different pesticides Real-time PCR technique was used to examine the expression of Alhsc70 in 2-d old adults treated with 4 different pesticides or extreme temperatures (4 and 40°C). Results indicated that the transcriptional expression of Alhsc70 in A. lucorum adults treated with extreme high temperature (40°C) was significantly higher than Ct controls (P < 0.01). Treated by extreme low temperature (4°C), the expression of Alhsc70 in A. lucorum adults were significantly lower than Ct controls (P < 0.05) (Fig. 7). Treated by cyhalothrin, imidacloprid or emamectin benzoate (induction dose = LD50), the expression of Alhsc70 in 2-d old adults were significantly higher than that in Ct controls, Ac controls or A. lucorum adults treated by cyhalothrin, imidacloprid or emamectin benzoate (induction dose = LD20), respectively (P < 0.01) (Fig. 7). Furthermore, the expression of Alhsc70 in A. lucorum adults treated by cyhalothrin (induction dose = LD50) was significantly higher than that adults treated by imidacloprid or emamectin benzoate (induction dose = LD50) (female P < 0.01, male P < 0.05). In addition, a significant difference of the Alhsc70 expression was not found between A. lucorum adults treated by chlorpyrifos (induction dose = LD50) and chlorpyrifos (induction dose = LD20) (P > 0.05). Treated by chlorpyrifos (induction dose = LD20, LD50), the expression of Alhsc70 in A. lucorum adults were significantly lower than Ct and Ac controls This article is protected by copyright. All rights reserved.
13
(female P < 0.05, male P
ORIGINAL ARTICLE Identification of heat shock cognate protein 70 gene (Alhsc70) of Apolygus lucorum and its expression in response to different temperature and pesticide stresses Yang Sun1,2, Jing Zhao1, Yang Sheng1, Ying-Fang Xiao3, Yong-Jun Zhang2, Li-Xin Bai1, Yongan Tan1, Liu-Bin Xiao1 and Guang-Chun Xu1 1
Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014,
China; 2Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 3Entomology and Nematology, Mid-Florida Research and Education Center, University of Florida, FL 32703, USA
This is an Accepted Article that has been peer-reviewed and approved for publication in the Insect Science but has yet to undergo copy-editing and proof correction. Please cite this article as doi: 10.1111/1744-7917.12193. This article is protected by copyright. All rights reserved.
1
Abstract Heat shock cognate protein 70 (Hsc70) is a very important stress-resistance protein of insects against environmental stresses. We employed fluorescent real-time quantitative PCR and western-blot techniques to analyze the transcriptional and translational expression profiles of AlHSC70 under extreme temperature (4°C and 40°C) or 4 pesticide stresses in Apolygus lucorum. The results showed that the expression of AlHSC70 were significantly induced by cyhalothrin or extremely high temperature (40°C) in both transcriptional and translational levels (P < 0.05), while the transcriptional and translational level of AlHSC70 decreased significantly in treatments of chlorpyrifos or extreme cold temperature (4°C) (P < 0.05). Moreover, after A. Lucorum treated by imidacloprid or emamectin benzoate, the expression of AlHSC70 were only up-regulated significantly in transcriptional level (P < 0.05), though obviously up-regulated in translational level of AlHSC70 were also showed. Therefore, this study confirmed that the Alhsc70 played important roles in response to both temperature and pesticide stresses, especially for cyhalothrin or extremely high temperature (40°C). In addition, the significant polynomial regression correlations between temperature and the Alhsc70 expression level were showed in all the nymph and adult stages (P < 0.01), indicating temperature was an important factor to affect the relative expression of Alhsc70. Key words A. lucorum; expression files; extreme temperature; hsc70; pesticide
Introduction Heat shock proteins (HSPs) belonging to a protein superfamily are rapidly synthesized in response to a series of environmental stressors (Feder & Hofmann, 1999; Sonoda & Tsumuki, This article is protected by copyright. All rights reserved.
2
2007). Among these HSPs, the HSP70 family represents one of the most highly conserved proteins (Mayer & Bukau, 2005), which also has several functions in Eukaryotic organisms including translocation, folding newly synthesized proteins, degradation of unstable, and misfolded proteins, as well as prevention and dissolution of protein complexes (Daugarrd et al., 2007). Genes encoding HSP70 traditionally are divided into two groups. The first group is the most abundantly induced proteins, which can be induced quickly under a variety of stress conditions, while returned to a normal expression level under non-stressful conditions. However, the second group is not stress-inducible and is generally referred to as being constitutively expressed or as Heat Shock Cognates protein 70 (HSC70) (Denlinger et al., 2001; Qin et al., 2003; Bahar et al., 2013). As an important member of HSP70 family, HSC70 played important roles in homeostasis and immunity by functioning in protein synthesis, folding, transporting and degradation (Hartl & Hayer-Hartl, 2002; Lee et al., 2009; Su et al., 2010; Stankowski et al., 2011). Some reports indicated that hsc70 in organism was presented under most conditions and may or may not be influenced by heat, cold, heavy metal pollutant or endocrine disrupting pollutant stresses (Shim et al., 2006; Yoshimi et al., 2009; Gkouvitsas et al., 2009; Zhang & Denlinger, 2010; Morales et al., 2011). However, recent evidence indicated that the general pattern of hsc70 expression is also variable in response to different development stages (Mahroof et al., 2005) or stressors, such as temperature stress (Sonoda et al., 2006a,b; Wang et al., 2008; Wang et al., 2012), insecticides (Yoshimi et al., 2009), heavy metals (Yoshimi et al., 2009; Wang et al., 2012), and food restriction (Shim et al., 2006; Wang et al., 2012). Mirid bug, A. Lucorum, currently is one of key pests in Bt cotton fields in China (Lu et al., This article is protected by copyright. All rights reserved.
3
2010), and their feeding resulted in extremely plants losses both yield and quality (Lu et al., 2010; Lu & Wu, 2008). Due to the strong temperature adaptability of both nymphs and adults, A. lucorum has spread over to most cotton planting regions in China (Lu et al., 2007; Lu et al., 2010; Lu & Wu, 2008), and calendar-based insecticide sprays are the sole management option for A. lucorum (Lu et al., 2007). To better understand how A. lucorum regulated its hsp in response to thermal and pesticide stresses, Sun et al. (2014) early studied the expression of hsp90 in A. lucorum treated with four highly-active pesticides (cyhalothrin, imidacloprid, chlorpyrifos, and emamectin benzoate) and thermal stresses, indicating that Alhsp90 of A. lucorum may be an important gene involving in the resistance or tolerance to both temperature and pesticide stresses. However, exception for hsp90, whether hsc70 was involved in the response to pesticide and thermal stresses on A. lucorum was still unknown. Therefore, in this study, we firstly identified the full-length cDNA of A. lucorum hsc70 (denoted as Alhsc70) by RT-PCR and RACE, and then investigated the transcriptional and translation responses of Alhsc70 in A. lucorum in response to different temperatures, extreme temperatures (4°C and 40°C) or pesticides by means of qRT-PCR and western blot techniques.
Materials and methods Collection, culture, and treatment of A. lucorum under different temperatures A colony of A. lucorum was established from about 900 adults collected from the broad bean fields of Dafeng and Dongtai City, Jiangsu Province, China. The colony was reared on sauteed green beans in the light incubator at the temperatures of 25 ± 1°C, RH 70% ± 5% This article is protected by copyright. All rights reserved.
4
humidity, and photoperiod 12 : 12 (L : D). According to the study reported by Sun et al. (2014) the tested temperatures were setup at 18, 21, 24, 27, 31, and 33°C, with 24°C as the control. More than 400 A. lucorum eggs were reared at each aforementioned temperature. A. lucorum samples collected were separated into nymph and adult stages. The nymph stage was divided into 1st, 2nd, 3rd, 4th, or 5th instars, and the adult stages were categorized based on new emergence (1-day-old), pre-mating (5-day-old) and post-mating (11-day-old). Each treatment sampled five A. lucorum adults or nymphs with 5 replicates for a total of 25 adults or nymphs were sampled at each stage or temperature. More than 120 2-d old female or male adults were treated at extreme temperatures (4 and 40°C) in insect rearing boxes with sauteed green beans as host for 1 h. Same sampling number of 2-d old female or male adults reared at 24°C were set as controls. Five female or male adults were collected each time after treatment, and this collection was repeated 12 times for a total of 60 adults at each temperature. These treatments were replicated two times, one was for real-time PCR, and the other was for western blot.
Bioassay and pesticide treatment For the pesticide treatments, the topical treatment technique reported by FAO (1980), Shi et al. (2011) and Sun et al. (2014) were adopted for the toxicity bioassay of cyhalothrin, imidacloprid, chlorpyrifos, emamectin benzoate, and acetone alone. The detail information about aforementioned 4 pesticides, such as the purity and manufactures of these 4 pesticides, the LD50 and LD20 value of each aforementioned pesticide to A. lucorum adults were obtained This article is protected by copyright. All rights reserved.
5
in our previous study reported by Sun et al. (2014) and listed in supplementary Table 1. Sixty female or male adults were treated with each aforementioned pesticide (Induction dose = LD20, LD50), respectively. The same batches of female or male adults reared at 24°C were set as CK controls (Ct), and the same batches of Ct controls adults treated with acetone alone were set as Ac controls. For pesticide treatments and Ac controls, 2-day old adults were treated with acetone (0.25 μL/A. lucorum). Mortality was assessed after 48 h treatment. Surviving adults after pesticides treatments, Ct controls and Ac controls were collected and stored at −80°C. These testaments, which adults were treated with each aforementioned 4 pesticides (Induction dose = LD50), Ct controls and Ac controls, were also performed two times, one was for real-time PCR, and the other was for western blot.
Cloning and sequencing the full length cDNA of Alhsc70 In this study, A. lucorum hsc70 (Alhsc70) fragment from 1 bp to 1605 bp of this 2333 bp full-length gene was first obtained from adult cDNA library that had been reported by Sun et al. (2014). And then, the 5' and 3' ends of Alhsc70 were obtained by rapid amplification of cDNA ends (RACE) and SMARTer™ RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA). Two pairs of Gene-specific Primers (GSPs) for RACE were used to obtain the full-length
cDNA
of
Alhsc70.
Two
AACCTGGCCCTGGTGATGGAAGTGT-3', GTTGTCAAAGTCCTCACCGCCCAAGT-3') CCATCGCCTACGGCCTCGACAAG-3',
5'GSPs
(5'GSP1:
5'-
5'GSP2: and
two
3'GSPs,
5'(3'GSP1:5'3'GSP2:5'-
CACAGTCCCCGCCTACTTCAACGACT-3') were designed based on the partial sequence This article is protected by copyright. All rights reserved.
6
obtained by adult cDNA library using Primer 5.0 software.
Bioinformatics analysis of Alhsc70 A sequence similar search both in the nucleotide and amino acid levels were performed with the BLAST program at the National Center for Biotechnology Information (http://www. ncbi. Nlm. Nih. gov/BLAST/). The inferred amino acid sequence was analyzed with the Expert Protein Analysis System (http://www.expasy.org/). Multiple alignment of AlHSC70 was performed with GeneDoc 3.2 software. Therefore, a phylogenic tree was constructed by ClustalX 2.0 and MEGA 4.0 based on the obtained AlHSC70 sequences and other known insect HSC70 amino acid sequences. Bootstrap analysis was used with 1000 replicates to estimate the confidence of branches produced by the neighbor-joining method.
Relative quantitation of AlHSC70 mRNA expression by real-time quantitative RT-PCR after A. lucorum treated by different temperatures and pesticides Total RNA of A. lucorum samples treated with different temperatures or pesticides was extracted using the SV Total RNA Isolation System (Promega, Madison, WI, USA). And then, cDNA was synthesized using MMLV Reverse Transcriptase (Promega, Madison, WI, USA), treated with Ribonuclease H (TaKaRa, Tokyo, Japan), and quantified by spectrophotometry. The SYBR Premix Ex Taq Kit (TaKaRa, Tokyo, Japan) were used to conduct the qRT-PCR reactions, and the qRT-PCR products of this Kit were limited from 100 bp to 300 bp. Therefore, the 279 bp length of Alhsc70 (GenBank accession no. KC119044) product and 177 bp length of β-actin (GenBank accession no. JN616391) product were designed for qRT-PCR. This article is protected by copyright. All rights reserved.
7
The primers used for qRT-PCR of Alhsc70 were 5'-CGCCGCCAAGAACACCTTG-3' and 5'-AATCCGCCGGGGAAACCT-3'. The endogenous reference gene β-actin of A. lucorum was used for data normalization with primers were 5'-ACCTGTACGCCAACACCGT-3' and 5'-TGGAGAGAGAGGCGAGGAT-3'. All the primers for qRT-PCR were designed by Primer 5.0 software. Dissociation curve analysis and gel electrophoresis showed that only the target gene was synthesized. Real-time fluorescence data of reactions containing SYBR Green I were carried out using the Bio-rad iCycler real-time quantitative RT-PCR detection system, and values were determined using iCycler iQ real-time detection system software (version 3.0a; Bio-Rad). Real-time reactions of each treatment were replicated 5 times, and non-template control reactions were performed in triplicate for each primer pair. The qRT-PCR cycling parameters were 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, 58°C for 15 s and then 95°C for 19s, and finally a dissociation curve from 60 to 95°C with an increment of 0.5°C per 5 s. To estimate the real-time quantitative RT-PCR amplification efficiency of primers, a standard curve of five dilution series (1 × 102, 1 × 101, 1 × 100, 1 × 10−1, and 1 × 10−2 ng) was constructed from purified cDNA fragments obtained from previous qRT-PCRs using the same primer set. The efficiency of primers was calculated using the formula E = 10−1/SLOPE. The amplification efficiency values of Alhsc70 and β-actin were 2.162 ± 0.091 and 2.115 ± 0.046, respectively. The relative gene expression levels of Alhsc70 after A. lucorum samples treated with different temperatures and pesticides were represented by relative quantification (RQ) values calculated by the 2-ΔΔCt method (Livak & Schmittgen, 2001).
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Western blot To identify AlHSC70 expression at protein level after extreme heat, extreme cold or different pesticide stresses, more than 30 of the 2-d female or male adults were selected to perform western blot for each sample according to our method (Sun et al., 2014). Total proteins were extracted using the Tissue Protein Extraction Reagent Kit (Nanjing Zoonbio Tech, Co., Ltd.). Total protein concentrations were determined using the bicinchoninic acid (BCA) method (Nanjing Zoonbio Tech, Co., Ltd.). Western blot analysis was performed according to Song et al. (2012) with small modification. Protein samples were electrophoresed on 10% SDS-polyacrlamide gel and electro-blotted to NC membrane (Bio-rad). Transfer was run at 100 mA for 3 h, using Tris/glycine buffer. Membranes were blocked with 5% nonfat powdered milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and incubated for 1 h at 37°C. The primary antibody against AlHSC70, which was produced from the AlHSC70 protein by Nanjing Zoonbio Tech, Co., Ltd., was used at 1:1000 dilutions and incubated for 1 h at 37°C. The AlHSC70 protein obtained by prokaryotic expressed from the cDNA of Alhsc70 ORF was also by Nanjing Zoonbio Tech, Co., Ltd. The membrane was washed three times in TBS-T, 5min each, and then incubated with HRP conjugated goat anti-rabbit IgG secondary antibody (Nanjing Zoonbio Tech, Co., Ltd.) diluted 1 : 5000 in blocking buffer for 1 h at 37°C. The membrane was washed three times with TBS-T, 5 min each, and then developed by using enhanced chemiluminescence advance kit (GE Healthcare). For each experiment, samples containing an equal amount of total protein were run on the same gel. Densitometric analysis of the immunoblots was performed using
a
desktop
scanner
(Hanwang
This article is protected by copyright. All rights reserved.
E60)
and
Image
J
free
software 9
(http://rsb.info.nih.gov/ij/index.html). Furthermore, the western blot for β-actin was also conducted with above method at the same time, and mouse anti-β-actin antibody using for western blot was purchased from Nanjing Zoonbio Tech, Co., Ltd. The optical density of immunoblots was determined by scanning densitometry and the result was presented in densitometric units. Translation was calculated by dividing the normalized AlHSC70 density by the normalized β-actin density (AlHSC70/β-actin ratio) (Song et al., 2012). The western blotting was replicated three times.
Statistical analyses All statistical analyses were executed using the software package SAS version 9.0. Statistical differences of Alhsc70 gene and protein expression in A. lucorum treated with different pesticide and temperature stresses were determined by the one-way analysis of variance, followed by the Tukey’s honestly significance difference (HSD) test (P < 0.05 and P < 0.01). The P-value, F-value, R-value, SE-value (Standard Error of regression function) and the shape of the regression figure were used to identify which mathematical model or function was the most suitable for revealing the regression correlation between relative expression of Alhsc70 and temperature.
Results Phylogenetic analyses By combining the SMART cDNA library of A. lucorum with RACE approaches, two fragments corresponding to the 5' and 3' ends of Alhsc70 cDNA were amplified. Finally, a This article is protected by copyright. All rights reserved.
10
nucleotide sequence of 2333 bp representing the complete cDNA sequence of Alhsc70 was obtained and deposited in GenBank (accession no. KC119044). The full-length cDNA of Alhsc70 was 2333 bp long, including a 5'-untranslated region (UTR) of 108 bp, a 3'-UTR of 254 bp, a canonical polyadenylation signal sequence AATAAA, a poly(A) tail, and an ORF of 1971 bp. The ORF encoded a polypeptide of 656 amino acids with a predicted molecular weight of 71.57 kDa and a theoretical isoelectric point of 5.38. In addition, Classical HSP70 protein signature motifs include IDLGTTYS (AA 10–17), IFDLGGGTFDVSIL (AA 200–213) and IVLVGGSTRIPKVQKL (AA 337–352). Three other typical motifs are also found in the Alhsc70: the first is a deduced ATP-GTP binding site, AEAYLGQK (AA 132–139);
the
second
is
a
putative
bipartite
nuclear
localization
signal
[(KRKYKKDLTT(Q)NKRALRRL (AA 249–266); KRALRRLRTA(S)CERAKRTL (AA 260–277)], which is needed for the selective translocation of HSP70 into the nucleus (Knowlton & Salfity, 1996); the third is a non-organellar consensus motif RARFEEL (AA 302–308). The sequence of the ATPase domain (AA 1–336) of HSP70 is highly conserved, while the C-terminal domain is less conserved than the ATPase domain (Figs. 1, 2) (Su et al., 2010). Furthermore, conserved motif EEVD was also found in the C-terminal of AlHSC70. In addition, 15 characteristic residues (IVLVGGSTRIPKVQKL, AA 337–352), which only presented in constitutive HSC70 amino acid sequence but not in inducible HSP70 were also found in AlHSC70 (Chen et al., 2006). Among the HSC70 proteins compared, the highest percentage identity of AlHSC70 was with HSP70 from Nilaparvata lugens (95% identity). The lowest percentage identity and similarity was Plutella xylostella HSC70 with HSC70 from Aedes aegypti (87% identity). This article is protected by copyright. All rights reserved.
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BLAST results from GenBank indicated a high percentage identity of AlHSC70 compared with other insect HSC70s, and the lowest percentage identity and similarity was those of AlHSC70 with HSP70 from Anopheles gambiae (85% identity, accession no. XP_315042). A phylogenetic tree was constructed with CLUSTAL X 2.0 and MEGA 4 based on 29 HSC70s from eight different insect orders (Fig. 3). Results of the phylogenetic tree showed that 24 HSC70s
belonged
to
Hemiptera,
Lepidoptera,
Blattodea,
Anoplura,
Coleopteran,
Hymenopera, and Orthoptera insects located in a close branch, and the sequence similarity among these HSC70s was at least 85%. HSC70s belonged to Diptera insects located in the branch far from that of the other seven insect orders. However, the sequence similarity among these 29 HSC70s was at least 85%, which may indicate that these HSC70 genes were derived from the same ancestral gene.
Quantitative analysis of Alhsc70 expression when A. lucorum was reared at different temperatures The expression of Alhsc70 in A. lucorum nymphs and adults reared at ~24°C were lower than other temperatures (Fig. 4), which indicated that the expression of Alhsc70 in A. lucorum nymphs and adults can be up-regulated by both cold shock (18°C) and heat shock (30°C and 33°C) significantly (P < 0.01) (Fig. 4). Moreover, the polynomial correlation with mathematical function y = ax2 + bx + c was regarded as the most suitable regression correlation for indicating the function relationship between relative expression of Alhsc70 and temperature among all these functions of software package SAS version 9.0, because of the lower SE-value, P-value and higher F-value, R-value. After A. lucorum culturing from 18 This article is protected by copyright. All rights reserved.
12
to 33°C, significant polynomial regression correlations between the relative expressions of Alhsc70 with temperatures were found in all the A. lucorum nymph stages and adult stages (Figs. 5, 6) (P < 0.01).
Transcriptional quantitative analysis of Alhsc70 expression when A. lucorum adults treated with extreme temperatures (4 and 40 °C) or 4 different pesticides Real-time PCR technique was used to examine the expression of Alhsc70 in 2-d old adults treated with 4 different pesticides or extreme temperatures (4 and 40°C). Results indicated that the transcriptional expression of Alhsc70 in A. lucorum adults treated with extreme high temperature (40°C) was significantly higher than Ct controls (P < 0.01). Treated by extreme low temperature (4°C), the expression of Alhsc70 in A. lucorum adults were significantly lower than Ct controls (P < 0.05) (Fig. 7). Treated by cyhalothrin, imidacloprid or emamectin benzoate (induction dose = LD50), the expression of Alhsc70 in 2-d old adults were significantly higher than that in Ct controls, Ac controls or A. lucorum adults treated by cyhalothrin, imidacloprid or emamectin benzoate (induction dose = LD20), respectively (P < 0.01) (Fig. 7). Furthermore, the expression of Alhsc70 in A. lucorum adults treated by cyhalothrin (induction dose = LD50) was significantly higher than that adults treated by imidacloprid or emamectin benzoate (induction dose = LD50) (female P < 0.01, male P < 0.05). In addition, a significant difference of the Alhsc70 expression was not found between A. lucorum adults treated by chlorpyrifos (induction dose = LD50) and chlorpyrifos (induction dose = LD20) (P > 0.05). Treated by chlorpyrifos (induction dose = LD20, LD50), the expression of Alhsc70 in A. lucorum adults were significantly lower than Ct and Ac controls This article is protected by copyright. All rights reserved.
13
(female P < 0.05, male P
