Characterization of T-complex polypeptide 1 (TCP-1) from the Chilo suppressalis HSP60 family and its expression in response to temperature stress

1. Introduction

Molecular chaperones are an evolutionarily-conserved family of proteins that are responsible for the folding and assembly of newly-synthesized proteins and the refolding of denatured proteins (Yam et al. 2008). Furthermore,chaperones can prevent the unfolding of denatured proteins and promote the aggregation of dissolved protein (Sonna et al. 2002). Heat shock protein, HSP60, is a ubiquitous,highly-abundant molecular chaperone that is induced in response to high temperature, hypoxia, infection, and trauma (Ohashi et al. 2000; Borges and Ramos 2005).HSP60 plays an important role in enhancing the ability of cells to tolerate stress and maintaining protein stability and functionality (Wang et al. 2012). HSP60 is further classified into types I and II (Chen et al. 2011); type II occurs in the cytosol of eukaryotes and includes T-complex polypeptide 1(TCP-1), which is conserved in eukaryotes, archaebacteria and eubacteria (Kubota et al. 1995). TCP-1 assists in the folding of newly synthesized proteins, such as tubulins and actins (Sternlicht et al. 1993), cyclin E (Won et al. 1998),and α-transducin (Farr et al. 1997).

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The rice stem borer, Chilo suppressalis (Walker)(Lepidoptera: Pyralidae) is a major pest of rice in Asia, North Africa and southern Europe (Luo et al. 2014). C. suppressalis occurs in both southern and northern China, which indicates a high tolerance to extreme temperatures (Guo et al. 2002).The mechanistic basis of thermal tolerance in C. suppressalis is unclear; genes encoding four large and seven small HSPs have been identified in C. suppressalis and were proposed to have roles in temperature stress (Sonoda et al. 2006; Cui et al. 2010a, b, c; Lu et al. 2014; Pan et al. 2017).

HSP60 has been previously characterized in C. suppressalis(Cui et al. 2010c), but its role in this insect pest is not entirely clear. In this study, we isolated and characterized Tcp-1 in C. suppressalis. The expression of Tcp-1 in various insect tissues and in response to thermal stress were also investigated.

2. Materials and methods

2.1. Insects

Populations of C. suppressalis were collected from a suburb of Yangzhou (32°39´N, 119°42´E), which was located in the Jiangsu Province, China. C. suppressalis was reared for three or more generations in an indoor breeding room maintained at (27±1)°C with a 16 h L:8 h D photoperiod and approximately 75% humidity as described previously(Shang et al. 1979).

其正负用来表示各明细费用占总费用构成的变动方向。当VSV>0，称为正向变动，说明某项目构成比增加；反之，则称为负向变动，说明某项目构成比减少。

2.2. RNA extraction and cDNA synthesis

Total RNA was isolated using the SV Total RNA Isolation System (Promega, USA) and combined with DNase digestion to eliminate DNA contamination. cDNA was synthesized from 1 μL of RNA using oligo(dT)18 primers(Fermentas, Canada) by rapid ampli fication of cDNA ends(3´- and 5´-RACE); these experiments were conducted using the TaKaRa RACE cDNA Ampli fication Kit (TaKaRa, Dalian,China) according to the manufacturer.

2.3. Cloning and characterization of Tcp-1

The deduced amino acid sequence of TCP-1 in C. suppressalis was highly similar to orthologous proteins in other insects. We used CLUSTALX and MEGA 7.0 to compare selected TCP-1 proteins using neighbor-joining, minimum evolution, maximum likelihood and maximum parsimony methods. The four phylogenetic trees were highly similar and each contained two clusters (Fig. 2). TCP-1 from C. suppressalis, Bombyx mori, Danaus plexippus, Papilio xuthus and Operophtera brumata fell into a single well-supported cluster with a high level of similarity (60–99%),indicating that TCP-1 in C. suppressalis is closely related to other Lepidopteran forms of the protein.

2.4. Sample preparation

In experiments focusing on differential expression in seven tissue types, fifth-instar larvae were selected with similar body sizes; each group contained ten larvae and experiments were repeated four times. Larvae were anesthetized on ice before dissection. Heads, epidermis,fat body, foregut, midgut, hindgut, and malpighian tubules were collected from larvae and rinsed with a 0.9% sodium chloride solution. The samples were frozen immediately in liquid nitrogen and stored at –70°C until needed for analysis.

Table 1 Primers used in this study1)

1) Tm, melting temperature; E, real-time qPCR efficiency (calculated from the standard curve).

Primer name Primer sequence (5´→3´) Tm (°C) E (%)For RT-PCR TCP-1 GARGTYGGWGATGGNACYAA GCCATRTCWCCRAAYTT For RACE PCR TCP-1-5´ TCGTGGTTACACCCAAGCGGAGCA 68.0 TCP-1-3´ CCAGCCGTTGTCAAAGGTATCAGA 68.0 For genomic ampli fication TCP-1 GCACATGAGGAGAACATT CTCGCGTAGTCCAACAAA For qPCR TCP-1 AATGGAAAGTGTTGAATCCCGT 60.1 107.8 AGTTGTCTGCCAGTGCCCGA H3 CTGCACCAAGCACTGGTGGA 56.0 107.5 TAGCGGCGGACTGGAAACG EF1 AAAATGGACTCGACTGAACCCC 56.6 94.0 TCTCCGTGCCAACCAGAAATA

2.5. Temperature treatments

In this study, degenerative primers based on a conserved region of Tcp-1 were used to amplify cDNAs derived from C. suppressalis larvae. The 5´ and 3´ flanking sequences were obtained by 5´- and 3´-RACE. After assembly, a 2 144 bp full-length cDNA sequence was obtained (Fig. 1)that contained start and stop codons at 99–101 bp (ATG)and 1 733–1 735 bp (TAA), respectively. The sequence contained a 98 bp 5´-UTR with a polyadenylation signal sequence (AATAAA), a 1 635-bp ORF, a 408-bp 3´-UTR,and a poly(A) tail at the 3´ end (GenBank accession number: MF471349) (Fig. 1). The deduced translational product contained 545 amino acids with a predicted mass of 59.42 kDa and isoelectric point of 5.29. TCP-1 had two signature sequences homologous to the TCP-1 family of proteins, namely RSAYGPNGMNKMI (residues 44–56) and QDAEVGDGT (residues 93–101). Genomic analyses indicated there were lacked introns in the Tcp-1 gene.

2.6. Quantitative real-time PCR analysis

Total RNA was extracted using the SV Total RNA Isolation System (Z3100, Promega, USA), followed by DNase treatment to eliminate DNA contamination. The integrity of RNA in all samples was verified by comparing the ribosomal RNA bands in ethidium bromide-stained gels and purity was evaluated using spectrophotometric measurements(BioPhotometer plus; Eppendorf, Germany) at 260 and 280 nm. Reactions were performed in a 20 μL volume containing 10 μL of iTaq™ Universal SYBR® Green Supermix (Bio-Rad, USA), 6 μL ddH2O, 1 μL of each genespecific primer (Table 1) and 2 μL of cDNA templates.Reactions were carried out using a CFX-96 Real-time PCR System (Bio-Rad, USA). The efficiencies of the target and reference genes were similar (Nolan et al. 2006; Bustin et al. 2009). The quantity of Tcp-1 mRNA was calculated using the 2–ΔΔCT method (Pfaffl 2001) and normalized to the abundance of histone 3 (H3) and elongation factor 1 (EF1)genes for tissues and thermal stress, respectively (Xu et al.2017). Expression in larvae maintained at (27±1)°C and in insect heads were used as controls as described previously(Schmittgen and Livak 2008). The homogeneity of PCR products was con firmed by melting curve analysis, which was read every 5 s per 0.5°C increment from 65 to 95°C.Treatments included four replicates, and each reaction was run in triplicate.

2.7. Phylogenetic analysis

（5）材料配套标准不完善。标准绝对不是独立存在的，航空用金属材料标准的有效实施需要一系列活动的高效配合，其中配套标准的制定完善度与实施力度是不可或缺的关键点之一。只有制定完善的配套标准，才能保证航空用金属材料标准全面有效地实施、推广。

ORF Finder Software (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to identify ORFs in the Tcp-1 sequence.Sequence analysis tools of the ExPASy Molecular Biology Server, including Translate, Compute pI/MW and Blast(Swiss Institute of Bioinformatics). ScanProsite (http://prosite.expasy.org/scanprosite/) was used to detect motifs in TCP-1. Amino acid sequences were used to estimate phylogeny using neighbor-joining, minimum evolution, maximum likelihood, and maximum parsimony methods. Phylogenetic trees were constructed with 1 000 bootstrap replicates using MEGA version 7.0 (Kumar et al.2016).

2.8. Statistical analysis

The expression of Tcp-1 was induced when temperature increased from 27 to 43°C (F6, 18=4.970, P=0.002)(Fig. 5). Tcp-1 expression was 1.35- and 1.29-fold higher than the control (27°C) after a 2-h exposure to 30 and 36°C, respectively. Expression of Tcp-1 declined when temperature exceeded 36°C, and the expression level at 43°C was significantly lower than that at 27°C (P=0.002;Fig. 5).

3. Results

3.1. Sequence analysis

Larvae (n=30) were con fined individually in glass tubes, and experimental groups were exposed to selected temperatures(–11, –9, –8, –6, –3, 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30,33, 36, 39, 42, and 43°C) for 2 h in a constant temperature subzero incubator (DC-3010, Jiangnan Equipment,Changzhou, China). Larvae were then allowed to recover at(27±1)°C for 2 h, after which surviving larvae were frozen in liquid nitrogen and stored at –70°C. The larvae exposed to 27°C were regarded as the control group. Each treatment included four or more surviving larvae.

推荐理由:作者曾获以色列布伦纳奖、以色列总理奖、美国犹太图书奖等奖项，这部小说被他视为自己创作成熟的标志。中文译本首次出版。小说讲述了1930年代巴勒斯坦的一个小村庄里，朱迪斯与她的三个爱慕者之间发生的故事。作者从宗教故事和神话传说中汲取灵感，并融入犹太乡村的风土人情，用魔幻现实主义的高超技法，将这个《雅歌》般的传奇娓娓道来。

3.2. Phylogenetic analysis

Degenerate primers were used to amplify the partial segments of Tcp-1 (Table 1). Full-length cDNAs of Tcp-1 were obtained using 5´- and 3´-RACE (SMARTer™ RACE,Clontech, USA) and the primers were shown in Table 1. The sequence of the Tcp-1 ORF was con firmed by 5´-RACE.Genomic DNA of C. suppressalis was extracted using the AxyprepTM Multisource Genomic DNA Kit (Axygen, USA).Based on the sequence of the full-length Tcp-1 gene, pairs of specific primers (Table 1) were designed to amplify Tcp-1 genomic fragments. These products were purified using a gel extraction kit (Axygen, USA), cloned into pGEM-T Easy vector (Promega, USA), and transformed into competent Escherichia coli DH5α cells for sequencing.

Fig. 1 The complete cDNA sequence and predicted amino acid sequence of the gene encoding T-complex polypeptide 1(TCP-1) in Chilo suppressalis. Two highly-conserved typical motifs of TCP-1 family were selected in the boxes. The asterisk indicates the translational termination codon.

3.3. Expression of Tcp-1 in different tissues of C. suppressalis larvae

Quantitative real-time PCR indicated that Tcp-1 was expressed differentially in the seven tissues/organs of C. suppressalis larvae (F6, 17=7.093; P=0.001) (Fig. 3).Expression of Tcp-1 was the highest in the hindgut and the lowest in malpighian tubules. Expression in larval heads was 4.30 times higher than expression in malpighian tubules.Tcp-1 expression was similar in the foregut, midgut and epidermis (Fig. 3).

3.4. Expression of Tcp-1 in C. suppressalis exposed to temperature stress

Tcp-1 expression was different from the control (27°C) in C. suppressalis larvae exposed to reduced temperatures(F14, 38=4.158; P＜0.001) (Fig. 4). Tcp-1 expression was 2.01-fold higher at 6°C than that of the control (27°C) (Fig. 4).Tcp-1 expression at –6, –3, 9, 12, 15, 18, and 21°C was nearly the same as those of the controls (27°C). Tcp-1 expression was 1.70-fold higher than that of the control(27°C) after a 2-h exposure to 24°C.

Homogeneity of variances among different groups was evaluated by Levene’s test. One-way ANOVA and a LSD test were used to detect significant differences among treatments, followed by Tukey’s tests and SPSS16.0 (Pallant 2007).

谢瑞天对我，确实很好，换句话说，如果对我不这么好，我也不会在他身上浪费那么多的时间。这个年头，有钱的男人大把，不是吗？

4. Discussion

The role of molecular chaperones in response to various stressors has been widely studied in eukaryotes.Our results demonstrated that Tcp-1 expression was the highest in the hindgut of fifth-instar larvae and the lowest in C. suppressalis heads. However, Kumar et al. (2015)reported that the expression of hsp60 in Lucilia cuprina was significantly increased in the malpighian tubules and fat body. One explanation is that hindgut and malpighian tubules reabsorb water, salts and other substances prior to excretion by the insect, and HSPs protect these tissues or organs from toxic injury. This may be also related to the function of TCP-1 in the folding of newly synthesized proteins(Yam et al. 2008), including tubulins and actins (Sternlicht et al. 1993), cyclin E (Won et al. 1998), α-transducin (Farr et al. 1997) and von Hippel-Lindau protein (Hansen et al.2002). Álvarez-Fernández et al. (2015) demonstrated that TCP-1 chaperonin complex as a key regulator of the actin cytoskeleton essential for the wound healing response in Drosophila.

Fig. 2 Phylogenetic tree of T-complex polypeptide 1 (TCP-1) proteins determined by the neighbor-joining method. Chilo suppressalis TCP-1 is labeled with black rectangle.

Fig. 3 Relative expression of Tcp-1 in different tissues (organs)of Chilo suppressalis as determined by quantitative real-time PCR. FG, foregut; MG, midgut; FB, fat body; MT, malpighian tubules; EP, epidermis; HE, head; HG, hindgut. Expression in insect heads was regarded as a control. Columns labeled with different letters indicate significantly different (P＜0.05) by one-way ANOVA and Tukey’s test. Error bars indicate SE.

In this study, Tcp-1 was cloned and characterized from the rice stem borer, C. suppressalis. The cDNA sequence encoded a 1 635-bp ORF, and the predicted translational product contained 545 amino acids. TCP-1 had an estimated mass of 59.42 kDa and an isoelectric point of 5.29. Tcp-1 contained no introns, which is also the case for hsp60 in humans and Chinese hamster (Venner 1990).However, hsp60 had five introns in Frankliniella occidentalis(Lu et al. 2016), indicating heterogeneity in this gene. An inverse correlation between intron size and gene expression has been suggested, and genes lacking introns or containing shorter introns showed higher levels of expression than genes containing multiple or longer introns (Comeron 2004).

Fig. 4 Relative expression of Tcp-1 in Chilo suppressalis larvae exposed to low temperatures. Expression at 27°C was regarded as a control. Columns labeled with different letters indicate significantly different (P＜0.05) by one-way ANOVA and Tukey’s test. Error bars indicate SE.

Fig. 5 Relative expression of Tcp-1 in Chilo suppressalis larvae exposed to elevated temperature (27–43°C). Expression at 27°C was regarded as a control. Columns labeled with different letters indicate significantly different (P＜0.05) by one-way ANOVA and Tukey’s test. Error bars indicate SE.

Heat shock proteins play an important role in environmental stress tolerance and heat adaptation (Frydenberg et al.2003; Hoffmann et al. 2003). The production of HSPs can affect insect growth and development and negatively impact many physiological processes (Krebs and Feder 1997;Feder and Hofmann 1999; Huang and Kang 2007). TCP-1, a member of the HSP60 family, was first shown to be induced by heat shock in human cells (Schena et al. 1996). In this study, expression of Tcp-1 in C. suppressalis was inhibited at 39 and 43°C, indicating that transcription had decreased.However, Cui et al. (2010c) previously demonstrated that hsp60 expression in C. suppressalis was the highest after a 2-h exposure to 36°C and declined at 39°C. Huang and Kang (2007) observed a similar induction pattern for hsp60 in Liriomyza sativae where expression was induced in response to heat shock and inhibited when temperatures exceeded 42.5°C. It has also been reported that heat shock can suppress activation of the mitogen-activated protein kinase (MAPK) pathway (Gao et al. 2014). Inhibition of the MAPK pathway can reduce the synthesis of ternary complex factors (TCFs), which further inhibit expression of TCP-1(Gendron et al. 2003).

1938年4月3日，熊式辉曾向蒋介石提出:“本党成立将近三十年，由革命党改为政党，现在却一不像美之共和民主等政党，二不像苏俄之共产革命专政党，三不像德意之纳粹党及法西斯党，实际并以上三种性质兼有而不俱全，以此来与民社等政党及凭借武力革命之共产党相周旋，一切政治施为，固难求其适应。”㉞蒋介石侍从室的高级幕僚唐纵在1944年4月28日的日记中也提到，“目今党何以不能在政治上起领导作用?党的上层干部，对于主义政策的认识，并不彻底。由革命到取得政权，思想和观念已为之大变，现在大家的观念是现实问题。上级干部在追求权位，下级同志在追求生活。主义、政治、革命，都已忘却了! 消失了!”㉟

Surprisingly, exposure to low temperatures did change Tcp-1 expression in C. suppressalis. Tcp-1 expression was the highest, which was 2.01-fold higher than the control after a 2-h exposure to 6°C. However, Huang and Kang (2007)observed that hsp60 expression at low temperatures (–20,–17.5, –15, –12.5, –10, –7.5, –5, –2.5, 0, 2.5, and 25°C)were nearly the same as the control (25°C). Difference in Tcp-1 expression in C. suppressalis at low temperature may be related to the structure and function of TCP-1 (Yam et al. 2008). Factors that contribute to cold hardiness in insects have been described, including cryoprotectants,supercooling points, antifreeze proteins, homeoviscous adaptation, and heat shock proteins (Storey and Storey 1991; Graether et al. 2000; Kostál et al. 2003). Kayukawa et al. (2005) clearly demonstrated that expression levels of Tcp-1 are highly correlated with the cold hardiness in Delia antiqua pupae. Since the production of HSPs may impact the survival and viability of offspring, it is important to understand the cost/benefit ratio balance in terms of hsp gene expression (Lu et al. 2014).

In summary, TCP-1 helps protect insects from cellular damage. The role of TCP-1 in adjusting actin and tubulin levels is worthy of further study and will advance our understanding of TCP-1 function in insect behavior and development.

5. Conclusion

We obtained Tcp-1 encoding heat shock protein for C. suppressalis, and its predicted amino acid sequence showed high similarities with published TCP-1s of other insects in Lepidoptera. When exposed to heat stress, the expression of Tcp-1 was induced. Our results indicated that Tcp-1 expression was differentially expressed in C. suppressalis tissues, and was impacted by temperature stress, and provided useful information in understanding the thermotolerance of C. suppressalis at the molecular basis.

Acknowledgements

This research was funded by the National Natural Science Foundation of China (31401733) and the Incubation Study Project of Science and Technology of Fuyang Normal University, China (2014KJFH02). We sincerely thank Dr.Carol L. Bender from Consulting Limited Liability Company,USA for editing and providing comments on the manuscript.We express our deep gratitude to the Testing Center of Yangzhou University, China.

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