Cloning of a new HSP70 gene from western flowerthrips, Frankliniella occidentalis, and expression patterns during thermal stress

Insects

Frankliniella occidentalis adults were originally collected from Zhejiang Academy of Agricultural Sciences in September 2008 and the adults reared in the laboratory according to Li et al. (2011). The experimental colony was fed on Phaseolus vulgaris maintained in a QHX-300BS-III climate chamber at 25 ± 1 °C, 70–80% RH, and a 16:8 h light: dark photoperiod.

High and low temperature treatments

Second instar larvae (n = 120) were collected, placed in glass tubes and exposed to various temperatures for 1 h. Larvae were exposed to cold (−6, −8, −10, −12, −14 °C) and hot (33, 35, 37, 39, 41 °C) conditions using a temperature controller (DC-3010, Ningbo, Zhejiang, China). The control group consisted of thrips maintained at 26 °C, and all treatments were replicated four times.

Recovery times

Adults (n = 200) were collected and placed together in glass tubes; two replicates of each sample were prepared. The adults were exposed to 40 °C for 1 h in a constant temperature water bath and allowed to recover at 26 °C for 0, 1, 1.5, 2 and 2.5 h. Treated and control samples were frozen in liquid nitrogen for 5 min and then stored at −80 °C. Each recovery period was replicated four times. The same protocol was used for second instar nymphs and pupae.

RNA extraction and cDNA synthesis

Total RNA was extracted from F. occidentalis adults using the SV Total RNA Isolation System (Promega, San Luis Obispo, CA, USA). The concentration and quality of RNA were analyzed by spectrophotometry (Eppendorf Bio Photometer Plus, Hamburg, Germany) and agarose gel electrophoresis. Total RNA (1 μg) was used as a template and oligo(dT)₁₈ primers were used to generate the first strand cDNA as recommended in the First Strand cDNA Synthesis Kit (Clontech, Mountain View, CA, USA).

Cloning full-length Fohsp706

Primers (Table 1) were designed to amplify DNA fragments of F. occidentalis based on sequences obtained from the transcriptome. PCR reactions were as follows: 94 °C for 3 min, 19 cycles of 94 °C for 30 s, 64–44 °C (decreasing by 1 °C/cycle) for 30 s, 72 °C for 1 min, and then 30 cycles of 94 °C for 30 s, 45 °C for 30 s, and 72 °C for 1 min, with extension at 72 °C for 10 min. Purified products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) and transformed into competent Escherichia coli DH5α cells for sequencing.

Gene-specific primers (Table 1) were designed to obtain 5′ and 3′ regions using the SMART RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) based on the sequence of partial fragments. PCR parameters were as follows: 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 68 °C for 30 s, and 72 °C for 3 min, followed by extension at 72 °C for 10 min. Bands of the expected size were cloned and sequenced as described above.

Sequence analysis of Fohsp706

Nucleotide and amino acid sequence similarities were evaluated using the BLAST program available at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/) was used to identify complete open reading frame. Amino acid sequences (Gasteiger et al., 2003) were deduced using ExPASy sequence analysis tools, and motifs were identified using ScanProsite software (http://www.expasy.org/tools/scanprosite). Homology searches were carried out using Blast programs of NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree of insect HSP70s was constructed by neighboring joining, minimum evolution, maximum likelihood, and maximum reduction methods using MEGA X (Kumar et al., 2018).

Amplification of genomic DNA

Genomic DNA of F. occidentalis adults was extracted according to AxyPrep instructions, and samples were stored at −20 °C. Based on the full-length cDNA sequence of Fohsp706 in F. occidentalis, multiple primers (Table 1) were designed to amplify the genomic sequence. PCR products were cloned and sequenced as described above. Homologous sequences were obtained using BlastN. After confirmation, full-length hsp70 genomic sequences and the DNA sequence of the new hsp70 gene (Fohsp706) were compared, and intron distribution was analyzed.

Quantitative real-time reverse transcriptase PCR (qRT-PCR)

Real-time quantitative cDNA was synthesized using instructions provided with the PrimeScript RT Reagent Kit (Bio-Rad, Berkeley, CA, USA), and primers were designed using Primer 5.0 software (Table 1). Reactions were conducted using a CFX-96 Real-Time PCR System (Bio-Rad, Berkeley, CA, USA), and each PCR reaction included three replicates. PCR reactions included iTaq Universal SYBR Green Supermix (2×) (Bio-Rad, Berkeley, CA, USA), one μL of each forward and reverse primer (10 μmol L^–1) (Table 1), two μL of cDNA template (2.5 × 10^–4 μg μL^–1), and six μL of ddH₂O. Quantitative real-time reverse transcriptase PCR conditions were as follows: 95 °C for 30 s, 40 cycles of 95 °C for 30 s, and 56.3 °C for 15 s; melting curve analysis was then performed to determine the specificity of PCR products.

Statistical analysis

Expression levels in each treatment were analyzed using the 2^–ΔΔCt method (Livak & Schmittgen, 2001) and differences were detected using Tukey’s multiple comparison method. All data were analyzed and processed using SPSS16.0 software, and the significance level was P < 0.05.

Characterization of a new hsp70 in F. occidentalis

A 257-bp fragment was amplified by PCR with internal primers (hsp706DP-F/R, Table 1) using F. occidentalis cDNA as a template and then cloned and sequenced. BlastN alignment revealed 75–91% identity with hsp70 in other insects, suggesting that the fragment encoded part of an hsp70 gene in F. occidentalis. The 5′-terminal (563 bp) and 3′-terminal (1519 bp) fragments of the putative F. occidentalis hsp70 were subsequently amplified by RACE. The full-length hsp70 was then obtained by splicing the 257-, 563-, and 1519-bp fragments together with DNAMAN (version 6.0, Lynnon Biosoft, America). The full-length sequence was verified by the cDNA sequence and was deposited in GenBank as Fohsp706 (accession no. MK603518).

The ORF Finder revealed that Fohsp706 contained a 5′ untranslated region (159 bp 5′ UTR), a 1917-bp ORF and 3′ untranslated region (1713 bp 3′UTR); the 3′UTR contained a typical polyA tail. ProtParam analysis tool (ExPASy) showed that Fohsp706 encoded 638 amino acids with a theoretical molecular weight of 70.1 kDa, a formula of C₃₀₅₁H₄₉₁₂N₈₇₆O₉₈₁S₁₆ and an isoelectric point of 5.31. Alanine (Ala) was the most prevalent amino acid (9.6% of total), followed by aspartic acid (Asp), which accounted for 7.7%. The predicted protein contained 94 negatively charged residues (Asp + Glu) and 81 positively charged residues (Arg + Lys). The overall stability index of the protein is 40.51 (≤40 is considered stable), the aliphatic index is 81.66, and the hydrophilicity index is −0.461 (non-hydrophilic). ScanProsite showed that the deduced FoHSP706 amino acid sequence contained three HSP70 signature sequences: IDLGTTYS (6–13 aa), IFDLGGGTFDVSVL (194–207 aa) and VVLVGGSTRIPKVQS (332–346 aa). The C-terminal end is a typical EEVD (Glu-Glu-Val-Asp), indicating that the protein exists in the cytoplasm of F. occidentalis (Fig. S1).

Genome structure of Fohsp706

A pair of primers, hsp706DNA-F/R (Table 1.) was designed to amplify the genomic copy of Fohsp706, and a 2022-bp sequence was obtained. In Table 2, the genomic structures of Fohsps from F. occidentalis were compared, except Fohsc705, the genomic structure of which was not available. The genomic form of Fohsp706 had no introns, which suggested that the gene might be inducible. In contrast, Fohsp701 (accession no. KC148536), Fohsp702 (accession no. KC430097), and Fohsp704 (accession no. MF377632) have 4, 7, and 4 introns, respectively; whereas, Fohsp703 (accession no. KY914546) has no introns. Therefore, this indicates that not only the number of introns, but also the distribution positions of introns in HSP70 gene of the same species may be different (Fig. 1).

Phylogenetic analysis of FoHSP706

ClustalW (Thompson, Gibson & Higgins, 2002) indicated that the deduced amino acid sequence of FoHSP706 was over 70% similar to other HSP70s (Fig. S2). Several HSP70 amino acid sequences, including the FoHSP703 mentioned above, were compared with FoHSP706. Phylogenetic trees of HSP70s were constructed using MEGA X and the neighbor joining, minimum evolution, maximum reduction, and maximum likelihood methods (Zuckerkandl & Pauling, 1965; Kumar et al., 2018). The tree was divided into five branches, and these six FoHSP70s were distributed in different branches (Fig. 2). Phylogenetic analysis showed that FoHSP706 has a close evolutionary relationship with HSP70 of Anaphothrips obscurus (Muller), which also belongs to Thripinae (Gao & Feng, 2018).

Expression of Fohsp706 in response to cold and heat shock

RT-PCR was used to study expression profiles of Fohsp706 in second instar larvae of F. occidentalis. qRT-PCR assays resulted in the production of single amplicons with efficiency values between 93.5 and 107.3% and an R² value of 0.980, which meets the basic requirements of real-time quantitative research. The relative mRNA levels of Fohsp706 were compared at −14, −12, −10, −8, −6, 26, 33, 35, 37, 39 and 41 °C. Fohsp706 was significantly induced in response to hot and cold temperatures as compared with the control (26°C). With respect to high-temperature stress, expression of Fohsp706 was highest at 35 °C (Fig. 3A); in response to cold stress, expression of Fohsp706 was highest at −8 °C (Fig. 3B).

In response to high-temperature stress (40 °C), the expression of Fohsp706 in F. occidentalis adults after recovery times of 0, 0.5, 1.5, and 2 h remained significantly induced in comparison to the control group. However, the recovery time of 1.0 h was not significantly different from the control group (Fig. 4A). Statistical analysis showed that different recovery times after high-temperature exposure had no effect on Fohsp706 expression in F. occidentalis adults (F_{5, 17} = 9.529, P < 0.001). Contrary to adults, Fohsp706 expression in pupae was significantly induced after the recovery time of 1 h, but not at other time intervals (Fig. 4B). Fohsp706 expression in larvae showed significant induction after recovery times of 0, 0.5, 1, and 2 h (but not 1.5 h) as compared to the control (Fig. 4C). Thus, different recovery times after high-temperature treatment impact Fohsp706 expression in F. occidentalis pupae and larvae.