Molecular Cloning and Expression Analysis of Heat Shock Protein 90 ( Hsp 90 ) of the Mud Crab , Scylla Paramamosain

Heat shock protein 90 (HSP90), functions as a molecular chaperone in protein biosynthesis play an important role in signal transduction, immune responses and embryogenesis. However, the function research of HSP90 in invertebrates is limited. To further understand the role and mechanism of HSP90 in immune and stress response, we have isolated the the fragment of HSP90 homologue from mud crab Scylla paramamosain through transcriptome sequencing of mixture of hepatopancreas and hemocytes, The full-length cDNA of HSP90 is harvested through RACE technology and it contains 73 bp 5’terminal UTR, 577 bp 3’terminal UTR and 2373 bp open reading frame which encoding a 790-amino-acid protein. BLASTP results demonstrate that SpHSP90 share high identity with Tribolium castaneum and Locusta migratoria and other reported crustacean (61%-73%). Phylogenetic tree based on HSP90 proteins show that SpHSP90 and Daphnia pulex form a cluster within the invertebrate group while other reported HSP90s of vertebrate are grouped into another branch. The tissue distribution was tested by quantitative real-time PCR and SpHSP90 was detected express in all test tissues, the results showed that HSP90 was constitutively expressed in Scylla paramamosain. The highest expression of HSP90 was found in hepatopancreas, the following was in heart, and the lowest expression of it was found in hemocyte. The expression patterns of SpHSP90 after challenged by the Staphylococcus aureus, Vibrio harveyi and WSSV were also analyzed by qRT-PCR and the result showed that the expression level of SpHSP90 was enhanced after challenged with the Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Vibro harveyi) and virus (white spot syndrome virus, WSSV). These results suggested that SpHSP90 involved in the immune response against invading pathogens. Furthermore, SpHSP90 was expressed by prokaryotic expression system and purified by His Bind resin chromatography.


Introduction
Heat shock protein90 (HSP90) is one kind of molecular chaperone that widely exists in cells and highly conserved, plays an important role in regulating cell cycle, body immunity and signal transduction, also have an influence on the occurrence and development of tumor, has great significance in clarifying the development of tumor (Picard, 2002;Wiech et al., 1992;Miyata et al., 1992;Holmes, 2008).HSP90 directly involve in protein assembly and transfer, stabling protein structure and regulating cell cycle, and indirectly participate in regulating many signal pathways by adjusting its receptor protein activity and maintaining structure stability of its receptor protein (Miyata et al., 1995;Schumacher et al., 1994;Nathan et al., 1997 ).HSP90 also have interaction with cytoplasmic proteins or nuclear, including regulatory tyrosine kinases, some serine/threonine kinases, transcription factors, cytoskeletal proteins, and βγsubunits of G proteins (Aligue et al., 1994).In recent years, many functions of HSP90 and the relationships between injury and infection have been constantly clarified, and the studies of HSP90 gene sequence, protein function and its role in the embryonic development process have caused wide public concern, including the research of this gene of many kinds of aquatic crustaceans (Gao et al., 2007;Li et al., 2012).It will be induced to express when heat shock responses occur under the stress from environment or pathogens and take part in relevant immune reaction, suggested that they play an important role in response to potentially stress conditions (Polla, 1991;Kregel, 2002;Feder et al., 1999;Jolly et al., 1999).
The Scylla paramamosain, green mud crab belongs to Arthropoda, Crustacea, Decapoda, Portunidae, is one of the most promising and valuable crabs for aquaculture in China (Estam, 1949).The culture area of the green mud crab in China expanded inrecent years (Li et al., 2004).However, With the enlargement of the cultivation scale and intensive culture, couple with the deterioration of ecological environment, Bacterial disease become more serious, even white spot syndrome virus (WSSV) began to infect this kind of crab now, which cause lots of economic losses (Lange et al., 2000;Pandey et al., 2000).Therefore, clarify the expression pattern of HSP90 after pathogen infection will be benefit to the farming of aquatic crustaceans.In the present study, we cloned a full-length cDNA sequence and characterized of a HSP90 from the green mud crab Scylla paramamosain.The immune relevant functions of HSP90 were validated.After being infected with bacteria or WSSV, the transcripts of HSP90 gene were up-regulated, These results suggest that HSP90 plays an important role in defending against bacterial and virus infections.
Healthy adult mud crabs Scylla paramamosain (average body weight: 300 ± 50 g) purchased from a commercial crab farm in Chongming county (Shanghai, China) were reared in 300 L tanks with continuous aeration for a week before processing.Water temperature, pH and salinity was maintained at 18°C, 8.0~8.2, and 10, respectively.

Immune Challenge and Sampling
Crabs were randomly divided into 5 groups including staphylococcus aureus group, vibrio harveyi group and WSSV group, PBS group as negative control of bacterial stimulation, and the supernatant of homogenate of healthy shrimp gill tissue group as negative control of WSSV stimulation.Each group contains three replicates.Each crab was injected with 50 µl staphylococcus aureus supernatant (5×10 7 cfu), or 50 µl vibrio harveyi supernatant (5×10 7 cfu) or 50 µl WSSV supernatant (5×10 5 virus particle) at the base of the right second leg.Crab from the corresponding control was injected with 50 µl PBS or healthy gill tissue supernatant.Hemocytes was collected from each crab at 0, 2, 6, 12, 24, 48 h post-injection, and mixed with an equal volume of pre-cooled anticoagulant solution (0.14 M NaCl, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid, 10 mM EDTA, pH 4.6).Subsequently hemocytes were centrifuged at 800 g at 4°C for 15 min, and hemocyte pellets were immediately suspended in Trizol for RNA extraction.In addition, hemocytes, heart, hepatopancreas, gill, stomach, intestine, muscle, gonad and connective tissue from healthy crabs were collected for SpHSP90 tissue distribution analysis.

RNA Extraction and cDNA Synthesis
Total RNA was isolated from healthy crabs hemocyte, heart, hepatopancreas, gill, stomach, intestines, muscle, connective tissue and gonad using Trizol following the protocol of the manufacturer, as well as isolated from the hemocyte of challenged crabs at 0, 2, 6, 12, 24 and 48h post-challenge, stored at -80°C.The quality was checked by spectrophotometry.Total RNA was reserve transcribed with the M-MLV RTasec DNA Synthesis Kit to get the first strand cDNA.The product was diluted properly and stored at -20°C till analysis.

Tissue Distribution and mRNA Expression Profiles Analysis
Transcript expression profiles of in nine different tissues including hemocyte, heart, hepatopancreas, gills, stomach, intestines, muscle, connective tissue and gonad from S. paramamosain were detected by real-time qRT-PCR using a pair of specific primers: SpHSP90RF (5′-GGACTGGACTCTGGTGAATGA-3′) and SpHSP90RR (5′-CTTGATGTTGTCTGTGCGTGT-3′).A pair of Sp18S primers, Sp18SF (5′-GCTTTCGCAGTAGTTCGTCTTG-3′) and Sp18SR (5′-CCTCGGTTCTATTTTGTCGGT-3′) designed from the 18S rRNA sequence (GenBank Number: FJ646616.1)were used as internal control.The qRT-PCR was performed in a volume of 20 µl containing 10 µl 2×SYBR Premix Ex Taq, 2 µl cDNA, 4 µl each primer (1 µM) and 4 µl reverse primer(1 µM).The amplified reaction was carried out under these conditions: a pre-denaturation of 10 min at 95°C, followed by 40 cycles of 95°C for 10s，60°C for 60s and a melt from 60°C to 95°C.The expression levels were calculated using 2 -△ CT method.The mRNA expression level of SpHSP90 in hemocytes after pathogen stimuli was measured by qRT-PCR with the same primers and methods described previously, the templates were synthesized cDNA from the total RNA extracted from hemocytes of S. paramamosain after challenged by Staphylococcus aureus, Vibrio harveyi and WSSV.The expression profiles after Pathogenic stimuli were analyzed by 2 -△△ CT .

Recombinant Plasmid Construction
A pair of primers: HSP90EXF (5′-CGCCATATGAACAAGGAGATCTTCCTG-3′) and HSP90EXR (5′-TACTCACTCGAGTCATTTCTTCCACCTCA-3′) used to obtain the HATPase_c and HSP90domains of SpHSP90.The thermal cycling conditions used were 95°C for 5 min, followed by 35 cycles of 95°C, 30s; 59°C, 45s; 72°C, 1 min 20 s and an extension of 72°C for 7 min.Restriction sites for Nde I and Xho I were engineered at 5′ and 3′ end of this expressed sequence by using this pair of primers.The product was purified using Agarose Gel DNA Purification kit and cloned into pET-30a vector.The transformants were selected on LB agar plates supplemented with Kanamycin (final concentration of 25 μg/mL).The inserted sequenced was confirmed by sequencing.

Protein Expression and Purification
The positive recombinant plasmid pET-30a-SpHSP90 was transformed into expression host strain Rosseta (DE3) for over-expression and induced by a final concentration of 1 mM Isopropyl β-D-1-Thiogalactopyranoside (IPTG) at 28°C for 10 h.thalli was collected by centrifugal, washed twice by PBS, then re-suspended in PBS and broken by the ultrasonic wave.The recombinant protein SpHSP90 with His-tag was purified by His Bind resin chromatography following the operating instructions.This recombinant protein was evaluated using SDS-PAGE and was further verified by Western blot, transferred the purified proteins containing in the gel onto a  Vol. 5, No. 7; and vertebrates with a box.The PCR normalize vel was detect Figure 4).

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The expre infection w rapidly fro normal gro control gro maximal l expression higher tha recovered  the expression profiles of SpHSP90 after injection with WSSV or bacteria.SpHSP90 expression levels increased after WSSV challenged, indicating that SpHSP90 may take part in immune reaction against WSSV.In addition, SpHSP90 expression levels were also up-regulated after the S. aureusor V. harveyi challenge.The higher increase of SpHSP90 expression levels was detected in V. harveyi challenged groups, which suggested that V. harveyi was probably a stronger inducer for SpHSP90 and alsoillustrated that V. harveyi may be apathogenic bacteria in S. paramamosain.(Zhang et al., 2005).According to a previous report, HSP90 was considered as an important immune related gene (Triantafilou et al., 2001).Similar to other HSP90s that were detected in many tissues, SpHSP90 mRNA was detectedin all the examined tissues (hemocytes, heart, hepatopancreas, gill, stomach, intestine, muscle, gonad and connective tissue), and showed the highest level in the hepatopancreas.
In summary, the cDNA sequence of SpHSP90 was reported for the first time, and the expression profiles of SpHSP90 mRNA suggested that SpHSP90 may play an important role in the immune system of S. paramamosain.All the data of this research would be beneficial to further clarify the role of SpHSP90 in immune defense and the specific mechanism still needs more studies to unscramble.