Molecular Characteristics Of HSPs Genes And Their Expression Pattens In The Golden Apple Snails, Pomacea Canaliculata (Gastropoda: Ampullariidae)

The golden apple snail, Pomacea canaliculata (Lamarck), originated fromSouth America, has been a serious pest of many agricultural crops in Asia now.Benefit from its strong adaptability, the snails can establish a new population veryquickly in the new environment. As a critical factor, the water temperature causedthe seasonal onset of copulatory and spawning behavior in the apple snails andplayed a critical role on the distribution and development of the snails. Variousenvironmental stressors (high temperature, pressure, hypothermia, hypoxia, etc.) canstimulate and increase rapidly the synthesis of a highly conserved class of proteins,heat shock proteins (HSPs). It is speculated that there is a relationship between thedifferences in temperature adaptation and population distribution of apple snails andits expressions of HSPs. The present study using molecular biology, proteomics andimmunohistochemistry techniques reported four major HSPs (HSP60, HSP70,HSC70and HSP90), and analyzed the gene structure, tissue specific expressionpatterns and their expression reacting to different temperature stress. Based on theseresults, it was found that the adaptation to high and low temperaturein the applesnails,was related to HSPs expression. The results are benefit to understand thedistribution, spread and hazard of apple snails1. The molecular cloning and characterizing of HSP60, HSP70, HSC70andHSP90gene in P. canaliculataComplementary DNAs (cDNAs) were synthesized from total RNAs by usingReverse Transcription Polymerase. Partial cDNA sequences encoding HSP60,HSP70, HSC70and HSP90were repectively amplified by four pairs of degeneratedoligonucleotide primers, the design of which were based on HSPs amino acidsequences homology. The5’ and3’ cDNA ends of four HSPs genes weresynthesized by Rapid Amplification of cDNA Ends (RACE). Then all cDNAfragments were spliced into the full-length cDNAs of HSP60, HSP70, HSC70andHSP90. The deduced amino acid sequences of HSP60, HSP70, HSC70and HSP90were closely matched to other HSPs in invertebrates and vertebrates respectively, especially in mollusks.For HSP60, the complete cDNA sequence was2447bp in length, which includeda short5’ untranslated region (UTR) of41bp, a much longer3’ UTR of684bp witha canonical polyadenylation signal sequence AATAAA, a poly (A) tail, and an openreading frame (ORF) of1722bp encoding a polypeptide of574amino acids with apredicted molecular weight of61.07kDa and theoretical isoelectric point of5.41.The full-length cDNA sequence of HSP70contained a short5’ UTR of90bp, alonger3’ UTR of280bp with a typical polyadenylation signal AATAAA and a poly(A) tail, and an ORF of1911bp encoding a polypeptide of637amino acids withcalculated molecular weight of69.97kDa and theoretical isoelectric point of5.53.The full-length cDNA sequence of HSC70contained a short5’ UTR of77bp, alonger3’ UTR of248bp with a an atypical polyadenylation signal ATTAAA and apoly (A) tail, and an ORF of1950bp encoding a polypeptide of650amino acidswith calculated molecular weight of71.41kDa and theoretical isoelectric point of5.43. The full-length cDNA sequence of HSP90contained a short5’ UTR of86bp,a longer3’ UTR of282bp with a typical polyadenylation signal AATAAA and apoly (A) tail, and an ORF of82.71bp encoding a polypeptide of726amino acidswith calculated molecular weight of82.71kDa and theoretical isoelectric point of4.70.Using combinations of primers designed from HSP60, HSP70, HSC70and HSP90full-length cDNA sequence, the full-length sequences of the four HSPs genes wereaquired. There are13introns in HSP60gene,9introns in HSC70and8introns inHSP90except HSP70, and each of them has the first intron in its5’ UTR. Analyzingthe introns sequence of HSP60, HSP70, HSC70and HSP90, several cis-actingelements with transcriptional activated functions, such as heat shock element (HSE)similar sequence, TATA box, CAAT box, were found. The introns may play animportant role in regulation of HSPs expression on apple snails.2. The tissue-specific expression and temperature-induced expression of HSP60,HSP70, HSC70and HSP90Quantitative real time RT-PCR experiments were used to evaluate the expressionpatterns of HSP60, HSP70and HSP90in gill, digestive gland, kidney and footmuscle under different temperature stress conditions. The results showed that under laboratory condition, the mRNA levels of HSP60, HSP70and HSP90in the snailswas not at the same level (HSP90> HSP60> HSP70) and exhibits tissue speciaficexpression pattern; Both under heat shock and cold shock conditions, HSP60transcription level of tissues did not change significantly or slightly declined, exceptfor up-regulated in some tissues; Under heat shock condition (36°C and39°C), thetranscription level of HSP70and HSP90was up-regulated significantly in all tissues,compared with the cold shock condition (9°C and6°C); HSP70was strictly heat-inducible with a very low mRNA level under laboratory condition, and when thesnails were under heat shock condition (especially36°C and39°C), HSP70mRNAlevel could be up-regulated to more than1000folds significantly. These resultsindicated HSP70and HSP90play a critical role in the temperature adaptation processof apple snails, and reflected the reason of the low survival rate when exposing to thelow temperature and high survival rate when exposing to the high temperature.In the qPCR analysis, HSC70mRNA was expressed constitutively undernon-stressed conditions, and was also heat-inducible. This suggested that a certainamount of PHSC70mRNA was present in most tissues both under stressed andnon-stressed conditions. With exposure to heat shock treatments, an increase ofHSC70mRNA was found in the gill, digestive gland, kidney and foot muscle fromthe snail. It was indicated that heat shock can induce higher expression of PHSC70,while cold shock cannot induce that. HSC70exhibited tissue-specifc expressionpattern, for the lowest protein levels detecting in the digestive gland and kidney ascompared with the gill and foot muscle under normal condition. These results mightsuggest that PHSC70plays a crucial role in the increased tolerance againstenvironmental stressors of apple snails.