Identification of Differentially Expressed Genes in Hepatopancreas of White Shrimp Litopenaeus Vannamei Induced by Long-term Low-salinity Stress
|School||Ocean University of China|
|Keywords||Litopenaeus vannamei Hepatopancreas Subtractive cDNA library Quantitive RT-PCR Salinity clone|
The Pacific white shrimp (L. vannamei), which is naturally distributed on thePacific coast of the Americas from the northern Mexico to northern Peru, has becomeone of the primary species being cultured in the East hemisphere. L.vannamei iscapable of tolerating a wide range of ambient salinity (0.5~40), and ishypo-osmoregulator and hyper-osmoregulator when the ambient salinity is above andbelow iso-osmotic point of718mOsm kg-1(equivalent to25). Recently, L. vannameihas become a promising cultivar for inland low salinity farming in most of the worldbecause of diseases and water pollution in near shore coasts, but there exit threeprominent problems: slow growth, low survival rate, low immunity and highsusceptibility to pathogenic bacteria of low salinity culture, which restrict thelarge-scale popularization. However, research on L.vannamei at long-term low salinityis still limited. Therefore, it is necessary to discuss effect of long-term low salinity onphysiology of L.vannamei for inland low salinity water farming. The research ofmolecular mechanism induced by long-term low salinity breeding in L.vannamei notonly provided some theoretical basis, but also established the check platform based onmolecular level. We applied suppression subtractive hybridization (SSH) method toconstruct the hepatopancreas cDNA library of L. vannamei exposed to long-term lowsalinity, then made the functional annotation and classification of assembled sequences,the results of cDNA library verified the differential genetic information induced bylong-term low salinity, and discussed the transcript level of high abundant differentialgenes at different salinities, which found the basis of long-term stress mechanism frommolecular level. The thesis including:1. To investigate the effect of long-term low salinity stress on gene expression ofhepatopancreas in shrimp, we applied suppression subtractive hybridization (SSH) injuvenile L. vannamei exposed to long-term low salinity. The shrimp (initial bodyweight,0.27±0.01g) cultured at salinity2and salinity30psu for56days respectively, forward and reverse subtractive cDNA libraries of hepatopancreas in shrimp culturedat these two salinities were constructed. A total of200(80from forward,120fromreverse) randomly selected clones with more than100nucleotides were selected forfurther analysis using bioinformatics tools after vector screening.19contigs and54singletons were generated from a total of73consensuses. The consensuses, on asequence homology search using BLASTX (NCBI), revealed that24.66%(18/73) ofthem had no significant match to reported sequences in the database, suggesting thatthey were found for the first time and probably associated with stress-regulatedfunction. The remaining75.34%(55/73) consensuses encoded proteins correspondedto a wide range of functions including immune-related proteins and enzymes,metabolism-related enzymes, ribosomal proteins, apoptosis-related proteins andtransfer proteins, which illuminated that the corresponding physiology responseinduced by long-term low salinity was very complicated. The cDNA subtractivelibrary could well reflect the gene information of L.vannamei induced by long-termlow salinity-stress, results of which provide the basic data to research of relationshipbetween environmental stress and gene expression of liver.2. The relative expression of5genes in reverse library were confirmed byquantitative real-time polymerase chain reaction (qRT-PCR) and compared with theshrimp induced by the short-term (24hours) low salinity stress (30→2psu) at thesame time, to investigate these genes responded in short-term hyposmotic stress basedon the result of SSH library. Results of qRT-PCR showed the5differential genes(hemocyanin, chitinase, ecdysteroid regulated protein, trypsin and chymotrypsin1) ofreverse library decreased2-、1.45-、11.11-、1.33-and1.54-fold, respectively, above ofwhich demonstrated the construction of subtractive cDNA library was successful. Thesignificant difference of5genes transcript level to long-term (56days) and short-term(24hours) salinity stress showed the different responsive mechanisms to salinity stress,above results enriched the gene information of environmental stress of crustacean.3. The objectives of this study were to examine the growth performance, survivaland the hepotapancreas hemocyanin, chitinase, ecdysteroid regulated protein, trypsinand chymotrypsin1mRNA level at different low salinities in order to understand therelationship growth performance and physiological status of long-term low salinitybreeding. Results indicated low salinity could, to some extent, reduce growthperformance and survival rate significantly; different genes had the different salinity -induced range; According to the sea water control group, the transcripts level ofhemocyanin，chymotrypsin1and salinity were positively correlated, so abovetranscripts level reflected the long-term salinity-stress strength.4. The L.vannamei COMT gene was cloned by SSH and RACE methods in thisstudy (GenBank number: JQ345478). The full-length of LvCOMT cDNA is910bp,which contains a single open reading frame (ORF) of666bp encoding a putativeCOMT protein of221amino acids. The predicted protein has a calculated molecularweight of74.9kDa and a pI of4.98as well as twelve phosphorylation sites, but nosignal peptide which suggests that the putative COMT is not secreted into thecirculating haemolymph and may be regulated by reversible phosphorylation ofparticular residues. LvCOMT shows high similarities with its homologues in otherspecies and was more close to the homologues of mammal of phylogenetic evolutionresults. Real-time PCR was used to detect the tissue-specific expression of COMT inL.vannamei. Experimental results show that the COMT mRNA in the hepatopancreastissue is highest and that in the gill is the lowest.5. The L.vannamei CRT gene was cloned by SSH and RACE methods in thisstudy (GenBank number: JQ682618). Its full-length cDNA of1866bp contained anORF of1221bp that coded for a protein of406amino acids with an estimatedmolecular mass of15.3kDa. Within the coding sequence, characteristic feature of CRTproteins were found, such as a calreticulin family tag (KHEQNIDCGGGYLKVF), thesignal peptide (MKTWVFLALFGVVLVES) and conservative HDEL endoplasmicreticulum recovery tags. LvCRT shows high similarities with its homologues in otherspecies and was more close to the homologues of insects of phylogenetic evolutionresults. Real-time PCR was used to detect the tissue-specific expression of CRT inL.vannamei. Experimental results show that the CRT mRNA in the muscle tissue islowest and that in the intestine is the highest.