Cloning And Expression Profile Of The Response To Stress Genes From Mud Crab, Scylla Paramamosain And Eriocheir Sinensis

Scylla paramamosain is one of the most precious marine crabs aquacultured inChina. The crab is prone to infection by microbes at various stages during its growth,leading to high mortality. The Chinese mitten crab (CMC) Eriocheir sinensis, one ofthe economical aquatic species, is widely reared in south of China. Recently, Eriocheirsinensis culture in China is sustaining a severe mortality problem, resulting in theintensive culture. Understanding the immune defence mechanisms of crab are believedto be effective to health management in crab aquaculture.In the present study, we used the methodology and rapid amplification of cDNAends (RACE) methods to isolate and clone the immune-related gene from Scyllaparamamosain. The identification of immune-related genes will show new light onimmunity and resistance research in Scylla paramamosain and Eriocheir sinensis. Theresults are as follows:1. Thioredoxin，the ubiquitous small proteins(12KD) with a redox activedisulfide bridge， are found to be important regulatory elements in some vitalbiochemical reaction of metabolism．A hepatopancreas cDNA library from the mudcrab Scylla paramamosain was constructed using the SMART technique. A completecDNA sequence that showed high identity with the conserved sequence of thethioredoxin gene was cloned from the cDNA library and subjected to furtherinvestigation. The full-length thioredoxin gene of Scylla paramamosain (SpTrx)consists of1336bp and contains a complete open reading frame of396bp. The geneencodes a polypeptide of131amino acids, constituting a predicted molecular weight of14.45kDa and a theoretical isoelectric point of8.08. The deduced protein shares87%identity with the ferritin protein of Portunus trituberculatus. Quantitative real-timePCR analyses revealed that the expression of thioredoxin was ubiquitous in differentorgans of Scylla paramamosain, including gill, muscle, heart, blood, testis, andhepatopancreas. The highest expression level was found in the hepatopancreas and gill, while blood tissue showed the lowest level. The expression of thioredoxin wasobviously up-regulated after Vibvio parahaemolyticus challenge and reached thehighest level after challenged for6h. This study provides useful information regardingthe structure and functions of thioredoxin and will play an important role in immunityand resistance research in Scylla paramamosain.2. Eriocheir sinensis is a euryhaline crab migrating from sea to freshwaterhabitats in juvenilehood. To investigate the gene expression profile under varioussalinities, quantitative real-time PCR (qRT-PCR) was performed to study the geneexpression of Na~+-K~+-ATPase, Hsp70(Heat Shock Protein70) and Hsp90in megalopaexposed to different salinities of0,2,5,10and15ppt. The results showed that bothlow and high salinities markedly stimulated the gene expression of Na~+-K~+-ATPase,Hsp70and Hsp90of Chinese mitten crab megalopa, and that salinity had differenteffects on Na~+-K~+-ATPase, Hsp70and Hsp90levels depending on the duration ofsalinity stress, implying that Na~+-K~+-ATPase, Hsp70and Hsp90in Eriocheir sinensismay play an important role in salinity tolerance.3. Crabs lack an acquired adaptive immune system and host defense is believed todepend entirely on innate, non-adaptive mechanisms to resist invasion by copper.Discovery of immune-related factors are helpful for understanding the molecularresponse of crabs to copper. In this study, six immune-related genes were analyzed bymultiplex genes expression analysis using the GenomeLab GeXP analysis system(Beckman Coulter). The expression levels of three detected genes（Toll-like receptor，thioredoxin and metallothionein) rose after challenged under low and high coppertreatments. This indicated that toll-like receptor，thioredoxin and metallothionein weremuch susceptive immune factor responding to copper. The expression level of Hsp70and Hsp90and small ubiquitin-like modifier-1haven’t fluctuated obviously under lowand high copper treatments.The multi-gene analysis presented a new approach forscreening of immune-related genes, and provided more information about overall viewof genes transcript alteration in response to copper challenge.