| The sea cucumber, Apostichopus japonicus, belongs to the phylumEchinodermata and commonly distributes along the Northwest Pacific shollow coastsin China, Japan, Russia and Korea. This species is one of the most aquaculturalspecies in China due to its nutritional and supposed medicinal properties. In recentyears, rapid expansion has led to a series of problems including disease outbreak,decrease disease resistance ability, slow growth rate and so on. These factors willinfluence the sustainable development of this industry. Therefore, investigating thegenetic diversity and immune-related gene defence responses in A. japonicus willprovide reference for this species genetic improvement and lay the foundation fordiseases prevention. In this study, ITS1gene and D-loop sequence were used to detectand compare genetic diversity and structure among3popilation of A. japonicus. Inaddition, the full-length of Allograft inflammatory factor-1(AjAIF-1) was clonedfrom A. japonicus and the temporal expression pattern of AjAIF-1transcript underbacterial challenge and physical injury were investigated. The results were as belows:(1) The internal transcribed spacer1(ITS1) was applied to assessed geneticdibersity and genetic differentiation in three wild populations of the sea cucumberApostichopus japonicus from Dalian China (CD), Rajin North Korea (KN) andVladivostok Russia. PCR products were cloned and sequenced. Two different lengthof ITS1sequence (517-519bp and524bp) were obtained. The mean contents of G+C(64.5%) were higher than A+T. The proportion of polymorphic sites was9.33%, inwhich4parisimony informative were found,40genotypes were identified from61sequences, among which,2genotypes were shared by3populations. Genetic diversityparameters showed high genetic diversity among populations. The fixation indices(FST) of analysis of molecular variance (AMOVA) within populations was88.65%,which show that the genetic differentiation of the three populations were weak or onlymoderately differentiated. Based on genetic distance, the molecular phylogenetic treewas constructed with NJ method, which showed that the closer linkage between KNpopulation and RV population, than their individual distances to CD population.(2) Mitochondrial D-loop sequences were analyzed to study the genetic structureof A. japonicus. In this study,71individuals from three natural populations werecollected fromCD, KN and RV.447-465bp D-loop sequences were obtained usingPCR method. The mean content of A+T (59.2%) was higher than G+C. The proportion of polymorphic sites was24.24%, in which parisimony informative siteswere more than53. Forty haplotypes were identified from71samples, among which7haplotypes are shared by three populations. Genetic diversity parameters showed highgenetic diversity among populations. Analysis of molecular variance (AMOVA) wasperformed by Arlequin3.5. The results showed that the genetic differentiation of thethree populations were weak or only moderately differentiated, whose differentiationtime was roughly estimated at19.68-22.1million years ago. The genetic distances ofamong-population were0.0374(KN and RV) and0.042(CD and RV). The molecularphylogenetic tree was constructed with the D-loop sequence of Qingdao (QD) andWeihai (WH) from GenBank with NJ method using software MEGA4.0. Populationsof QD and WH clustered into a small branch then with CD, while KN and RVclustered into other group. The results were consistent with their geographicalisolation mode.(3) A full length cDNA coding allograft inflammatory factor-1(AIF-1) wascloned from A. japonicus by rapid amplification of cDNA ends, which named asAjAIF-1. The full-length cDNA of AjAIF-1is1541bp with a477bp open readingframe (ORF) encoding158amino acids, which included two EF-hand Ca2+-bindingmotifs. The deduced amino acid sequence had a pupative size of17.92kDa and thetheroretical isoeletric point was4.91. Real-time quantitative PCR was carried out tomeasure AjAIF-1mRNA expression in different tissues and monitor mRNAexpression patterns in the coelomocytes after Vibrio splendidus challenge andphysical injury. AjAIF-1was widely expressed in all tested tissues (body wall,intestine, respiratory tree, tube feet, coelomocytes and longitudinal muscle), with thehighest expression in respiratory tree. AjAIF-1expression in the coelomocytes wasup-regulated after the V. splendidus challenge and physical injury, and all reached itsmaximum level at4h post-stimulation, and decreased gradually thereafter.Forty-eight hours after bacterial challenge or tissue injury, AjAIF-1levels incoelomocytes were similar to those of the control. |
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