Abstract:Rotifer and artemia are indispensable living food in aquatic larval animal breeding. In order to evaluate bacterial communitiy change in rotifer and artemia enriching process with probiotics adding for turbot larva breeding, their microflora were analyzed using high-through sequencing technique based on illumina HiSeq platform. The results revealed that the diversity of experimental group with probiotics addition was higher than control group, which was the normal process in breeding. During rotifer enrichment process, the bacterial community structures of control group at different time were obviously different, and the dominant bacteria species varied greatly. However, bacterial community structures in different experimental groups were similar, and the dominant bacteria species were more diverse. Lactococcus sp., Pseudoalteromonas sp. and Alteromonas sp. as main bacteria appeared in each experimental group. In artemia enrichment process, bacterial communities of each control group were highly similar. The dominant bacteria of Cobetia sp. relative abundance reached 54%-65.2% in control group. With probiotic adding, bacterial community of each experimental group were still highly similar, but Cobetia sp. declined to 4.3%-25.3%, Pseudoalteromonas sp. and Alteromonas sp. became the new dominant bacteria. In conclusion, our findings showed that the bacterial community structure of living food for aquatic animal larva changed obviously and trended to be stable with probiotic adding, and the bacteria species in them became more uniform.

Abstract:The swimming crab (Portunus trituberculatus) is a commercially important marine crab in China. It is distributed in China, Japan and Korea and has the largest production in the Yellow Sea and Bohai Sea. It has the characteristics of fast growth, high edible value, breeding profit, etc. Salinity is closely related with osmoregulation of aquatic crustacean animal. Adaptation of aquatic crustacean animal to the changes of the salinity is mainly controlled by the osmotic pressure of the blood. The osmotic pressure level of the aquatic crustacean animal is mainly determined by the content of the inorganic ions and free amino acids in the blood. In addition, it is also affected by the metabolism of carbohydrate, lipid, protein, ammonia and other substances in the blood. In order to investigate the change regulation and characteristics of serum biochemical indicators of “Huangxuan No.1” P. trituberculatus during salinity stress, four salinity gradients (salinity 5, 10, 20, 50) experimental groups and a control group of salinity 30 were set up in the experiment. The samples of the experimental groups were respectively taken at 3, 6, 9, 12, 24, 48, 72 h and the samples of the control group were taken at 0, 3, 6, 9, 12, 24, 48, 72 h. The results showed that the values of serum glucose (GLU), triglyceride (TG) and total protein (TP) of the experimental groups declined firstly and then became stable, and were significantly lower than the control group. The values of serum GLU, TG and TP of the control group were stable during 0-72 h. While the contents of serum GLU, TG and TP of the experimental group were stable, the larger difference of the salinity between experimental and control group, the greater decline of the contents of serum GLU, TG and TP. Maximum decline of serum GLU and TP content was greater than the maximum decline of serum TG content. The values of serum GLU of experimental groups declined quickly during 0-9 h. The values of serum TG and TP declined during 0-12 h. The maximum decline of the value of serum TP was less than the maximum decline of the value of serum GLU in salinity 20 experimental group. The maximum decline of the value of serum TP was greater than the maximum decline of the value of serum GLU in salinity 5, 10, 50 experimental groups. The values of serum urea(UREA) of the experimental groups rose firstly and then became stable, and were significantly higher than the control group. The value of serum UREA of salinity 5 experimental group was near the value of serum UREA of salinity 50 experimental group. However, the values of serum TP of salinity 10 and 50 experimental groups were near each other. It could be concluded that GLU, TP and TG in serum of P. trituberculatus were closely related to osmoregulation. Serum GLU and TP of P. trituberculatus were main materials for energy supply and the serum GLU supplied energy firstly in the osmoregulation process. Partial content increase of serum free amino acid of P. trituberculatus could be derived from other tissues in high salt water environment.

Abstract:In order to select the best method to remove microorganism of Pyropia haitanensis, we treated P. haitanensis thallus with different combinations of ampicillin, gentamicin sulfate and kanamycin. Meanwhile, we explored the relationship between the growth of P. haitanensis thallus and the change of microflora. The results showed that low concentration of gentamicin sulfate (10 μg/mL) or high concentration of kanamycin (800 μg/mL) could inhibit the growth of P. haitanensis thallus. In contrast, ampicillin of each concentration in this paper had a promoting effect on the growth of P. haitanensis thallus. We chose the combination of ampicillin (500 μg/mL), gentamicin sulfate (500 μg/mL) and kanamycin (300 μg/mL) treating P. haitanensis thallus for 24 hours. The combination of antibiotics could obviously inhibit the microbes and had no side effects on the growth of Pyropia. The discs in different strains of P.haitanensis, different-day-old and culture density of P. haitanensis, different salinity and concentration of medium were all growing faster in antibiotics treated groups than control group. Moreover, two special bacteria strains, whose morphology of colonies and strains were different between antibiotics treated groups and control group, were found after comparing bacteria strains isolated from antibiotic treated groups and control group. Based on the above results, a certain type of microorganism multiply rapidly which inhibit growth of the other microorganism that were benefited for the growth of P. haitanensis thallus. When the microorganism that inhibit growth were inhibited with antibiotics, the probiotics became the advantage group and promoted the growth of blades. The probiotics was not sensitive to antibiotics, but with the change of culture condition, the effect of antibiotics on the strains that inhibited the growth of Pyropia became weaker, thus they multiplied rapidly, as a result, hindering the growth of Pyropia thallus.

Abstract:Pseudomonas plecoglossicida has been reported as the causative agent of the visceral granulomas disease in farmed Larimichthys crocea. The pathogenic strain NB2011 encodes a typical type Ⅲ secretion system, which is present in many Gram-negative pathogens, and may be an important virulence factor of this strain. ExsA, the transcription regulator of this system, is essential for the type Ⅲ expression and function. In this study, the mutant strain ΔExsA NB2011 was obtained by the method of double homologous recombination whose internal sequence of the ExsA gene of NB2011 was replaced by a kanamycin resisting gene. Adhesion, internalization of macrophage J774, and intracellular replication of the mutant strain were detected, LD₅₀ of artificial challenge was tested, and the ultrastructure pathology of infected fish was investigated. Compared to the wild strain, the number of the internalized mutant strain in the macrophages was a little lower, the number decreased in several hours and was too low to be detected in 12 h; while the number of wild strain increased after 12 h post internalization; LD₅₀ of the mutant strain increased to over 190 times the wild strain; TEM observation results showed the wild bacteria survived and replicated in macrophages of the infected fish, while no mutant bacteria were observed in the fish tissue during the experiment time. The results indicated a dramatic virulence attenuation of ΔExsA NB2011. The strain may serve as a good candidate for vaccine development.

Abstract:Comparison of the expression of fish-derived lactic acid bacteria expression system in oral vaccines was made under different immune process, to determine fish source Lactobacillus expression system immune program for immunization of juvenile rainbow trout. In this study, we constructed L. plantarum L1212 in recombinant protein expression IPNV VP2-VP3, then the recombinant strain pPG612-VP2-VP3/ L1212 was coated with pellet feed and immune Oncorhynchus mykiss juvenile fish. Immunization program was divided into continuous immunization group: the group that is immune for a single day after immunization for 1 day; the group that strengthens the immune 1 times after 4 days of continuous immunization 32 d and the group that strengthens the immune 1 times after interval immunization 32 d. After immunization, tail artery blood collection is carried out, and the serum antibody titers are measured by indirect ELISA. After immunization for 66 days, IPNV is injected abdominal cavity and the relative immune protection rates were calculated for each group. To determine the pellet feed in accordance with 1 mL/g ratio and 108 CFU/mL recombinant bacteria pPG612-VP2-VP3/ L1212 and 2.5% sodium alginate mixed 20℃ drying preparation of oral vaccine; the titer and challenge protection rate of serum antibody in interval immunization group were significantly higher than those of other groups, and the serum antibody titer and challenge protection rate in the twice-immunization group were significantly higher than those of the group immunized once. The immunization program and the immunoprotection rate of the juvenile O. mykiss were collected by the recombinant bacteria. The immunization program was superior to the immunization program for continuous immunization and protected the invasion of IPNV.

Abstract:The western stock of the winter-spring cohort of neon flying squid, Ommastrephes bartramii, is widely distributed in the Northwest Pacific Ocean, and its distribution and abundance are mediated by the climate variability and environmental conditions. Based on the fishery data during 1995-2011 from the Chinese Squid-jigging Science and Technology Group and the environmental data including the anomalies of sea surface temperature (SSTA), sea surface height (SSHA) and mixed layer depth (MLDA), we analyzed variability in the spatio-temporal distribution of environmental factors under different climate modes (warm and cold phases of the Pacific Decadal Oscillation, PDO). The results suggested that the catch per unit effort (CPUE) was high during the warm PDO phase and low during the cold PDO phase. Significant interannual variations were found in the SSTA, SSHA and MLDA, which corresponded to the variability of the warm/cold PDO phases. A negative correlation was found between the PDO index and the SSTA as well as the SSHA with time-lags of -9- -10 and -20-17 months, respectively, both with the highest correlation coefficient at the time-lag of 0 month; while the MLDA was positively correlated with the PDO index with a time-lag of -6-5 months, and the highest correlation coefficient occurred at the time-lag of -1 month. The empirical orthogonal function method was used to analyze the major mode of spati-temporal variability of SSTA, SSHA and MLDA, the first five modes of EOF for each environmental factor contributed to 78.73%, 32.82% and 64.57% of variation, respectively. Our findings suggested that shifts in the climate mode largely drove the variations in the environmental factors on the squid fishing ground, which led to significant abundance variability of O. bartramii in the Northwest Pacific Ocean.

Abstract:MicroRNAs, small non-coded RNA of about 18-25 nucleotides, regulate target genes expression by partially complementary binding to 3'UTRs (untranslated regions) and are involved in many biological processes. Recently, microRNAs have been studied broadly in teleosts. This paper briefly reviews the biological property of microRNAs and the post-transcriptional regulation they confer, focusing on microRNAs expression pattern and the discovery of microRNAs in teleost fish by next-generation sequencing. The function studies about microRNA in teleost cover osmotic stress regulation, reproduction, development, growth, immunity, metabolism and other biological processes. Results show that miR-200 and miR-30 family members control the cellular ion balance and salt resistance. Dicer1 is essential to the development of zebrafish, its deletion is lethal and the organogenesis will be impaired. MiR-20a regulates the morphogenesis of head, eye, spinal cord, somite during the gastrula stage; miR-92 controls the formation of endoderm; miR-10 regulates the formation of body axis; miR-219 affects the apoptosis of head and tail, and miR-122, miR-30 and miR-145 respectively adjust maturation of liver cells, the development of liver and intestinal tract. The development of heart is under the control of miR-138 and miR-143; miR-126, miR-150, miR-451 regulate the development and maturation of thrombocytes and hemocytes; miR-10 and miR-126 regulate the formation of blood vessel; miR-200 and miR-183 each regulate the development of olfaction and auditory system. Multiple microRNAs are involved in the process of organ regeneration. MiR-133a/b and miR-206 inhibit the proliferation of myocyte; miR-214/499/199/3906 are involved in the process of quick-twitch and slow-twitch muscle differentiation; miR-430 promotes the generation of primordial germ cell, and microRNAs are also reported to be involved in multiple physiological processes of energy homeostasis, innate immunity, hypoxia adaptation, body color regulation, stress responses, intermuscular bone formation. This paper summarizes those results and will benefit the understanding of the present situation of the microRNAs research in teleost and may be helpful for the promotion of the study in noncoded RNA.

Abstract:In order to explore the seasonal variation of the eco-groups in the coastal waters of central Fujian Province, we conducted three comprehensive marine surveys during December 2009 and April and August 2010, and the species composition and eco-groups were discussed by using non-metric multidimensional scaling (NMDS) and analysis of similarity (ANOSIM). The results showed that a total of 40 species was identified in winter, warm-water-species were the dominate eco-groups. But the ratio of warm-temperature-species were the highest (30%) among three seasons, and accounting for 17.65% and 16.96% of the biomass and individual density respectively. There were 39 species of fish in spring, and eco-groups are still dominated by warm-water-species. The biomass and individual density of the warm-water-species accounted for 86.48% and 94.52% of the total amount respectively. In summer, fish species accounted for 61, and warm-water-species appeared in large number by 55 species, accounting for 97.17% and 99.35% of the biomass and individual density respectively. Affected by Zhejiang-Fujian coastal water and Taiwan Strait warm current water alternately, species turnover rate and the number and density of warm water species were increased gradually, and accompanied by the estuarine species replaced by coastal species from winter to summer. The phenomenon was related to fish spawning, feeding and wintering migration, when Zhejiang-Fujian coastal water and Taiwan Strait warm current water occupied this area. It is concluded that the seasonal variation of eco-groups and species compositions in the coastal waters of central Fujian Province may be affected by the changes of water masses.

Abstract:To analyze daily variations in Anguilla japonica glass eel catch as related to ecological factors at Jingjiang section of the Yangtze River, we conducted daily monitoring program at Jingjiang section of the Yangtze River during the 2012 fishing season. A generalized additive model (GAM) was applied to evaluate the relationships between daily catch and their ecological variables, including water temperature, the tidal range, turbidity, lunar phase, tide phase and pressure. Fishing was performed at Jingjiang section of the Yangtze River from late January to early April. The total fishing period catch per boat ranged from 221 ind. to 443 ind. with a mean±SD of (344.8±83.4) ind. The daily catch differed by month. The mean daily catch was only 0.4 ind./d in January, and the abortive haul rate was 90.9%; while mean daily catch was up to 10.4 ind./d in April, and the abortive haul rate was only 10.0%. Stepwise GAM building using the AIC and adjusted pseudo-R² showed that turbidity, pressure and lunar phase did not affect daily catch of A. japonica glass eels significantly, but three predictor variables, tide phase-month interaction, water temperature and tidal range were highly significant. In addition, tide phase-month interaction, water temperature, and tidal range account for 42.4%, 19.1% and 13.1% of the variance in the data, respectively, and these three variables also showed a positive relationship with the daily catch. Statistical results also showed that the daily catch fluctuated with tide phase, since high catches appeared at times corresponding to the spring tide of the full moon or the new moon, while, low catches appeared at times corresponding to the neap tide during quarter moons. Moreover, glass eels were caught when the minimum water temperature was 6.3℃, and a greater catch was found in optimal water temperature ranging between 10 and 15℃. Overall, the proportion of total catches for spring or neap tide period was 76.8% and 23.2%, respectively. Furthermore, our results suggested that during glass eels upstream migration using ‘selective tidal stream transport’, some individuals were captured in the mouth zone of the Yangtze estuary, and some individuals preferred to stay near the river section of the estuary, whereas others migrated upstream. In our study, water temperature, tidal range and tide phase were the important ecological factors affecting the fluctuations in upstream migration of glass eels.

Abstract:From May 21 to 23 and on May 27, 2014, during light falling net fishing operations in the central Nansha Islands waters, the samples of meso-and micro-zooplankton were collected and analyzed to investigate their changes before and after the light trap. The results showed that zooplankton were sensitive to artificial light at night; the community of zooplankton was affected by artificial nighttime light. A total of 178 zooplankton species belonging to 16 groups were recorded before and after the traps. The results show that the number of species increased after traps. The succession rate of species compositions was estimated at 41%, except Cladocera, all the 15 groups of zooplankton showed significant changes in species composition. Except for the Protozoa which showed a decline in species number, all groups showed a tendency of increased species number after traps. The results also indicated that most of the zooplankton species are sensitive to light traps, either positive or negative, and the occurring frequency of dominant species decreased obviously after light traps. The dominant species composition was changed after light traps (replacement rate was 50%). Before light traps, the Copepoda was the main dominant species. The dominant degree of Appendiculata was dramatically enhanced after light traps. The dominant species of meso-and micro-zooplankton in Nansha Islands trended towards cumulative distribution. All dominant species were sensitive to artificial light. The adaptation to light was different between species. Most dominant species had high adaptation to light; their aggregation intensity was enhanced after light traps, such as Oikopleuridae. The aggregation intensity of Farranula gibbula and Corycaeus agilis was reduced after light traps. At first, the aggregation intensity of Copepoda larvae and Acartia negligens was enhanced, but then the intensity was significantly reduced. Biomass and density of zooplankton were significantly increased in the overall trend, with the largest increase occurring during the initial trap, then declined, but still remaining higher than before the trap. As increase in nekton feeding intensity led to the fall of zooplankton. Although the species richness and diversity were significantly improved by the light trap, the structure of the zooplankton community did not change. Zooplankton species react to artificial light, and the difference between groups is determined by the biological and physiological characteristics of different feeding strategies. Artificial light pollution is a global environmental issue, the ecological impacts of which are only now beginning to be examined in detail. Current knowledge of these impacts in marine ecosystems is insufficient. Although the light trap increases production, but it is important to pay attention to and strengthen the ecological effects of science, and strictly limit the intensity of light trapping with appropriate restrictions on the development of light trap fisheries.

Abstract:To assess the effects of dietary xanthophyll on the growth and body color of discus fish (Symphysodon sp.), xanthophyll was added into the basal diet (beef heart hamburg) with the levels of 0, 50, 100, 200, 300 and 400 mg/kg, respectively. The six experimental diets were fed to discus fish with an initial body weight of (10.3±0.3) g for 8 weeks. The results showed that the supplementation of 50-200 mg/kg xanthophyll had no significant effects on the weight gain rate, but the feed coefficient ratio significantly increased and the weight gain rate significantly decreased when the xanthophyll supplementation reached 200 or 300 mg/kg respectively. After 4 or 8 weeks’ feeding, the skin lightness (L^*) showed no significant differences among groups, but the skin yellowness (b^*) increased with the increase of dietary xanthophyll. The b^* value and xanthophyll content in skin of all xanthophyll-supplemented groups in the 8^th week were higher than those in the 4^th week. The b^* values in the 4^th week and 8^th week, and the xanthophyll content in whole fish in the 8^th week remained relatively stable when the xanthophyll supplementation reached 300 mg/kg. In the 4^th week, the skin redness (a^*) showed no significant difference among groups, but in the 8^th week, the a^* values significantly decreased when the xanthophyll supplementation reached 100 mg/kg. The hepatic total antioxidant capacity was significantly strengthened by the supplementation of 300 or 400 mg/kg xanthophyll. The above results showed that dietary xanthophyll could effectively improve the yellowness of the skin and strengthen the hepatic total antioxidant capacity of discus fish, but the high inclusion of xanthophyll reduced the growth of the fish. The supplementation level of xanthophyll was suggested to be 200-300 mg/kg.

Abstract:The purpose of the present study is to reveal the effects of different salinity on body weight and survival of families of Litopenaeus vannamei. A total of 4348 shrimps from 72 families were raised for 60 d in the 30 salinity and 15 salinity environment. We analyzed the genetic parameters of body weight (BW) and survival rate, and genotype by environment interaction (G×E) of L. vannamei at the 30 and 15 salinity. The results showed that at the 30 salinity, both the average body weight and survival rate were higher than those at the 15 salinity. In this study, the heritabilities of BW at the 30 and 15 salinity were (0.51±0.08) and (0.30±0.07), respectively, which were all high. After conversion, the heritabilities of survival were 0.22 and 0.39, which varied from medium to high. After integrating the data in the 30 and 15 salinity, the heritability of BW and converted heritability of survival were (0.45±0.07) and 0.31, respectively, which were both high. The estimates of genetic parameters might be over-estimated because the common environmental effect was not included in the estimation model due to convergence problem. The results showed there were high genetic correlations (0.81-0.90) for BW and survival between the two salinity. Ratios of G×E variance to additive genetic variance were all less than 0.5, G×E effects for BW and survival were not significant. These results suggested that although there were differences in growth and survival for families of L. vannamei between the 30 and 15 salinity, the G×E was not significant and re-ranking effect was low. Therefore, there is no need to establish a separate breeding line of L. vannamei for each salinity from 15 to 30 salinity environment.

Abstract:According to supply strategies for the parent selection during the breeding of Chlamys farreri, the relationship between the morphological characters and wet weight was studied using age one(324 individuals)and age two (230 individuals) which came from the same mix family. The traits included the shell length (x₁/cm), shell height (x₂/cm), shell width (x₃/cm) and wet weight (y/g). The result showed that the correlation coefficients between shell character and the wet weight were all very significantly different. The shell length and shell height showed the greatest correlation coefficients to the wet weight of age one and age two (0.939 and 0.808) respectively. The result of the path coefficient analysis showed that the shell length and shell height also showed the highest correlation coefficients to the wet weight and gave predominant direct effect and determinacy on the wet weight for scallop of age one (0.532) and age two (0.451) respectively. The results of the determinant coefficients were consistent with the results of the path analysis. The shell height and shell length showed the lowest correlation coefficients to the wet weight of age one (0.163) and age two (0.191). The multiple regression equations were obtained to estimate wet weight as age one: y=-10.527+0.287x₁+0.087x₂+0.409x₃, R²=0.926; age two: y=-68.609+0.254x₁+0.719 x₂+2.008x₃, R²=0.830. This paper provides theoretical basis and perfect measures for the selection of the parents scallop in the breeding.

Abstract:In order to investigate the function of cold inducible RNA-binding protein (CIRP) of large yellow croaker Larimichthys crocea in cold acclimatization, CIRP gene was cloned and the expression changes were analyzed by real-time quantitative PCR (qRT-PCR). The full-length of cDNA sequence of CIRP is 1211 bp, encodes 182 amino acid residues and the protein molecular weight is 19 ku. Amino acid sequence alignment showed that CIRP was conserved in teleost, especially the RNA recognition motif (RRM) at N-terminal was highly conserved. The phylogenetic tree analysis showed that CIRP of L. crocea had the highest identity to sablefish Anoplopoma fimbria (81.5%), CIRP of L. crocea and other teleost formed a clade. During chronic cold stress (water temperature slowly decreased from 12℃ to 6℃), the CIRP expression of skin and muscle significantly increased with the temperature decreasing, in which the expression levels at 6℃ rose 11.56 and 9.03 times compared to 12℃. In gill, brain and heart, the expression of CIRP increased at first and then decreased, and the maximum appeared at 8℃ which were 5.07, 7.69 and 2.83 times compared to 12℃. The expression level significantly decreased with the temperature increasing in intestine, kidney, liver and spleen, and the decreasing amplitude at 6℃ were 80.0%, 65.6%, 91.2% and 55.6% compared to 12℃ respectively. During acute cold stress (from 12℃ to 8℃ immediately and maintained for 4 h), the expression levels of CIRP in gill, skin, brain, liver and heart increased first and then decreased, reflected the stress and adaptation process of L. crocea to cold. Compared to 12℃, the CIRP expression in muscle and intestine were always at a lower level during acute cold stress. The difference of tissue expression of CIRP between chronic and acute cold stresses suggested that there were multiple regulation mechanisms replying to cold stress in L. crocea. The present study indicated CIRP was involved in L. crocea responding to cold stress, and the results could provide fundamental data for further research on the mechanism of response to low temperature in L. crocea.

Abstract:To investigate the effects of dietary arachidonic acid (ARA) on the sex steroid hormone production and the gene expressions of the key proteins in steroidogenesis in gonads of turbot Scophthalmus maximus broodstocks, three isonitrogenous and isolipidic (lipid content 13%) experimental diets were formulated to contain different ARA levels: the control diet without ARA supplementation (C, 0.72% ARA of total fatty acids) and two diets with low (5.63% of total fatty acids, ARA-L) or high ARA (15.03% of total fatty acids, ARA-H) level. The experimental diets were randomly assigned to 9 tanks of 3-year-old turbot (25 fish in each tank). Fish were reared in an indoor flowing seawater system and fed to apparent satiation twice daily. At the end of the feeding trial, sex steroid hormone production was assayed and gonad samples from females and males were collected to determine the gene expressions of sex steroid-synthesizing proteins. The results showed that compared to the control group, ARA-H significantly reduced the estradiol production in females but in males ARA-L significantly reduced the testosterone production. In ovaries, ARA-H significantly reduced the mRNA expression of follicle stimulating hormone receptor, but ARA-L significantly increased the mRNA expression of 17α-hydroxylase. In testes, ARA-L significantly reduced the mRNA expression of steroidogenic acute regulatory protein and 17α-hydroxylase, but significantly increased the mRNA expression of aromatase. Dietary ARA supplementation increased the ARA accumulation but decreased the EPA accumulation in fish tissues. The ARA concentration in ovaries was higher than that in testes. In conclusion, certain content of dietary ARA inhibited the estradiol and testosterone production in immature turbot broodstock. In ovaries, this inhibition may be attributed to the inhibition on follicle stimulating hormone receptor, but in testes it may be attributed to the inhibition on steroidogenic acute regulatory protein and 17α-hydroxylase.

Abstract:Heat shock protein 90 (HSP90), a molecular chaperone in cell, which can regulate multiple signaling pathways, plays a key role in the process of cell differentiation, development and transportation. To explore the role of the promoter in regulating the expression of heat shock protein 90, based on the HSP90 gene cDNA of Haliotis diversicolor from our lab, its 5' flanking region was cloned by genome walking and Tail-PCR techniques. Results showed that there is an 809 bp intron between translation initiation site (ATG) and first exon (94 bp). The length of 5' flanking region is 2800 bp before the first exon and 2811 bp before the predicted transcriptional start site (A). A TATA-box was located in the upstream -30 bp of the transcriptional start site (A). Potential transcription factor binding sites include ATF, TBP, Sp1, Oct 1, C/EBPalpha, NF-1, NF-kappaB, GATA-1, and Sox-2, etc. A CpG island was found by the CpG island prediction software, whose length is 131 bp. Eight firefly-luciferase reporter gene vectors with different deletions of HdHSP90 gene were constructed and transiently transfected into 293T cells, and the activity of dual-luciferase reporter gene was detected. The results showed that the core promoter is located between -98 to 83 bp, and the three transcription factors between -624 to -539 bp, Oct-1, C/EBPalpha, and NF-1, can inhibit the gene transcription.

Abstract:Autophagy is a cellular process for degradation of damaged proteins and organelles via forming autophagosome. Microtubule-associated protein 1 light chain 3 B (LC3B) is a marker protein for autophagy detection. However, in many lower animals, autophagy detection is limited for a lack of available specific LC3B antibody. In the present study, the coding sequence of LC3B of Mytilus galloprovincialis was cloned and inserted into a pET-32a prokaryotic expression vector. To obtain a high-level and soluble expression of recombinant MgLC3B-His protein, the IPTG concentration and induction time were investigated. Then, the purification conditions of this recombinant protein were also studied. Our results showed that MgLC3B was inserted in the pET-32a prokaryotic expression vector successfully. The recombinant MgLC3B-His protein was induced at 20℃, 150 r/min with IPTG concentration of 0.6 mmol/L after 10 h culture. A single-band recombinant protein was obtained after affinity purification. After immune injection, and the rabbit polyclonal antibody was also obtained, with a titer of 25 600. Therefore, our results will be helpful for the investigation of autophagy in mussels and similar species.