Variation Of Bacterial Community Diversity In Mizuhopecten Yessoensis And The Mariculture Zones Of Different Seasons,and The Expression Of Gene LITAF

To study the seasonal changes of bacteria numbers and classes in sea surface water,10 m layer of water and adult scallop gills in the Da Changshan Island Dalian Changhai Japanese scallop(Mizuhopecten yessoensis)aquaculture farm.The plate method(PC Act)and PCR-DGGE method was used.Real-time quantitative PCR was carried out to measure LITAF mRNA expression in the scallop.In the surface water,10 m layer of water and adult scallop gills,the highest number of heterotrophic bacteria and pathogenic vibrios are all appeared from May to July,and the highest number of heterotrophic bacteria were 7.9×105 cfu/mL,5.9×105 cfu/mL and 1.2×105 cfu/g;the maximum value of the number of pathogenic vibrios were 1.6×102 cfu/mL,1.7×102 cfu/mL,8.7×10 cfu/g;then the lowest values 1.8×105 cfu/mL,8.0×104 cfu/mL,2.7×104 cfu/g and 7.4×10 cfu/mL,6.7 X 10 cfu/mL,3.4×10 cfu/g were appeared from November to January.From a seasonal point of view,is generally highest in summer and lowest in winter,spring and fall somewhere in between,but the the number of pathogenic vibrio in scallop gills is got the highest values in the spring.Polymerase Chain Reaction/Denaturing Gradient Gel Electrophoresis(PCR-DGGE)was used to investigated the predominant bacterial in the three kinds of samples.38 major bands in the DGGE profiles were eluted from the polyacrylamide gels,cloned,and sequenced for each band.Six major phylogenetic lineages were identified:γ-Proteobacteria,α-proteobacteria,Bacteroidetes,ε-Proteobacteria,Firmicutes,Cyanobacteria;belonged to 14 genus,they were Pseudoalteromonas,Vibrio,Alistipes,Halomicronema,α-Proteobacteria,Synechococcus,Cyanobacterium,Bacteroidetes bacterium,Microbulbifer Bacillus,Arcobac-ter,Flavobacterium,Brumimicrobium,Phaeobacter.with Vibrio(23.68%)being the predomi-nant bacteria.In scallop gills,the samples from summer and autumn both have the greatest abundance bands,there are 19 bands;in the 10 m layer of water,the samples from summer has the greatest abundance bands,there are 17 bands;in the surface water,is summer too,16 bands.So we can find that in these three kinds of samples,the bacterial community in summer and autumn are more variously than that in spring and winter.Based on the expressed sequence tag(EST)of Japanese scallop(Mizuhopec-ten yessoensis),the cDNA of LITAF gene was amplified using rapid amplification of cDNA ends(RACE)approach,and the gene have four alternative splicing isoforms.Real-time quantitative PCR was carried out to measure the four alternative splicing isoforms(named as MyLITAFAS1,MyLITAFAS2,MyLITAFAS3,MyLITAFAS4)of LITAF mRNA expression in adult tissues and after bacteria(Vibrio anguillarum)challenged,also in the seasonal scallop samples.The expression level of MyLITAFAS1 mRNA with the highest in the heatopancreas,MyLITAFAS2,MyLITAFAS3 are in the adductor muscle,MyLITAFAS4 only expression in the kidney.MyLITAFAS1 expression showed significant difference(P<0.05)between the control and bacteria challenged specimens at 24 h,and MyLITAFAS2,MyLITAFAS3 are at 48 h.These results suggest that the LITAF should be a member of the LITAF family,and may play an important role when the scallop subjected to pathogen.