Li Jiale

Sequence variation of Ribosomal DNA Internal Transcribed Spacer 1 of Hyriopsis cumingii from the Five Freshwater Lakes of China

The internal transcribed spacer 1(ITS-1)was amplified from Hyriopsis cumingii in the five large lakes of China:Lake Poyang,Lake Dongting,Lake Taihu,Lake Chaohu and Lake Hongze,and 430bp lengths of homoiogical fragments of the ITS-1 was sequenced from 78 individuals of ten stocks.The homological fragment analysis showed that the average contents of G+C were obviously higher than that of A+T.The nucleotide diversity of ITS of Lake Poyang was the highest,while that of Lake Chaohu was the lowest.The genetic distances among the six stocks from Lake Poyang were very near,which was from 0.0071 to 0.0092.The genetic distance among the five stocks from five large lakes were relatively far,which was from 0.0752 to 0.1381.The phylogenetic tree of ITS-1 built with MEGA 3.1 showed that the six stocks from Lake Poyang clustered together independently,and had a relative closer relationship with Lake Chaohu.The phylogenetic relationship between Lake Dongting and Lake Hongze was relatively close and clustered independently.The stocks of Lake Taihu was an independent one,which diverged from those of Lake Pongyang and Chaohu.

M、F型 CO II基因在雌雄三角帆蚌不同发育时期的表达差异研究

与传统的母系遗传不同，双壳贝类线粒体有M和F型2种mtDNA，称为双单亲遗传(doubly uniparental inheritance, DUI)。为了探究DUI的形成机制，实验利用RACE方法得到三角帆蚌M、F-type CO Ⅱ基因的cDNA全长；荧光定量检测各时期各组织中M、F-type CO Ⅱ基因的表达。结果发现，①M-type CO Ⅱ基因的cDNA序列全长1244 bp，F-type CO Ⅱ基因的cDNA序列全长808 bp，M型3′端较F型有一段546 bp的特异性延长序列；跨膜结构预测显示M-type CO Ⅱ基因3′特异性延伸区域有4个跨膜结构，推测这段延伸序列具有一定的功能性；②早期幼龄(胚胎期-5月龄)三角帆蚌的组织团中，M、F-type CO Ⅱ基因均能检测出，且同时期中F-type CO Ⅱ基因的表达量显著高于M-type CO Ⅱ；③6月龄蚌各组织中，F-type CO Ⅱ基因在各组织中表达量明显高于M型的表达量，而M-type CO Ⅱ基因在性腺中表达明显高于其他组织；④一龄雌性各组织中，M、F-type CO Ⅱ基因均有表达；而一龄雄性各组织中，M-type CO Ⅱ基因仅在性腺中表达；⑤二龄雌性各组织中，M、F-type CO Ⅱ基因均有表达；二龄雄性各组织中，F-type CO Ⅱ基因除性腺外的其他组织中均有低量表达，M-type CO Ⅱ基因仅在性腺中表达，且表达量较高。研究表明，在三角帆蚌雌雄个体发育过程中，M-type mtDNA在组织中选择性降解或聚集。

Effects of alkalinity and pH on survival, growth, and enzyme activities in juveniles of the razor clam, Sinonovacula constricta

In order to clarify the possibility of rearing razor clams (Sinonovacula constricta) in inland saline water (ISW) and to facilitate their breeding under these stressful conditions, we performed semi-static acute and chronic toxicity tests to determine the effects of carbonate alkalinity (CA) and pH on the survival and growth rate, and critical metabolic enzyme activity in juvenile of S. constricta (JSC). (1) Acute toxicity test. As the water CA increased from 1.22 to 45.00 mmol L-1, the survival rate decreased significantly, which was exacerbated by the increase in the pH. When the water CA was set at 2.5 mmol L-1, the 48 h lethal concentration 50% (LC50) for JSCs with respect to pH was 9.86. When the water pH was 9.0, 9.5, and 10.0, the 48 h LC50 values for JSCs with respect to CA were 10.38, 8.79, and 3.11 mmol L-1, respectively. (2) Chronic toxicity test. Four experimental groups comprising the control, CAS, pHS, and CA-pHS were designated according to the target ISW data. After 3 months of stress, the JSC survival rate in each group exceeded 85%, but survival was significantly lower in the CA-pHS group than the control group (p < 0.05) in the first month. For the JSCs in various groups, the shell length growth rate (SGR) and weight gain (WG) rate were significantly lower in the CA-pHS group than the other groups (p < 0.05 for SGR; p < 0.001 for WG) in the first month. However, the difference in the growth rate among groups decreased in the next 2 months. For the JSCs in the CA-pHS group, the oxygen consumption, ammonia-N excretion, Na+/K+-ATPase, aspartate aminotransferase, and superoxide dismutase levels were significantly higher than those in the other groups during the first month, but there were no significant differences between the groups subsequently. The acetylcholinesterase and lysozyme levels did not differ significantly among groups during stress for 3 months. The integrated biomarker response index showed that stressors comprising high pH and CA could be tolerated well by JSCs over long periods of stress. These results indicate that water CA and pH together affect the survival, growth, and physiological activity of JSCs. S. constricta is suitable for culture in ISW.

克氏原螯虾泛素结合酶 E2r 基因的克隆及表达分析

为了解泛素结合酶E2r基因在克氏原螯虾性腺发育中的作用，利用RACE技术得到了cDNA全序列，命名为Pc-UBE2r。该基因序列全长为3 671 bp，包含泛素结合酶的特有结构域，以及半胱氨酸残基活化位点。开放阅读框为729 bp，编码242个氨基酸，预测蛋白分子量约为27.6 ku。BLAST比对发现，克氏原螯虾UBE2r基因与节肢动物UBE2r基因具有较高的同源性。系统进化分析表明，Pc-UBE2r基因与日本囊对虾聚为一枝。qRT-PCR研究表明，Pc-UBE2r基因在性腺中的表达量显著高于其他组织（P<0.05）。在卵巢中，Pc-UBE2r基因的表达量在未发育期最低，在卵黄发生前期表达量快速增长，随后表达量逐渐降低。在精巢中，Pc-UBE2r基因的表达量在精母细胞发生期达到最高水平。组织原位杂交结果显示，Pc-UBE2r基因在卵巢中均匀分布在未发育期的卵母细胞细胞质中，随后逐渐迁移到卵母细胞细胞核以及滤泡细胞周围。在精巢中，Pc-UBE2r基因主要分布在精母细胞的周围。组织总蛋白Western blot检测发现，UBE2r蛋白在精巢和卵巢中的表达量也显著高于其他组织（P<0.05）。综上所述，我们推测Pc-UBE2r基因在克氏原螯虾配子发生和性腺发育方面发挥了重要作用，本研究将为虾蟹类性腺发育调控的分子机制奠定基础和提供依据。

三角帆蚌活化蛋白激酶C受体1基因( HcRACK 1)的克隆及表达分析

为了探究 RACK 1基因在三角帆蚌免疫系统中的调控机制,采用RACE技术克隆得到三角帆蚌 RACK 1基因(命名为 HcRACK 1)的cDNA全序列,并对该序列进行分析。结果显示, HcRACK 1基因全长为1249 bp,其中开放阅读框957 bp,编码318个氨基酸。蛋白质结构域分析显示 HcRACK 1含有RACK1家族特有的7个WD40结构域。运用荧光定量PCR技术分析 HcRACK 1在正常组织中及受到嗜水气单胞菌侵染和重金属镉暴露后相关组织表达量的变化。结果显示, HcRACK 1在各个组织中均有表达,其中闭壳肌中表达量最高;嗜水气单胞菌侵染后, HcRACK 1在血淋巴中的表达量逐渐上升,24 h时达到峰值,随后下降;暴露在100 μg/L浓度的重金属镉中, HcRACK 1在肝胰腺中的表达量逐渐上升,在第3天达到峰值;血淋巴在第2天达到峰值,随后下降;鳃中 HcRACK 1的表达量无明显变化。上述结果表明, HcRACK 1与细菌及镉引起的机体的氧化应激反应有关,并能参与到机体的免疫反应中。

Effects of biofilms on settlement of plantigrades of the mussel Mytilus coruscus

The effect of biofilms developed in the aquariums on settlement of plantigrades of the mussel Mytilus coruscus was examined in the laboratory.Various aspects of biofilm biology were investigated including age,dry weight,chlorophyll a(Chl.a)content,bacterial and diatom densities.Bacterial community in biofilms was analyzed by the DGGE fingerprint technique.The correlation between the inducing activity of the biofilms and dry weight,bacterial and diatom densities,Chl.a content was also investigated.With the increase of biofilm age,dry weight,bacterial and diatom densities increased significantly and the maximum values were,respectively,0.87 mg/cm2,1.5×107/cm2 and 1.0×106/cm2 after 28 d.Furthermore,above three parameters were correlated with biofilm age.Chl.a content reached the maximum of 2.22 μg/cm2 after 14 d and no correlation between Chl.a content and biofilm age was observed.Plantigrades of M.coruscus preferred to settle on the biofilmed surfaces rather than the surfaces unfilmed.Overall the settlement of plantigrades on biofilms that formed in the aquariums increased with biofilm age,and the maximum of 76% settlement was obtained after 28 d.The enhanced settlement correlated to dry weight,bacterial density and diatom density,and their corresponding correlation coefficients were 0.717,0.711 and 0.754.In contrast,there was no correlation between inducing activity of biofilms and Chl.a content.Moreover,Shannon diversity index of bacteria community in DGGE profiles increased up to 28 d following immersion.The shift in bacterial community composition over time may be responsible for the higher settlement of plantigrades of M.coruscus on the older biofilms,indicating that bacterial community play an important role in the settlement of plantigrades in this species.

Primary study on neuronal development of the embryo and early larvae of the mussel Mytilus coruscus

In the present study,the development of FMRFamide-,serotonin-containing cells in the mussel Mytilus coruscus was examined using immunocytochemical and histofluorescent techniques.During the embryonic development,no FMRFamide-like immunoreactive(lir)and serotonin-lir sensory cell appeared.Neurogenesis started during the trochophore stage at the apical extreme with the appearance of one FMRFamide-lir cell and one serotonin-lir sensory cell.The numbers of FMRFamide-lir cell increased at 36 hours postfertilization(hpf)and appeared on the two sides of apical extreme.Fibres of two lateral FMRFamide-lir apical cells projected posteriorly and reached developing foot.The location of the first FMRFamide-lir apical cells in the most ventral apical region did not change during early development.At 48 hpf,three FMRFamide-lir apical cells,two lateral FMRFamide-lir apical cells and two cells of developing foot appeared.The FMRFamide-lir fibres projected and the basal neuropil formed.The earliest serotonin-lir cell appeared at 18 hpf at the anterior extreme of the apical region of developing apical organ.At 30 hpf and 36 hpf,the serotonin-lir cell extended a short basal fibre into the region of the developing neuropil of the apical organ.The numbers of the serotonin-lir cell increased to 2-3 at 42 hpf and appeared in the apical region of the D-shaped larvae.Four serotonin-lir cells were detected at 48 hpf and these cells projected basal fibres into the compact apical neuropil.

Molecular characterization and expression analysis of calreticulin cDNA from Hyriopsis cumingii

In order to study genes related to pearl formation in freshwater pearl mussel and their regulation mechanism,a 1 437 bp cDNA sequence of calreticulin gene from Hyriopsis cumingii(HcCRT)was obtained by rapid amplification of cDNA ends(RACE).It consisted of a 231 bp 5′-untranslation region(UTR),a 615 bp 3′UTR and a 591 bp open reading frame(ORF).The inferred amino acids sequence was composed of 196 amino acids,including a signal peptide of 21 amino acids and a mature peptide of 175 amino acids.The molecular weight of the peptide was predicted to be 22.4 ku,with a theoretical isoelectric point of 5.01.Amino acid sequence analysis showed that there was no obvious amino acid sequence of membrane domain.Results of the ProtScale online analysis showed that the protein was hydrophilic protein.Homology analysis indicated that HcCRT amino acid had a conservative sequence of calreticulin family and was highly conserved with Danio rerio(77%),Crassostrea gigas(70%),Pinctada fucata(70%).The sequence analysis showed that HcCRT shared two potential calreticulin family signature motifs with CRT from other species,KHEQNIDCGGGYLKVF and IMFGPDICG.The prediction results of H.cumingii HcCRT protein secondary structure and tertiary structure indicated that the protein contains the alpha helix and beta folding.Real-time quantitative PCR(RT-PCR)showed that HcCRT is expressed in a wide range of tissues including the mantle,blood,gill,foot,liver,kidney,intestine and muscle,with the highest level of transcripts in mantle,followed by blood,while there is rare expression in other tissues.These data suggested that HcCRT might be involved in pearl formation of H.cumingii.This study which was first time to obtain HcCRT gene from H.cumingii played an important role in exploring pearl formation of H.cumingii.

Isolation and genetic diversity analysis of microsatellite DNA in Macrobrachium nipponense

Microsatellite-enhanced genomic library of oriental river prawn Macrobrachium nipponense of Lake Taihu was constructed using repeat-enrichment method with biotin-labeled oligos [(CA)15 and (CT)15]and streptavidin magnetic beads. From a total of randomly selected 120 clones,83 clones with more than ten motif repeats were positive. And the sequence size ranges from 191 to 580 bp with an average of 311 bp. The (CA/ GT)n and (AG/TC)n were the abundant microsatellite motifs with a percentage of 59. 09% and 20. 45% in the isolated microsatellites. Other 17 types of repeat sequences were detected,including repeats consisting of (GC)n,(AAG)n,(ACAA)n and (GGCAGA)n. Characterization of these microsatellites showed that 25. 75% of sequences were classified as perfect type,20. 45% as imperfect type and 53. 80% as compound type. Genetic diversity of 4 wild stocks of M. nipponense in 4 sites of Lake Taihu,i. e. Wujiang (WJ),Binhu (BH),Yixing (YX) and Wuxing (WX),were investigated using 12 microsatellite DNA loci from the isolated primers . The allele numbers,effective allele numbers,observed heterozygosity and expected heterozygosity under Hardy-Weinberg equilibrium were estimated to characterize genetic diversity. The result showed that all the 12 loci in the 4 stocks were highly polymorphic. Tests of departures from Hardy-Weinberg equilibrium indicated the loci except Mni86、Mni103 and Mni114 in each stock with significant heterozygosity deficiency. FST and AMOVA analysis across all stocks and loci indicated that there was low level of divergence among 4 stocks. The UPGMA clustering tree based on DA genetic distance demonstrated that Wujiang and Wuxing stocks have near relationship,and they have a distant relationship with Wuxi and Yixing stocks.