Li Mingyun

[Cloning, physical and chemical property analysis of the Japanese sea bass Wap65-2 gene and its expression following Vibrio harveyi infection].

Wap65-2（warm temperature acclimation related 65 kDa protein-2）是鱼类中发现的一种血浆糖蛋白，与细菌感染性免疫应激紧密相关。该研究首次克隆了花鲈Wap65．2基因全长cDNA序列。它由1601个核苷酸组成，包含一个大的开放阅读框，预期编码一个由436个氨基酸组成、相对分子质量为4．87x10。的前体蛋白，N端19个残基为信号肽序列。序列和系统进化树分析表明，花鲈Wap65．2与欧洲海鲈进化关系最近，两者氨基酸同源性高达80．7％。健康花鲈Wap65—2基因mRNA主要在肝中表达，心和肌肉中少量表达。qRT-PCR分析揭示，哈维氏弧菌（Vibrio harveyi）感染感染12h后，花鲈肝中Wap65．2基因mRNA表达显著上调，24h时达到峰值，为健康对照的6．89倍。原核表达花鲈Wap65．2并制备抗血清。Westernblot分析表明，哈维氏弧菌感染12h后，花鲈血清中Wap65．2含量显著增加，36h时达到最大值，为健康对照的5．33倍。综上所述，花鲈Wap65—2基因的表达与其细菌感染性免疫应激紧密相关。

Expression of OmpK gene of Vibrio alginolyticus and development of a detection method of indirect ELISA.

Vibrio alginolyticus is the main Vibrio pathogen in aquaculture in the south of China,the number of which is the largest in marine Vibrio class.In the present study,the outer membrane protein K gene(OmpK)of V.alginolyticus strain ATCC17749 was amplified by PCR and cloned into high efficient expression vector pET28a.The results of sequencing and restriction enzyme analysis combined with agarose gel electrophoresis showed that the open reading frame sequence was correct and fully consistent with what had reported.The recombinant plasmid of pET-28a-OmpK was successfully constructed and transformed into E.coli BL21 pLys E.The fusion protein was expressed under the IPTG inducing condition,and the expression product was purified by an affinity chromatographic method.Mouse anti-OmpK poly-clonal antibodies(PAbs)were obtained via applying protein OmpK as antigen to immune mice.Western-blotting analysis proved the recombinant protein has a good reactive ability against OmpK positive serum.Then,an indirect Enzyme-Linked Immunosorbent Assay(ELISA)for the rapid diagnosis of V.alginolyticus has been developed using the PAbs.The lowest V.alginolyticus suspension was 104 CFU/mL.Cross reactions of antisera with other bacteria were detected,and all results were negative.The results indicated the suitability and simplicity of the test as a rapid,field diagnostic tool for V.alginolyticus and it can be used for the rapid detection of V.alginolyticus.Further investigation will be focused on the development of an indirect ELISA Kit for the detection of V.alginolyticus.

Studies on the growth characteristics and heterosis of genealogies of Pseudosciaena crocea.

In this study,the growth characteristics of four Pseudosciaena crocea groups including the selfing of DD(Daiquyang♀×Daiquyang♂),the selfing of GG(Guanjingyang♀×Guanjingyang ♂),their direct cross DG(Daiquyang ♀×Guanjingyang ♂),and back-cross GD(Guanjingyang ♀ × Daiquyang ♂) were determined.Results after 526 days of grow-out showed that the final mean weights were higher for the GG(330.514 g) and the GD(336.694 g) than those of the DD(278.975 g) and the DG(243.297 g).The 1-year-old DD grew in a lower rate,but after the winter,the growth rate of DD accelerated significantly:the growth rate in length was higher than the other three groups while the weight gain was not significantly different from the GG and the GD.That is,the growth potential of the DD at later stage was high.The order of the fullness on 526th day was DDDGGDGG.A superior cross GD was constructed,which displayed significant heterosis for growth and body shape.It was concluded that the GD had excellent economic and breeding potential.The growth curves(DD:W=0.020 6L2.942 7,R2=0.994 9;GG:W=0.013 9L3.099 4,R2=0.978 5;DG:W=0.022 9L2.913 6,R2=0.990 5;GD:W=0.017 7L3.007 9,R2=0.994 9) fitted according to weight and body length had the high degree.The weight could be estimated by their body length,and this growth curves model could be applied in the production.

Molecular cloning, sequence analysis and stress-related changes of the heat shock protein 60 gene in Neobenedenia melleni

Heat shock protein 60 is an essential chaperone that can maintain the natural structure and function of mitochondrial proteins. Here, we successfully cloned the full length cDNA of HSP60 from Neobenedenia melleni, designated as NmHSP60. Real-time quantitative PCR and Western blot were used to analyze the expression change of NmHSP60 under different temperature and salinity. Compared with the typical 25 Degrees Celsius, expressions decreased dramatically in eggs and adults at 18 Degrees Celsius. Conversely, at 32 Degrees Celsius, expression increase dramatically in adults. Compared with salinity 24, expressions were significantly down-regulated in adults at salinity 18, and up-regulated in eggs at salinity 30. Experimental results suggest that NmHSP60 may play an significant role in N. mellenis adaptation to adverse environmental conditions.

大黄鱼冷诱导结合蛋白(CIRP)基因cDNA克隆及低温胁迫对其时空表达的影响

为研究冷诱导结合蛋白(CIRP)在大黄鱼低温适应过程中的作用，采用同源克隆技术获得了大黄鱼 CIRP 基因，并通过实时荧光定量PCR (qRT-PCR)技术检测了低温胁迫下大黄鱼各组织中 CIRP 基因的表达变化。结果显示，大黄鱼 CIRP 基因cDNA序列全长1211 bp，推测编码一个由182个氨基酸组成、分子量为19 ku的蛋白。序列比对显示硬骨鱼类 CIRP 基因序列较为保守，特别是N端的RNA识别基序(RRM)高度保守。系统进化树分析显示大黄鱼 CIRP 与银鳕相似性最高(81.5%)，所有硬骨鱼类聚为一个大支。慢性低温胁迫中(12 °C缓降至6 °C)，皮肤和肌肉中 CIRP 基因的表达量呈随温度降低而升高的趋势，6 °C时表达量分别为12 °C时的11.56倍和9.03倍；鳃、脑和心脏中 CIRP 基因表达量均呈先显著上升后显著下降的趋势，其峰值均出现在8 °C时，表达量分别为12 °C时的5.07、7.69和2.83倍；肠、肾脏、肝脏和脾脏中的表达量随温度降低而下降，6 °C时的表达量最低，与12 °C时相比降幅分别为80.0%、65.6%、91.2%、55.6%。急性低温胁迫(由12 °C骤降至8 °C并胁迫4 h)条件下，鳃、皮肤、脑、肝脏、心脏中 CIRP 基因表达量均呈先升高后降低的变化，反映了机体对低温胁迫的应激、适应过程；肌肉和肠中的表达量则始终显著低于胁迫前。慢性和急性低温胁迫中各组织 CIRP 表达量变化的差异提示，大黄鱼机体在低温胁迫响应和适应中有着多种调节机制。研究表明， CIRP 基因参与了大黄鱼应对低温胁迫的过程。

大黄鱼( Larimichthys crocea )新品种“东海1号”体长相关的DArT标记筛选

大黄鱼（ Larimichthys crocea ）是我国重要的海产经济鱼类。因多年未加选育的累代养殖，养殖大黄鱼生长缓慢，性早熟、免疫力低下，亟需对养殖大黄鱼进行遗传改良。多样性芯片技术（Diversity arrays technology，DArT）具有高通量和低成本的显著特点，不需要明确物种的基因组DNA序列信息，因而广泛应用于动植物遗传图谱制作和遗传多样性分析。本研究旨在采用DArT技术鉴定与“东海1号”大黄鱼体长相关分子标记。首先随机测得“东海1号”大黄鱼199个个体体长，均值为13.45cm，符合正态分布（ P >0.05），“极端大群体”和“极端小群体”之间差异显著（ P Pst I/ Alu I、 Pst I/ BanI I、 Pst I/ Bsp 1286I、 Pst I/ Bst NI、 Pst I/ Hae Ⅲ、 Pst I/ Rsa I、 Pst I/ Taq I）分别进行酶切，获得基因组代表性DNA片段并用于文库构建。根据多态性克隆数和多态性率确定 Pst I/ Rsa I酶切组合为最优降低基因组复杂度方法。之后从 Pst I/ Rsa I基因组代表性DNA片段文库中扩增获得3360个片段，以此为多样性芯片探针进行芯片点制，杂交筛选获得18个大黄鱼体长相关DArT候选标记。经过新群体样本再次验证，仍有8个DArT标记与体长相关。本研究有助于选育生长性状优良的大黄鱼群体。