金鱼胰蛋白酶原基因的克隆及镉和过氧化氢暴露对其基因表达的影响

为深入研究胰蛋白酶在鱼类中的蛋白结构和生理功能, 利用RT-PCR和RACE方法, 从金鱼肝胰脏中成功克隆获得了一种全长864 bp的胰蛋白酶原cDNA序列(gfTryp)。gfTrypc DNA包含21 bp的5′-非翻译区、114 bp的3′-非翻译区和729 bp的开放读码框, 编码242个氨基酸组成的胰蛋白酶原(gfTryp)。gfTryp含有15个氨基酸的信号肽和5个氨基酸(LDDDK)的激活肽。氨基酸序列分析表明, gfTryp具备胰蛋白酶原的保守结构特征, 如含有催化三联体氨基酸(His-57、Asp-102和Ser-195), 12个半胱氨酸, 位于底物结合口袋底部Asp-189和口袋开口处的Gly-216、Gly-226等, 提示其可能具有保守的蛋白消化功能。RT-PCR结果显示, gfTryp mRNA在所检测的各个组织中均有表达, 其中在肝胰脏、肠和脂肪中表达量为最高。进一步研究发现, 相较于摄食前, 肝胰脏gfTryp mRNA在金鱼摄食后显著升高。在0.5和5 μg/mL镉暴露处理后, 肝胰脏gfTryp mRNA显著升高(与未处理组相比, 分别约为3.2 和 4.7倍)。随着镉浓度增加到10 μg/mL后, gfTryp mRNA表达量下降。经100 μmol/L过氧化氢处理3h、6h、12h和24h后, 金鱼肝胰脏gfTryp mRNA的表达水平均显著下降, 在6h达到最大效应(约为对照组的0.21倍)。研究结果证实了重金属镉和过氧化氢处理能调控胰蛋白酶原基因表达, 为进一步探讨鱼类消化生理提供了新的视角。     

Molecular cloning, characterization and expression of cDNA encoding translationally controlled tumor protein (TCTP) from Jatropha curcas L

A cDNA encoding translationally controlled tumor protein (TCTP) of Jatropha curcas L., JcTCTP, was isolated from an endosperm cDNA library. JcTCTP consisted of a 5?? untranslated region (UTR) of 526 bp, a 3?? UTR of 377 bp and an open reading frame (ORF) of 507 bp, encoding a protein of 168 amino acid residues, which contained two signature sequences of TCTP family. Its deduced amino acid sequence was similar to the other known plants TCTPs in a range of 77.4?C92.3%. Expression of JcTCTP was the highest in the stem, endosperm at embryo formation stage and embryo of J. curcas tissues, and the lowest in the endosperm at seminal leaf embryo stage and flower, demonstrating a pattern of temporal and spatial specific expression.     

Cloning and characterization of a hsp70 gene from Asiatic hard clam Meretrix meretrix which is involved in the immune response against bacterial infection

In the present study, a 71.43 kDa heat shock protein cDNA was cloned from Asiatic hard clam Meretrix meretrix. The cDNA was 2292 bp, containing an open reading frame (ORF) of 1959 bp, which encodes a protein of 652 amino acids with a theoretical molecular weight of 71.43 kDa and an isoelectric point of 5.32. Based on the amino acid sequence analysis and phylogenetic analysis, this hsp70 cDNA is a member of cytoplasmic hsc70 (constitutive genes) subfamily in the hsp70 family, and is designated as MmeHsc71. Quantitative RT-PCR was carried out to compare the spatial and temporal expression patterns of MmeHsc71 in the mRNA level between control clams and Vibrio parahaemolyticus-infected clams. Spatially, MmeHsc71 mRNA was found in all tested tissues, including foot, hepatopancreas, mantle and gill. MmeHsc71 mRNA expression level in hepatopancreas and gill displayed a significant increase in vibrio-challenged clams at 24h post-infection compared to control clams (P < 0.05). Temporally, there was a significant increase of MmeHsc71 mRNA level in hepathopancreas of vibrio-challenged clams compared to control clams at 6, 12, and 24h post-challenge, respectively. The result of quantitative immunofluorescence also indicated that there was obvious increase of MmeHsc71 in hepatopancreas of vibrio-challenged clams compared to control clams in protein level at 24h post-infection. The results suggested that MmeHsc71 may play an important role in mediating the immune responses of M. meretrix to bacterial challenge.     

Molecular cloning and characterization of the translationally controlled tumor protein from <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis>

The translationally controlled tumor protein (TCTP) is a multi-functioning protein that performs vital roles, particularly in various complicated life processes. In this study, a new TCTP cDNA was cloned from Fenneropenaeus chinensis and hence was designated as Fc-TCTP. Its length is 711 bp, and it is characterized by 507-bp open reading frame that encodes a deduced 168-amino acid protein, including a TCTP domain. Moreover, this study analyzed the expression patterns of this gene when it responds to infection specifically with Vibrio anguillarum and the white spot syndrome virus (WSSV). Based on the results, Fc-TCTP was present in all the analyzed tissues. Additionally, Fc-TCTP’s expression level decreased after having been infected by bacteria, but was upregulated in the hepatopancreas after having been exposed to WSSV. Likewise, the Fc-TCTP protein was upregulated during its exposure to the virus. These results suggest that Fc-TCTP could well be involved in the antiviral response in F. chinensis.     

三角帆蚌热休克蛋白60基因克隆及其表达分析

利用3'RACE技术, 对高通量转录组测序所得三角帆蚌HSP60基因（hcHSP60）长片段（2629 bp）进行了3'末端克隆, 经拼接得到hcHSP60 cDNA全长序列。采用多种分子生物学软件对hcHSP60 cDNA全长序列进行了特征分析, 并采用RT-PCR技术检测了其组织分布及经冷热应激后的表达变化。结果显示, hcHSP60 cDNA全长为2807 bp, 其中开放阅读框为1707 bp, 编码568个氨基酸, 预测分子量大小为61.04 ku, pH 7.0时的理论等电点为5.63。序列中不存在信号肽与跨膜结构。氨基酸序列保守性分析表明, hcHSP60氨基酸序列与光滑双脐螺HSP60同源性最高（达82%）, 而与牙鲆、白云金丝鱼HSP60的同源性最低（为75%）。RT-PCR检测结果表明, hcHSP60在肝胰腺中的表达水平最高, 而在血液中的表达水平最低。30℃与35℃水温处理三角帆蚌4h后, 各组织中hcHSP60表达水平明显上升, 表明hcHSP60可能在三角帆蚌耐热应激反应中起着重要作用。       

镉胁迫对河南华溪蟹两种C型凝集素免疫应答的影响

C型凝集素是一类可以和糖类结合的蛋白质, 是先天性免疫系统中重要的模式识别受体。其中, 经典C型凝集素依赖Ca2+对糖类进行识别。Ca2+可作为细胞内第二信使, 参与多种信息传递。而重金属镉可导致细胞钙稳态失调, 干扰细胞内与Ca2+相关的信息传递。研究旨在探明镉胁迫对河南华溪蟹(Sinopotamon henanense) ShLec21和ShLec23两种C型凝集素免疫应答的影响。利用RACE方法, 克隆了ShLec21和ShLec23, 并进行了系统进化分析; 利用实时荧光定量PCR的方法, 研究了ShLec21和ShLec23的组织表达模式和镉联合嗜水气单胞菌(Aeromonas hydrophila)胁迫后肝胰腺和血淋巴中ShLec21和ShLec23表达模式。结果显示: ShLec21 cDNA全长863 bp, 编码152个氨基酸残基; ShLec23 cDNA全长681 bp, 编码164个氨基酸残基。ShLec21和ShLec23分别聚类为无脊椎动物的两个分支。ShLec21和ShLec23在血淋巴、鳃、肝胰腺、肠道、肌肉、卵巢和精巢中表达广泛, 但二者均主要在肝胰腺中表达。在胁迫条件下, 单独镉胁迫对肝胰腺和血淋巴中ShLec21和ShLec23表达量无显著影响; 在单独嗜水气单胞菌感染后, 肝胰腺中ShLec21和ShLec23表达量分别显著(P<0.05)与极显著(P<0.01)下调, 血淋巴中ShLec23表达量显著(P<0.05)下调; 而在镉胁迫后嗜水气单胞菌感染过程中,ShLec21和ShLec23表达量在肝胰腺和血淋巴中显著(P<0.05)或极显著(P<0.01)上调。研究结果表明, 河南华溪蟹ShLec21和ShLec23在响应嗜水气单胞菌感染过程中的表达, 在一定程度上能够被镉胁迫所上调。