三疣梭子蟹HMGBa基因克隆及其应答不同病原入侵的表达特征

为研究三疣梭子蟹(Portunus trituberculatus)高迁移率族蛋白B (High-mobility group box protein, HMGB)在其先天免疫中发挥的功能, 利用RACE技术首次克隆得到了三疣梭子蟹HMGBa基因, 命名为PtHMGBa。其cDNA序列全长1030 bp, 其中5′端非编码区(UTR)为94 bp, 3′端非编码区(UTR)为255 bp, 开放阅读框(ORF)为681 bp, 编码一个含有227个氨基酸, 分子量25.82 kD, 理论等电点为5.94的蛋白质。PtHMGBa蛋白包含2个HMG盒结构域和一个酸性尾部结构域。分析表明, 三疣梭子蟹HMGBa氨基酸序列与凡纳滨对虾(Litopenaeus vannamei) HMGBa相似度最高。实时荧光定量PCR结果显示, PtHMGBa基因在血细胞和肝胰腺的表达量最高, 在眼柄中表达量最低。在副溶血弧菌和WSSV感染过程中, PtHMGBa基因在肝胰腺和血细胞中均出现了表达上调。其中, 经副溶血弧菌感染后, 该基因在上述2种组织中分别于48h和6h达到表达量的峰值; 经WSSV感染后, 该基因在2种组织中均在12h达到表达量的峰值。结果表明PtHMGBa基因参与了三疣梭子蟹抵御外来病原的免疫响应, 研究为深入开展三疣梭子蟹和其他甲壳动物的免疫调控机理提供了科学依据。     

三疣梭子蟹Δ6脂肪酸去饱和酶基因全长cDNA克隆与组织表达

旨在探讨三疣梭子蟹高度不饱和脂肪酸自身合成能力,探究三疣梭子蟹HUFA生物合成途径。采用cDNA末端快速扩增(RACE)技术克隆得到三疣梭子蟹△6去饱和酶cDNA全长序列,并利用荧光定量PCR技术进行肝胰腺、肠道、鳃等8种组织的表达分析。通过分析序列表明,基因序列全长2875bp,其中5'非编码区长465bp,3'非编码区长1078bp,开放阅读框(ORF)长1332bp,编码443个氨基酸;并且编码的蛋白序列具有典型的去饱和酶特性:3个组氨酸保守区,一个N端细胞色素b5结构域以及一个血红素结合的HPGG结构域。荧光定量PCR结果显示,Δ6脂肪酸去饱和酶基因在三疣梭子蟹多个组织中均有表达,在肝胰腺中表达量最高,其次是肠道和肌肉,心脏中表达最少。结果表明三疣梭子蟹具有△6去饱和酶。     

青花菜雄性不育相关基因BoDHAR的克隆与表达分析

以一个与甘蓝显性核不育相关的差异表达片段的序列为信息探针,通过在NCBI与TAIR网站数据库中进行同源EST序列搜索,经人工拼接、RT-PCR、PCR 克隆与序列分析,获得了青花菜脱氢抗坏血酸还原酶DHAR dehydroascorbate reductase 基因的 cDNA 与 DNA 全长序列,命名为BoDHAR。并利用双链接头介导 PCR 的染色体步行技术（genome walking）克隆了其上游 644bp 的5′端序列。所获的BoDHAR基因全长 1486bp,存在两个内含子,DNA 编码区序列633bp,编码210个氨基酸;序列分析表明：BoDHAR与同源基因AT1G195701cDNA 序列有 82.3% 的一致性,推导的氨基酸序列有 79.6% 的一致性;编码的水溶性蛋白存在多个磷酸化位点;5′端上游区存在明显的转录调控序列。半定量RT-PCR结果表明：BoDHAR 在可育系花蕾中的表达量明显高于不育系花蕾,在花药中的表达明显高于其它部位。     

三疣梭子蟹Na+/H+-exchanger基因克隆鉴定及在盐度胁迫下的表达分析

为研究Na+/H+-exchanger基因在三疣梭子蟹（Portunus trituberculatus）盐度胁迫过程中的功能作用,克隆了三疣梭子蟹Na+/H+-exchanger基因并进行表达分析。结果显示,Na+/H+-exchanger基因（GenBank:KU519329）全长4233 bp,5和3非编码区（UTR）长分别为519和753 bp,开放阅读框（ORF）长2961 bp。编码986个氨基酸,预测蛋白质分子量和等电点分别为110.8 kD和7.42,具有信号肽和典型的Na+/H+-exchanger蛋白结构域,含12个跨膜螺旋;三疣梭子蟹Na+/H+-exchanger基因与普通滨蟹（Carcinus maenas）同源性最高,达到87.2%,系统进化分析也显示该序列与普通滨蟹聚为一支;表达分析显示,三疣梭子蟹Na+/H+-exchanger基因在鳃中表达量最高;在低盐（盐度5、10和20）胁迫过程中,Na+/H+-exchanger基因在0-12h上调表达明显,在24-168h间表达量呈下降趋势;在高盐（盐度50）胁迫初期（0-12h）,该基因表达量相对稳定,之后（24-168h）显著下调表达。研究表明低盐显著诱导Na+/H+-exchanger基因的高表达,推测三疣梭子蟹Na+/H+-exchanger基因在低盐环境下发挥重要的渗透调节功能。     

三疣梭子蟹NF-κB家族基因Relish和Dorsal的克隆及表达特征

为研究Relish和Dorsal在三疣梭子蟹免疫过程中所起到的作用, 研究利用RACE技术克隆获得三疣梭子蟹Relish(Pt-Rel)、Dorsal基因(Pt-Dor) cDNA全长, 并通过实时荧光定量PCR技术分析了Pt-Rel和Pt-Dor基因在健康蟹不同组织及其原代培养的血淋巴细胞在感染不同微生物后的表达情况。结果显示, Pt-Rel cDNA长3254 bp, ORF长2949 bp, 编码983个氨基酸, Pt-Dor cDNA长2348 bp, ORF长1911 bp, 编码637个氨基酸; 蛋白结构预测分析发现Pt-Rel和Pt-Dor均包含RHD (Rel homology domain)及IPT (Immunoglobulin-like fold, Plexins, TranscriPtion factors)Rel/NF-κB家族蛋白经典结构域。Pt-Rel和Pt-Dor与其他节肢动物Relish、Dorsal氨基酸序列具有很高的相似性; 在系统进化分析中Pt-Rel和Pt-Dor分别与中华绒螯蟹等甲壳动物的Relish、Dorsal聚在一支, 而昆虫类聚在另一支。Pt-Rel和Pt-Dor在检测的6种组织中均有表达, 且2个基因均在血淋巴细胞中表达量最高。蟹血淋巴细胞体外感染实验结果表明, 不同病原微生物对2个基因表达的影响非常相似, 假丝酵母在2h明显诱导了Pt-Rel和Pt-Dor基因的表达, 而金黄色葡萄球菌及溶藻弧菌在感染4h后使Pt-Rel和Pt-Dor基因的表达显著上调。上述研究结果表明Pt-Rel和Pt-Dor基因很有可能参与了三疣梭子蟹的抗感染免疫过程。     

日本血吸虫琥珀酸脱氢酶铁硫蛋白全长cDNA的克隆、表达及保护性免疫评价

根据GenBank日本血吸虫(Schistosoma japonicum)琥珀酸脱氢酶铁硫蛋白(SjSDISP)不完整的表达序列标签(BU804141)以及日本血吸虫成虫cDNA文库载体多克隆位点邻近核苷酸序列设计引物, 以日本血吸虫成虫cDNA文库为模板, 采用锚式PCR策略, 对SjSDISP cDNA不完整的3′端和5′端进行扩增、测序, 用序列比对拼接电子软件比对, 基于重叠区进序列合并, 获得1071 bp的SjSDISP全长cDNA. 序列分析推断该片段含有编码SjSDISP基因的完整阅读框, 编码278个氨基酸残基. 将其编码基因克隆到原核表达载体pQE30上, 在大肠杆菌M15中获得准确、高效表达, 表达产物分子量约为32 kD. 用日本血吸虫成虫抗原免疫血清对表达产物进行Western blot检测, 在预测位置上出现明显的识别条带. 用纯化的重组蛋白rSjSDISP免疫小鼠, 进行动物免疫保护性评价, 在虫荷、每克肝卵、每克粪卵和每雌子宫内卵数方面, 与佐剂对照组比较差异均具有显著性(P<0.05, P<0.01). 结果表明, 日本血吸虫琥珀酸脱氢酶铁硫蛋白酶(SjSDISP)全长cDNA成功克隆并在大肠菌中得到表达; 表达产物具有良好的抗原性和动物免疫保护效果, 是一种潜在的具有部分免疫保护性的抗日本血吸虫病疫苗候选分子.     

三疣梭子蟹脂多糖-β-1,3-葡聚糖结合蛋白基因的克隆及表达模式分析

脂多糖-β-1,3-葡聚糖结合蛋白(LGBP)作为一种模式识别受体在甲壳动物的先天免疫中占有重要地位。利用SMART RACE技术,从三疣梭子蟹(Portunus trituberculatus)血细胞中克隆得到一条LGBP基因。该基因cDNA序列全长为1378bp,包括1095bp的开放阅读框(ORF),共编码365个氨基酸,在基因的5'端还含有138bp的非编码区(UTR),3'端含有144bp的UTR。预测的成熟肽相对分子质量为39825.24k,等电点为4.49。同时,用生物信息学方法对该基因的序列和二级结构进行了初步分析。RT-PCR结果显示:LGBP基因在被检测的组织,包括血细胞、肝胰腺、心脏、鳃和肌肉中都有表达。用金黄色葡萄球菌(Staphyloccocus aureus)和溶藻弧菌(Vibrio alginolyticus)刺激三疣梭子蟹后,发现在实验48h内,混合细菌刺激组血细胞中LGBP基因的表达量明显高于生理盐水对照组,推测该基因在抵抗细菌感染的免疫过程中发挥着积极的作用。     

芒果生长素反应因子类蛋白的cDNA克隆和表达

通过SSH法获得了一个与不定根形成相关的差异表达的cDNA片段,其推导的氨基酸序列与拟南芥的生长素反应因子(ARF)类蛋白具有较大的同源性,因此将它命名为MiARF。用所设计的基因特异引物进行3′RACE扩增获得包含完整读码框架(ORF)的MiARF1（GenBank登录号为AY255705）和MiARF2（GenBank登录号为AY300808）。MiARF1全长为3272bp,其中,ORF含2523bp,5′非翻译区（5′UTR）含285bp, 3′非翻译区(3′UTR)含464bp。由该序列所推导的氨基酸序列与拟南芥ARF2（BAB10162）的ID值为64%, E值为0,在DNA结合区域（DBD）、III和IV区域的同源性更高,ID值均大于80%。MiARF2cDNA全长为1474bp,其中ORF含981bp,5′非翻译区含285bp, 3′非翻译区含208bp,由该序列所推导的氨基酸序列与拟南芥ARF2（BAB10162）的ID值为84%,E值为e-151。 MiARF2仅具有DBD保守区并与MiARF1的基本相同,但缺乏III和IV区域。Virtural Northern 杂交表明：MiARF2在生根的组织中表达水平高, 而在非生根的组织中未见表达;MiARF1在生根及非生根的组织中均有表达。     

绵羊tnp2基因的克隆及其在体外共培养系统中圆形精子细胞的转录分析

过渡蛋白2基因（tnp2）是圆形精子细胞特异表达的基因。为了开展绵羊圆形精子细胞标记基因的研究,根据GenBank上已公布的牛的cDNA序列设计引物,采用RT-PCR和分子克隆方法,克隆了蒙古绵羊tnp2基因cDNA部分编码区序列。DNA 序列测定结果与牛的核苷酸序列比对,同源性为95.3%。根据绵羊tnp2基因的cDNA序列设计引物,对共培养的四月龄绵羊睾丸生殖细胞进行RT-PCR鉴定。结果显示体外共培养的绵羊睾丸生殖细胞一直到第十周后仍有圆形精子细胞产生。绵羊tnp2基因的cDNA克隆和序列测定为进一步研究绵羊精子发生过程奠定了基础。     

三疣梭子蟹腺苷酸转移酶(ANT)基因的克隆与分析

腺苷酸转移酶(Adenine nucleotide translocase,ANT)是线粒体内膜上负责能量分子传导的转运蛋白,在能量代谢中起着关键作用。为了研究ANT基因在甲壳动物蜕皮中的作用,采用RT-PCR和c DNA末端快速扩增技术(RACE技术)克隆得到三疣梭子蟹ANT基因的序列全长(Gen Bank登录号:KM921660),该序列全长1 414 bp,包括132 bp的5'端非编码区,352 bp的3'段非编码区,具有930 bp的完整开放阅读框(ORF),编码309个氨基酸。将该ANT基因序列推导的氨基酸序列与已公布的其他物种ANT序列进行系统进化树分析发现,三疣梭子蟹ANT基因与其他甲壳动物ANT基因聚为一支,其中与拟穴青蟹ANT一致性高达96%。通过氨基酸序列比对发现,三疣梭子蟹ANT序列具有3个保守的线粒体穿膜功能结构域,是形成能量分子传导的转运通道,催化细胞质中ADP和线粒体内ATP间进行跨膜交换。采用实时荧光定量PCR技术,分析三疣梭子蟹ANT基因的组织差异表达,结果表明ANT基因在三疣梭子蟹肌肉(Ms)中的表达量最高,在其他组织中表达量均较低,具显著差异(P0.05);在三疣梭子蟹蜕皮周期中,肌肉中ANT基因的表达量A期最高(P0.05),然后下降,至C期最低,随后又逐渐上升至D1期,在D1期出现第2个峰值后再逐渐下降。研究结果说明ANT基因与三疣梭子蟹肌肉活动密切相关,可能在蜕皮调控中发挥重要作用。     

Cloning of profilin (FcPFN) from the shrimpFenneropenaeus chinensis, a highly expressed protein in white spot syndrome virus (WSSV)-infected shrimp

We isolated and characterized the profilin (FcPFN) cDNA from hemocytes ofFenneropenaeus chinensis, a unique shrimp species from the Yellow Sea. The FcPFN cDNA consists of 830 bp and encodes a polypeptide of 125 amino acids, having a predicted isoelectric point of 5.06. The deduced amino acid sequence of FcPFN shows 36% and 90% amino acid sequence identity to the profilin genes of Pacific white shrimpLitopenaeus vannamei and black tiger shrimpPenaeus monodon, respectively. The FcPFN mRNA was highly expressed in hemocytes and hepatopancreas and moderately in muscle of normal shrimp. The higher expression of FcPFN mRNA is observed in shrimp infected with the white spot syndrome virus (WSSV), which is a major concern in all shrimp-growing regions of the world. These results suggest a potential role for FcPFN in viral host defense mechanisms.     

Molecular cloning, characterization and expression analysis of two apoptosis genes, caspase and nm23, involved in the antibacterial response in Chinese mitten crab, Eriocheir sinensis

Apoptosis is a central regulatory feature of the immune system, and the most common form of death among immunological cells. However, the function of apoptosis, within the innate immune system of invertebrates, remains largely unknown. For this reason, we investigated the immune functionality of two apoptosis genes, caspase and nm23, in the Chinese mitten crab (Eriocheir sinensis), which is a commercially important and disease vulnerable aquaculture species. The entire length caspase and nm23 cDNA genes were cloned using PCR, based on an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The caspase cDNA contained an 1119 bp open reading frame that encoded a putative 372 amino acid protein, while nm23 cDNA contained a 456 bp open reading frame that encoded a putative 151 amino acid protein. Comparison, with other reported invertebrate and vertebrate sequences, revealed the presence of conserved enzyme active sites that were common among caspase and nm23 superfamilies. In brief, caspase and nm23 mRNA expression in E. sinensis were (a) both detected in all tissues, including the hemocytes, heart, hepatopancreas, gill, stomach, muscle, intestine, brain and eyestalk, and (b) responsive in hemocytes, gill and hepatopancreas to a Vibrio anguillarum immuno-challenge all appeared sharp increase. Collectively, the data presented here demonstrate the successful isolation of caspase and nm23 apoptosis genes from the Chinese mitten crab, and their role in the innate immune system of an invertebrate.     

三叶木通花粉前纤维蛋白基因的克隆与表达

以三叶木通花蕾为材料,采用RT-PCR、3-′RACE方法克隆了三叶木通花粉前纤维蛋白基因,命名为Atf-Pro(GenBank登录号GQ478584)。结果表明:AtfPro的cDNA全长735 bp、阅读框393 bp、编码131个氨基酸,有1个342 bp的3′端非翻译区。预测分子量约为14.081 kD,等电点4.74。氨基酸和核苷酸序列的同源性分析发现,AtfPro基因属于植物花粉profilin基因家族的新成员。RT-PCR定性分析表明,AtfPro基因在三叶木通花蕾、花药、雌花花瓣和柱头组织中均有表达,但在幼叶、茎尖、根尖组织中低水平表达或不表达,生殖器官中的表达时期从花序分化发育开始到开花散粉结束。     

A C-type lectin like-domain (CTLD)-containing protein (PtLP) from the swimming crab Portunus trituberculatus

C-type lectins play important roles in the non-self innate immune system of invertebrates. In this study, we isolated the full-length cDNA of the C-type lectin like-domain (CTLD)-containing protein, designated PtLP, from the hepatopancreas of the swimming crab Portunus trituberculatus, one of the most common edible crabs of East Asia. The PtLP cDNA consists of 923bp and encodes a polypeptide of 164 amino acids containing a well-conserved C-type lectin like-domain (CTLD). The deduced amino acid sequence of PtLP shows 29-36% amino acid sequence identity to other crustacean C-type lectin sequences. A phylogenetic analysis revealed that PtLP is in a large cluster together with black tiger shrimp PmAV, a gene involved in virus resistance of shrimp, and all of the C-type lectins from the various shrimps. Quantitative RT-PCR analysis showed that the PtLP mRNA was expressed highly in hepatopancreas and moderately in gills, hemocytes, and ovary of normal swimming crabs.     

Molecular cloning, characterization and expression analysis of macrophage migration inhibitory protein (MIF) in Chinese mitten crab, Eriocheir sinensis

Macrophage migration inhibitory factor (MIF) as a multi-functional cytokine mediating both innate and adaptive immune responses, however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of MIF in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full-length MIF cDNA (704 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a E. sinensis cDNA library. The MIF cDNA contained a 363 bp open reading frame (ORF) that encoded a putative 120 amino acid (aa) protein. Comparisons with other reported invertebrate and vertebrate MIF sequences revealed conserved enzyme active sites. MIF mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in hepatotpancreas, and (b) responsive in hemocytes, hepatopancreas and gill to a Vibrio anguillarum challenge, with peak exposure observed 8 h, 12 h and 12 h post-injection, respectively. Collectively, data demonstrate the successful isolation of MIF from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.     

Antimicrobial tachyplesin peptide precursor. cDNA cloning and cellular localization in the horseshoe crab (Tachypleus tridentatus)

The hemocytes of the horseshoe crab have been found to contain a new family of Arthropodous antibiotics, termed the "tachyplesin family." These peptides are composed of 17-18 amino acid residues with a carboxyl-terminal arginine alpha-amide. We report here the entire cDNA sequence coding for the tachyplesin precursors and their distribution in various tissues of the horseshoe crab. Sequence analysis of the cloned cDNAs revealed that the tachyplesin precursors consist of 77 amino acids with 23 residues in a presegment, and that there are two types of mRNAs corresponding to the isopeptides tachyplesins I and II. Both precursors contain a putative signal peptide, a processing peptide sequence and a carboxyl-terminal amidation signal "Gly-Lys-Arg" connected to the mature tachyplesin peptide. Moreover, an unusual acidic amino acid cluster, Asp-Glu-Asp-Glu-Asp-Asp-Asp-Glu-Glu-COOH, is present in the carboxyl-terminal portions of both precursors. These results suggest that the two types of tachyplesin precursors are first synthesized as preproproteins and are then incorporated into the intracellular organelle, accompanied by various processing events. Northern blot analysis on a total RNA from various tissues of the horseshoe crab revealed that the tachyplesin precursors are expressed mainly in hemocytes and cardiac and brain tissues. Tachyplesin was immunohistochemically localized in the smaller dense granules rather than the typical large granules present in abundance in the hemocytes.     

Molecular cloning and characterization of a catalase gene from Zhikong scallop Chlamys farreri

Catalase is one of the central enzymes involved in scavenging the high level of reactive oxygen species (ROS) by degradation of hydrogen peroxide to oxygen and water. The full-length catalase cDNA of Zhikong scallop Chlamys farreri (denoted as CfCAT) was identified from hemocytes by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The nucleotide sequence of CfCAT cDNA consisted of 3146bp with a 5' UTR of 103bp, an unusually long 3' UTR of 1519bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1521bp encoding a polypeptide of 507 amino acids with predicted molecular weight of 57.5kDa. The deduced amino acid sequence of CfCAT has significant homology to catalases from animals, plants and bacteria. Several highly conserved motifs including the proximal heme-ligand signature sequence RLFSYNDTH, the proximal active site signature FNRERIPERVVHAKGGGA, and the three catalytic amino acid residues of His(72), Asn(145) and Tyr(355) were identified in the deduced amino acid sequence of CfCAT. The CfCAT was demonstrated to be a peroxisomal glycoprotein with two potential glycosylation sites and a peroxisome targeting signal of ANL that was consistent with human, mouse and rat catalases. The time-course expression of CfCAT in hemocytes was measured by quantitative real-time PCR. The expression of CfCAT increased gradually and reached the highest point at 12h post-Vibrio infection, then recovered to the original level at 24h. All these results indicate that CfCAT, a constitutive and inducible protein, is a member of the catalase family and is involved in the process against ROS in scallop.     

Two types of coagulogen mRNAs found in horseshoe crab (Tachypleus tridentatus) hemocytes: molecular cloning and nucleotide sequences

The complete cDNA sequence coding for the coagulogen present in the horseshoe crab (Tachypleus tridentatus) hemocytes was determined. Clones carrying cDNA fragments for coagulogen were isolated from a cDNA library of the hemocyte mRNA using synthetic oligodeoxyribonucleotides as probes. The nucleotide sequence analyses of the cloned cDNAs revealed that the hemocyte coagulogen consists of 175 amino acids with 20 amino acids in a presegment, and that there are two types of mRNAs for coagulogen. The two mRNAs exhibited three nucleotide substitutions, two of which were in their protein-coding regions, resulting in two amino acid replacements. Subsequently, two molecular species of coagulogen, named coagulogens type I and type II, were identified by tryptic peptide mapping of the mature proteins isolated from the hemocyte lysate. These results suggest that the two types of coagulogens are first synthesized as preproteins and are incorporated into the granules that are abundantly present in the hemocytes with liberation of the signal peptides.