中华鳖转铁蛋白基因序列特征及表达研究

转铁蛋白是呼吸链的关键因子, 参与体液和免疫系统的调节, 具有抗菌抗病毒等功能。研究从嗜水气单胞菌诱导的中华鳖肝脏SMART cDNA文库中, 分离克隆了中华鳖转铁蛋白基因cDNA和gDNA全序列, 对其序列特征、组织表达及原核表达进行了分析研究。结果显示, 中华鳖转铁蛋白对应全长cDNA的基因组DNA全长为19100 bp, 由17个外显子和16个内含子组成; cDNA全长为2359 bp, 开放阅读框为 2094 bp, 编码697个氨基酸; 转铁蛋白的分子量为76.6 kD, 包含2个糖基化位点, 存在4个二硫键形成区域的缺失, 两个同源结构域的相似性为37%, 信号肽位于第119位点。荧光定量PCR结果显示, 正常组中华鳖转铁蛋白基因在肝脏中的表达量最大; 以嗜水气单胞菌刺激后, 其在心、肝、脾和肾脏组织中的表达, 均有一个上调后减少的趋势。此外, 实验还进行了重组转铁蛋白原核表达和Western blot 检测鉴定, 为深入研究中华鳖转铁蛋白功能的提供了基础数据。       

Molecular Characterization and Expression Analysis of VSIG4 from the Asian Yellow Pond Turtle, Mauremys mutica

V-set and immunoglobulin domain-containing protein 4 (VSIG4), a member of the immunoglobulin superfamily, plays an important role in the immune system. This study isolated and characterized a cDNA encoding VSIG4 (MaVSIG4) from the Asian yellow pond turtle (Mauremys mutica). The MaVSIG4 cDNA is 1840 bp long and contains an open reading frame of 1,182 bp that encodes a polypeptide of 372 amino acids. The genomic sequence of MaVSIG4 spans 7,682?bp, with six exons and five introns. The phylogenetic tree shows that MaVSIG4 is most closely related to Gallus gallus VSIG4. The expression analysis by real-time PCR reveals that MaVSIG4 is ubiquitously expressed in various healthy tissues, with a higher expression level in the liver. After immune stimulation, the expression level of MaVSIG4 sharply decreased in the liver, heart, and kidney at 12?h (P?<?0.01). These results provide a basis for further study of the function of MaVSIG4 in the turtle’s immune system.     

Molecular cloning and expression analysis of myd88 from oriental weatherfish (Misgurnus anguillicaudatus) in response to bacterial challenge

Myeloid differentiation factor 88 (Myd88) plays an important role in both innate and adaptive immune response. In this study, the full-length complementary DNA (cDNA) of myd88 from Misgurnus anguillicaudatus was characterized. The myd88 cDNA is 1333 bp in length and contains an 855 bp open reading frame encoding a predicted protein of 284 amino acids. The predicted protein possesses typical Myd88 domain structural features including a death domain in the N-terminus, and box 1, 2, and 3 motifs of the Toll/IL-1 receptor domain in the C-terminus. Quantitative real-time PCR (qRT-PCR) revealed that myd88 messenger RNA (mRNA) was ubiquitously expressed in all examined tissues, especially highly in brain, kidney, blood, intestines and liver. qRT-PCR and western blotting were used to determine the mRNA and protein levels of Myd88 after Aeromonas veronii challenge, respectively. The Myd88 was remarkably upregulated in response to infection of A. veronii. These results suggested that Myd88 may play a vital role during the immune response of M. anguillicaudatus against bacterial infection.     

Molecular cloning and characterization of a transferrin cDNA from the white-spotted flower chafer, Protaetia brevitarsis.

A full-length cDNA clone with high homology to insect transferrin genes was cloned by screening a Protaetia brevitarsis cDNA library. This gene (PbTf) had a total length of 2338 bp with an open reading frame (ORF) of 2163 bp, and encoded a predicted peptide of 721 amino acid residues. Like known cockroach, termite, and beetle transferrins, PbTf appears to have residues comprising iron-binding sites in both N- and C-terminal lobes. The deduced amino acid sequence of the PbTf cDNA was closest in structure to the beetle Apriona germari transferrin (68% protein sequence identity). Northern blot analysis revealed that PbTf exhibited fat body-specific expression and was upregulated by wounding, bacterial or fungal infection and iron overload, suggesting a functional role for PbTf in defense and stress responses.     

A tandem-repeat galectin involved in innate immune response of the pearl oyster Pinctada fucata

The cDNA of a tandem-repeat galectin from the pearl oyster Pinctada fucata (designated PfGal) was cloned by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of PfGal was 1386 bp, consisting of a 5′ untranslated region (UTR) of 26 bp, a 3′ UTR of 313 bp, and an open reading frame (ORF) of 1047 bp encoding a polypeptide of 348 amino acids with a predicted molecular weight of 38.09 kDa and theoretical isoelectric point of 8.49. Similar to other tandem-repeat galectins, PfGal contained two tandem carbohydrate recognition domains (CRDs), with typical conserved motifs which were important for carbohydrate recognition, and it appeared to possess neither a signal peptide nor a transmembrane domain. Fluorescent quantitative real-time PCR analyses indicated that PfGal mRNA was highly expressed in hemocytes, digestive gland and mantle, and its expression was increased in all studied tissues after Vibrio alginolyticus challenge. The temporal expression of PfGal mRNA in hemocytes challenged by V. alginolyticus was clearly time-dependent and reached the maximum level at 6 h post-challenge, and then recovered to the original level. These results collectively indicated that PfGal may be involved in the immune response against bacterial infection and clearance of bacterial pathogens in P. fucata.     

Cloning and blood‐meal dependent induction pattern of apolipophorin‐III from Anopheles sinensis

Apolipophorin‐III is known to play a role in transporting lipids in insects, and much attention has been paid to lepidopteran insects' apolipophorin. Thus, we were interested in examining the effects of blood‐meal on the expression pattern of apolipophorin‐III in mosquitoes. This led us to clone and partially characterize the full‐length cDNA of apoLp‐III (AnsiApoLp‐III) from Anopheles sinensis. Analysis of AnsiApoLp‐III cDNA shows that the 728‐bp sequence has a 582‐bp protein‐coding region with 94 bp of putative 5′ untranslated region and 152 bp of 3′ untranslated region. The deduced amino acid sequence begins with a methionine codon at position 95 and extends to position 674, encompassing a polypeptide of 193 amino acids. AnsiApoLp‐III has the highest identity (63%) to Culex quinquefasciatus apoLp‐III. Temporal expression pattern analysis shows that although AnsiApoLp‐III was expressed at all developmental stages, it was highly detected at egg and adult stages in the female mosquitoes. In addition, we found out that AnsiApoLp‐III was induced in An. sinensis adult females after uptaking a blood‐meal. Spatial expression patterns of AnsiApoLp‐III shows that AnsiApoLp‐III mRNA was strongly induced at day 1 and gradually decreased from day 1 to day 4 in the ovaries. Most interestingly, AnsiApoLp‐III mRNA in the Malpighian tubule was strongly induced at day 1, decreased during days 1–3, and then became elevated again at day 4. These data suggest that blood‐meal influences AnsiApoLp‐III mRNA induction in ovaries and Malpighian tubules. It remains to further elucidate the biological roles of AnsiApoLp‐III in these organs.     

Molecular cloning and characterization of a novel mannose-binding lectin gene from Amorphophallus konjac

A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root.     

Expression analysis of cathelicidin and NK‐lysin in Dabry’s sturgeon (Acipenser dabryanus)

Antimicrobial peptides (AMPs) are a conserved component of the innate immune response in many species. In the present study, the cDNA sequences encoding two AMPs (cathelicidin and NK‐lysin, comprising 1,576 and 606 bp, respectively) were cloned from Dabry's sturgeon (Acipenser dabryanus). Phylogenetic analysis demonstrated that the two AMPs were clustered together with homologous protein sequences from other fish. NK‐lysin was highly expressed during early embryonic development, suggesting maternal transmission. Tissue distribution analysis showed that cathelicidin had the highest expression in the liver and NK‐lysin was most abundantly expressed in the spleen. In response to Poly I:C treatment, the expression of cathelicidin was upregulated at 12 and 24 hr post induction (hpi), but downregulated at 72 hpi. NK‐lysin mRNA expression increased after treatment with Poly I:C, reaching a peak at 24 hpi. Lipopolysaccharide treatment also induced the expression of two antimicrobial peptide genes. Lipopolysaccharide treatment significantly upregulated the expression of cathelicidin at 6, 24, and 48 hpi, and upregulated NK‐lysin expression at 6 and 12 hpi. These results suggested that two AMPs could participate in the immune response induced by poly I:C or LPS stimulation.     

Identification and characterization of a pig FKBP5 gene with a novel expression pattern in lymphocytes and granulocytes

Abstract

The immunophilins are an important group of regulatory molecules in the immune system. FKBP5, expressed throughout mammals and in fish and birds, functions in both physiological and pathogenic pathways, including innate immunity and steroid-based diseases. In this study, we cloned the first porcine FKBP5 from Rongchang pig by the rapid amplification of cDNA ends technique. The full-length cDNA is 4097?bp, with an open reading frame of 1371?bp that codes for a 457-aa protein. Western blotting detected the porcine FKBP5 protein at highest levels in thymus, followed by spleen and lung. Immunohistochemistry detected the porcine FKBP5 protein in lymphocytes and granulocytes of the blood, and flow cytometry identified greater expression in unactivated (vs. activated) T lymphocytes. Finally, the expression level of porcine FKBP5 in the granulocytes was found to decline significantly from the time of birth to one-year-old. These collective data suggest that the newly identified porcine FKBP5 may function in activation of T cells in pig and in innate immunity in the newborn pig in particular.     

Molecular characterization,recombinant expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and biological activity of (<Emphasis Type="Italic">S</Emphasis>)-Tetrahydroberberine oxidase from <Emphasis Type="Italic">Corydalis saxicola</Emphasis> Bunt

(S)-Tetrahydroberberine [(S)-THB] oxidase is the last enzyme of benzylisoquinoline alkaloids pathway which catalyzes the dehydrogenation of four hydrogen atoms of (S)-THB to produce berberine, the final step of berberine biosynthesis. A (S)-THB gene, designated as Cs(S)-THBO (Genbank accession No. HQ393909), was cloned from a Corydalis saxicola cDNA library by rapid amplification of cDNA ends. The full-length of cDNA of Cs(S)-THBO was 1127 bp with an open reading frame of 699 bp that predicted to encode a 232-amino acid polypeptide, with a predicted molecular mass of 25.20 kDa. Cs(S)-THBO was the first (S)-THBO gene found in C. saxicola. Real-time quantitative PCR analysis indicated that Cs(S)-THBO was constitutively expressed in roots, stems, leaves and flowers of C. saxicola, and with the highest expression level in roots. The results of treatment experiment for plant defense responses revealed that expression of Cs(S)-THBO had a prominent diversity. Recombinant Cs(S)-THBO protein expressed in Escherichia coli strain BL21 (DE3) was active. The results of feeding experiment and HPLC–DAD–ESI–MSⁿ analysis showed that Cs(S)-THBO had the function of catalyzing (S)-tetrahydroberberine to berberine.     

巴西橡胶树鲨烯合酶基因的克隆与序列分析

鲨烯合酶(SQS )是植物甾醇和三萜化合物生物合成途径中的关键酶。以巴西橡胶树为试验材料,提取胶乳总 RNA,利用 RT-PCR 以及 RACE 的方法克隆橡胶树鲨烯合酶 cDNA 编码区片段,并进行序列分析。结果表明：橡胶树鲨烯合酶 cDNA 编码区为1239 bp,编码413个氨基酸,命名为 HbSQS 。荧光定量分析表明鲨烯合酶基因在不同组织里表达水平存在明显差异,且受乙烯调控。     

Cloning and expression analysis of a muscarinic cholinergic receptor from the brain of ant,Polyrhachis vicina

Muscarinic acetylcholine receptors (mAchRs) are the predominant cholinergic receptors in the central and peripheral nervous systems of animals. They also have been found in various insect nervous systems. In this article, a full‐length cDNA of a pupative mAchR (PmAchR) was obtained from the brains of ant Polyrhachis vicina by homology cloning in combination with rapid amplification of cDNA ends. PmAchR encodes a 599‐amino acid protein that exhibits a high degree of homology with other mAchRs. Real‐time quantitative RT‐PCR analysis showed that PmAchR is differentially expressed in the brains of workers, males, and females. By in situ hybridization, it is revealed that PmAchR is widely expressed in different soma clusters of the brain, including the mushroom bodies, the antennal lobes, as well as the optic lobes (OL), and the most intensely staining is found in Kenyon cells. Nonetheless, there are more positive nerve fibers in the OL of males' brains than in females' and workers' brains. © 2011 Wiley Periodicals, Inc.