植物凝集素的分子生物学研究

植物凝集素是一类具有高度特异性糖结合活性的蛋白,含有一个或多个可与单糖或寡聚糖特异可逆结合的非催化结构域。它的糖结合特异性主要针对外源寡糖,主要生理功能是介异植物的防御反应。到目前为止已克隆了２２２个植物凝集素基因。作者就植物凝集素的分类、性质、功能、凝集素基因的克隆和凝集素的翻译后加工过程作一综述。     

Molecular cloning, characterization and expression of a C-type lectin cDNA in Chinese mitten crab, Eriocheir sinensis

C-type lectins are pattern-recognition proteins which are functionally important for pathogen recognition and immune regulation in vertebrates and invertebrates. In this study, a lectin cDNA named as Es-Lectin was cloned and characterized from the Chinese mitten crab, Eriocheir sinensis. The full-length sequence of this Es-Lectin cDNA was 651 bp, including an open reading frame of 483 bp encoding 160 amino acids. The predicted molecular weight of the Es-Lectin was 11.8 kDa. A typical signal peptide of 21 amino acids was deduced at the N-terminus of the predicted protein. This Es-Lectin belongs to a C-type lectin and contains six cysteines, a conserved EPN motif (Glu-Pro-Asn) and an imperfect WND (Trp-Asn-Asp) motif (FND, Phe-Asn-Asp). This Es-Lectin had 55% and 32% identity with other two C-type lectins in E. sinensis, and 29-36% homology with decapods. Although the Es-Lectin was also expressed in gill, hepatopancreas, intestine, muscle and stomach, its expression in haemocytes was the greatest. The expression of Es-Lectins in haemocytes increased at 1.5 h after the Aeromonas hydrophila challenge. After a slight decrease, the Es-Lectin expression in haemocytes significantly increased at 48 h post-challenge. The diverse distribution of Es-Lectin and its enhancement by bacterial challenge indicate that C-type lectins are important in the innate immune response to bacterial infection, and can be activated for innate immune response in crab at the initial stage after pathogen infection.     

Comprehensive analysis of medaka major histocompatibility complex (MHC) class II genes: Implications for evolution in teleosts

The major histocompatibility complex (MHC) class II molecules play central roles in adaptive immunity by regulating immune response via the activation of CD4 T cells. The full complement of the MHC class II genes has been elucidated only in mammalian species to date. To understand the evolution of these genes, we performed their first comprehensive analysis in nonmammalian species using a teleost, medaka (Oryzias latipes). Based on a database search, cDNA cloning, and genomic PCR, medaka was shown to possess five pairs of expressed class II genes, comprising one IIA and one IIB gene. Each pair was located on a different chromosome and was not linked to the class I genes. Only one pair showed a high degree of polymorphism and was considered to be classical class II genes, whereas the other four pairs were nonclassical. Phylogenetic analysis of all medaka class II genes and most reported teleost class II genes revealed that the IIA and IIB genes formed separate clades, each containing three well-corresponding lineages. One lineage contained three medaka genes and all known classical class II genes of Ostariophysi and Euteleostei and was presumed to be an original lineage of the teleost MHC class II genes. The other two lineages contained one nonclassical medaka gene each and some Euteleostei genes. These results indicate that multiple lineages of the teleost MHC class II genes have been conserved for hundreds of millions of years and that the tightly linked IIA and IIB genes have undergone concerted evolution.     

Cloning and functional characterization of a GNA-like lectin from Chinese Narcissus (Narcissus tazetta var. Chinensis Roem)

A full-length cDNA encoding Narcissus tazetta lectin (NTL) was isolated from Chinese narcissus (N. tazetta var. Chinensis Roem). The open reading frame (ORF) was 519 bp long and encoded 172 amino acids with a theoretical isoelectric point of 5.27 and a calculated molecular mass of 18.6 kDa. Conserved domain analysis indicated that it possessed three D-(+)-mannose-binding sites, presumed to be similar to those of Galanthus nivalis agglutinin (GNA)-like lectins. A recombinant (glutathione S-transferase) GST-NTL fusion protein of around 40 kDa was successfully synthesized in vitro. Lysates of cells expressing this recombinant protein exhibited significant hemagglutinating activity [418 hemagglutinating units (HU)], as did the purified protein (265 HU). Sugar specificity assays suggested that mannose is the only sugar that significantly inhibits this hemagglutinating activity, confirming that NTL is a member of the GNA-like lectin family. NTL is highly transcribed in flowers, leaves and roots, but less so in scales. However, similar levels of the NTL protein were observed in all four of these organs by western blotting. A fluorescent NTL-GFP (green fluorescent protein) fusion protein was found to be primarily localized in the vacuole of transformed onion epidermal cells, indicating that NTL may be a vacuolar storage protein. This is the first study in which the function of NTL has been examined and provides a considerable body of data concerning its physiological role in Chinese narcissus. The results obtained may be useful in the molecular engineering of plants with enhanced tolerance of biotic and abiotic stresses. Moreover, they may be relevant to medical applications of lectins.     

Characterization of the major histocompatibility complex class II genes in miiuy croaker

Major histocompatibility complex (MHC) has a central role in the adaptive immune system by presenting foreign peptide to the T-cell receptor. In order to study the molecular function and genomic characteristic of class II genes in teleost, the full lengths of MHC class IIA and IIB cDNA and genomic sequence were cloned from miiuy croaker (Miichthys miiuy). As in other teleost, four exons and three introns were identified in miiuy croaker class IIA gene; but the difference is that six exons and five introns were identified in the miiuy croaker class IIB gene. The deduced amino acid sequence of class IIA and class IIB had 26.3-85.7% and 11.0-88.8% identity with those of mammal and teleost, respectively. Real-time quantitative RT-PCR demonstrated that the MHC class IIA and IIB were ubiquitously expressed in ten normal tissues; expression levels of MHC genes were found first upregulated and then downregulated, and finally by a recovery to normal level throughout the pathogenic bacteria infection process. In addition, we report on the underlying mechanism that maintains sequences diversity among many fish species. A series of site-model tests implemented in the CODEML program revealed that positive Darwinian selection is likely the cause of the molecular evolution in the fish MHC class II genes.     

盐度胁迫对幼体大海马基因转录表达的影响

为探究大海马(Hippocampus kuda Bleeker)幼体在高盐、低盐胁迫条件下的基因表达水平的变化规律,对实验条件下的大海马幼体的肝脏样品进行了转录组测序。对照组(CK, 25‰)、高盐(HS-test, 31‰)和低盐(LS-test, 17‰)胁迫组共获得71794个单基因簇(Unigenes), N50为1780 bp,平均长度为820.71 bp。高盐胁迫组与对照组比较,共获得2740个差异表达基因(DEGs),其中495个DEGs上调, 2245个DEGs下调;与对照组相比,低盐胁迫组共获得3715个DEGs,其中1854个DEGs上调, 1861个DEGs下调。高/低盐胁迫组DEGs经KEGG数据库富集发现,高/低盐度胁迫均能导致大海马幼体体内氨基酸代谢、免疫代谢、能量和脂肪酸代谢相关基因受到影响。其中,低盐胁迫时能量代谢和氨基酸代谢的相关基因显著上调,高盐胁迫时脂肪酸代谢的相关基因显著下调,而高/低盐胁迫时免疫代谢的相关基因都显著上调。从经过盐度胁迫的大海马幼体的肝脏转录组中分别筛选到免疫相关基因Gst、Hsp70、Hsp90、Sod、Bcl-2、Gadd45...     

活血化瘀方抗糖尿病心肌病的药理调控机制研究

目的:从基因表达谱的角度探究活血化瘀方抗糖尿病心肌病的药理调控机制。方法:采用链脲霉素建立糖尿病心肌病大鼠模型后,随机分组并对其中一组给予活血化瘀方灌胃,检测生理数据,并取样进行基因表达水平检测。针对芯片数据,初处理后依次进行:基因表达差异分析、基因表达与性状相关性分析、基因连接性差异分析和加权的基因共表达网络分析,最后整合上述多种分析结果,寻找关键基因。结果:成功建立糖尿病心肌病大鼠模型,并得到一系列从不同角度分析的结果:一千多个差异表达基因,五千多个差异连接性基因,鉴定出三个共表达网络中的关键模块以及三个关键基因。结论:活血化瘀方通过调节与糖尿病心肌病或者糖尿病密切相关的生物分子及其活动,同时综合作用于多种基础的细胞生理活动,从而影响糖尿病心肌病的发展。     

EST analysis of the immune-relevant genes in Chinese amphioxus challenged with lipopolysaccharide

It is generally accepted that the adaptive immune system is only present in vertebrates but not in invertebrates. Amphioxus is the most basal chordate and hence is an important reference to the evolution of the adaptive immune system. Here, a cDNA library of lipopolysaccharide-challenged amphioxus was constructed in order to identify immune genes. A total of 3024 expressed sequence tags (ESTs) were examined and 63 out of 398 annotated genes (16.3%) appeared related to immunity. Most of them encode cell adhesion molecules or signal proteins that are involved in immune responses. Although the key molecules such as TCR, MHC, Ig or VLR involved in the adaptive immune system were not identified in our database, we demonstrated the presence of histocompatibility-relevant genes and lymphocyte immune signaling-relevant genes. These findings support the statement that amphioxus presents some components that may be recruited by adaptive immune processes.     

Expression of putative immune response genes during early ontogeny in the coral Acropora millepora

Background

Corals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals.

Methodology/Principal Findings

Patterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes) were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR). Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A.millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria) during winnowing processes as symbioses are fine-tuned.

Conclusions/Significance

Our data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of these genes in alloimmune-challenged corals are needed to further clarify emerging evidence of a complex innate immunity system in corals.     

Expression analysis of lipopolysaccharide-and/or concanavalin A/phorbol myristate acetate-stimulated black rockfish peripheral blood leukocytes using a cDNA microarray to identify up-regulated genes

The responses of peripheral blood leucocytes (PBLs) from the black rockfish Sebastes schlegelii stimulated with lipopolysaccharides (LPS) and/or concanavalin A/phorbol myristate acetate (Con A/PMA) were investigated using a cDNA microarray consisting of 5,088 clones. This analysis showed that 254 unique genes were more than twofold upregulated, and they were selected for sequencing. Among the mitogens, 84 genes were more than twofold upregulated in LPS-stimulated PBLs, and 112 genes were induced in Con A/PMA-stimulated PBLs. Moreover, 58 other genes were more than twofold upregulated in PBLs stimulated with both LPS and Con A/PMA. Other genes were not significantly induced. Overall, these results suggest that certain genes in black rockfish have important roles in physiological and pathological processes. Furthermore, the microarray analysis suggested a promising tool for immune mechanisms in teleost fish.     

文昌鱼抗金葡菌感染差减文库的构建及免疫相关基因分析

适应性免疫的起源一直是免疫学研究的关键问题.文昌鱼被认为是最接近于脊椎动物的祖先 自从被发现以来一直是研究脊椎动物起源与进化机制的经典模式动物.为了在文昌鱼中寻找适应性免疫系统的分子证据,采用金黄色葡萄球菌感染文昌鱼以调查免疫的起源.应用抑制性差减杂交(SSH)技术,通过对差减文库克隆序列的测定,共获得588个表达序列标签(EST).对这些EST进行生物信息学分析和进一步功能分类,发现了一些免疫上调基因,如免疫调控基因、凋亡相关基因、细胞黏附相关基因、转录相关基因、信号传导相关基因等,以及一些非免疫相关基因;这些基因在文昌鱼中绝大多数为首次报道.金黄色葡萄球菌差减文库的成功构建,为调查文昌鱼抗细菌感染的分子事件提供了重要线索,对于这些新发现基因的进一步研究将有助于深入了解免疫系统起源与进化的机制.     

Animal lectins: a historical introduction and overview

Some proteins we now regard as animal lectins were discovered before plant lectins, though many were not recognised as carbohydrate-binding proteins for many years after first being reported. As recently as 1988, most animal lectins were thought to belong to one of two primary structural families, the C-type and S-type (presently known as galectins) lectins. However, it is now clear that animal lectin activity is found in association with an astonishing diversity of primary structures. At least 12 structural families are known to exist, while many other lectins have structures apparently unique amongst carbohydrate-binding proteins, although some of those "orphans" belong to recognised protein families that are otherwise not associated with sugar recognition. Furthermore, many animal lectins also bind structures other than carbohydrates via protein-protein, protein-lipid or protein-nucleic acid interactions. While animal lectins undoubtedly fulfil a variety of functions, many could be considered in general terms to be recognition molecules within the immune system. More specifically, lectins have been implicated in direct first-line defence against pathogens, cell trafficking, immune regulation and prevention of autoimmunity.     

病毒感染草鱼胸腺的EST分析和免疫相关基因的鉴定

以感染草鱼(Ctenopharyngodon idellus)出血病病毒(GCHV)的草鱼胸腺为材料,构建了草鱼胸腺的SMARTcDNA文库.筛选文库获得到1933条有效EST序列.BLASTX分析显示,583条序列在公共数据库中能找到同源基因(E-value≤1.00E 10-3,Identities≥30%),另外1350条序列则找不到显著同源性.已知基因按具体功能可划分为6类,大部分与细胞内的各种生理过程、细胞结构以及免疫防御相关.研究结果从分子水平上表明鱼类的胸腺在机体感染病毒的免疫反应中发挥重要作用,同时也表明胸腺组织在病毒感染后可能表达很多目前还不清楚功能的新基因.     

Maturation of the mouse oocyte-cumulus cell complex: stimulation by lectins.

We have used carbohydrate-binding proteins, or lectins, as tools to investigate the physiological phenomena associated with the preovulatory maturation of the oocyte-cumulus cell complex. Certain lectins are mitogens, and since other mitogenic agents such as growth factors are known to stimulate meiotic maturation and cumulus expansion, we tested the ability of lectins to provoke these physiological responses. Cumulus cell-enclosed oocytes (CEO) from primed mice were maintained in meiotic arrest in vitro with dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) and treated with one of eleven different lectins. With the exception of pokeweed mitogen (PWM), all of the mitogenic lectins tested were able to induce germinal vesicle breakdown (GVB) in meiotically arrested oocytes, and this action required the presence of the somatic cumulus cells; in fact, either there was no effect or maturation was suppressed when cumulus cell-free oocytes (denuded oocytes; DO) were treated with lectins. None of the nonmitogenic lectins stimulated meiotic maturation in either CEO or DO. The mitogenic lectin concanavalin A (Con A) also induced maturation in CEO when meiotic arrest was maintained with hypoxanthine, guanosine, or 3-isobutyl-1-methylxanthine. The kinetics of spontaneous oocyte maturation in inhibitor-free medium were not altered by Con A. Only the mitogenic lectins that induced meiotic maturation stimulated cumulus expansion, with Con A the most active lectin. The actions of Con A on the maturation of the oocyte-cumulus cell complex were inhibited by methyl-alpha-D-mannopyranoside as predicted by its sugar-binding specificity. These results demonstrate that (1) lectins can stimulate maturation of the mouse oocyte-cumulus cell complex; (2) mitogenicity is associated with the positive activity of the lectins; and (3) cumulus cells mediate the stimulatory action of lectins on oocyte maturation, while inhibition of GVB occurs at the oocyte level. These data support the idea that common signals mediate the mitogenic and maturation-inducing actions of lectins.     

大白菜生物技术研究进展

综述了国内外在大白菜(Brassica rapa ssp.pekinensis)单倍体培养、基因工程和分子标记方面的最新研究成果.重点讨论了大白菜小孢子培养的影响因素、取材要点、培养条件、植株再生和倍性鉴定以及大白菜的转基因载体选择、受体系统建立、遗传转化与转基因植株鉴定等.对小孢子培养及转基因研究的原理、方法和关键技术进行了总结.展望了大白菜生物技术研究的应用前景.     

Cloning and characterization of the lectin cDNA clones from onion,shallot and leek

Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells.cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level.Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5–13 kDa) after post-translational modifications.Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.     

Traditional Chinese medicine Bu-Shen-Jian-Pi-Fang attenuates glycolysis and immune escape in clear cell renal cell carcinoma: results based on network pharmacology

Clear cell renal cell carcinoma (ccRCC) is the most common malignant type of kidney cancer. The present study aims to explore the underlying mechanism and potential targets of the traditional Chinese medicine Bu-Shen-Jian-Pi-Fang (BSJPF) in the treatment of ccRCC based on network pharmacology. After obtaining the complete composition information for BSJPF from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, we analyzed its chemical composition and molecular targets and then established a pharmacological interaction network. Twenty-four significantly differentially expressed genes and nine pathways mainly related to tumor proliferation were identified and screened. Functional enrichment analysis indicated that the potential targets might be significantly involved in glycolysis and the HIF-1 signaling pathway. To further confirm the effect of BSJPF on ccRCC cell proliferation, a BALB/c xenograft mouse model was constructed. Potential targets involved in regulating glycolysis and the tumor immune microenvironment were evaluated using RT-qPCR. VEGF-A expression levels were markedly decreased, and heparin binding-EGF expression was increased in the BSJPF group. BSJPF also inhibited tumor proliferation by enhancing GLUT1- and LDHA-related glycolysis and the expression of the immune checkpoint molecules PD-L1 and CTLA-4, thereby altering the immune-rejection status of the tumor microenvironment. In summary, the present study demonstrated that the mechanism of BSJPF involves multiple targets and signaling pathways related to tumorigenesis and glycolysis metabolism in ccRCC. Our research provides a novel theoretical basis for the treatment of tumors with traditional Chinese medicine and new strategies for immunotherapy in ccRCC patients.