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.     

Functional screening for salinity tolerant genes from <Emphasis Type="Italic">Acanthus ebracteatus</Emphasis> Vahl using <Emphasis Type="Italic">Escherichia coli</Emphasis> as a host

Salinity reduces plant growth and crop production globally. The discovery of genes in salinity tolerant plants will provide the basis for effective genetic engineering strategies, leading to greater stress tolerance in economically important crops. In this study, we have identified and isolated 107 salinity tolerant candidate genes from a mangrove plant, Acanthus ebracteatus Vahl by using bacterial functional assay. Sequence analysis of these putative salinity tolerant cDNA candidates revealed that 65% of them have not been reported to be stress related and may have great potential for the elucidation of unique salinity tolerant mechanisms in mangrove. Among the genes identified were also genes that had previously been linked to stress response including salinity tolerance, verifying the reliability of this method in isolating salinity tolerant genes by using E. coli as a host.     

Generation and analysis of expressed sequence tags from the mangrove plant, Acanthus ebracteatus Vahl

Salinity is a major abiotic stress that greatly affects plant growth and crop production. Sodium ions in saline soil are toxic to plants because of their adverse effects on potassium nutrition, cytosolic enzyme activities, photosynthesis, and metabolism. It is important to identify genes involved in salinity tolerance from mangrove plants that survive under saline conditions. In this study, a total of 864 randomly selected cDNA clones were isolated and sequenced from the primary cDNA library of Acanthus ebracteatus. Among the 521 readable sequences, 138 of them were assembled into 43 contigs, whereas 383 were singletons. Sequence analyses demonstrated that 349 of these expressed sequence tags showed significant homology to functional proteins, of which 18% are particularly interesting as they correspond to genes involved in stress response. Some of these clones, including putative mannitol dehydrogenase, plastidic aldolase, secretory peroxidase, ascorbate peroxidase, and vacuolar H⁺-ATPase, may be related to osmotic homeostasis, ionic homeostasis, and detoxification.     

Molecular Cloning and Characterization of a NAC-like Gene in “Navel” Orange Fruit Response to Postharvest Stresses

A cDNA subtraction library had been constructed to identify differentially expressed genes in peel pitting of citrus fruit. Based on the sequence of a cDNA fragment homologous to NAC gene family, the full-length cDNA of 1,203 nucleotides was cloned from “navel” orange by rapid amplification of cDNA ends. It was designated as CsNAC, encoding a protein of 305 amino acids. The calculated molecular weight of the CsNAC protein was 35.2 kDa, and theoretical isoelectric point was 6.72. Sequence comparison showed that the CsNAC protein had a strikingly conserved region at the N terminus, which is considered as the characteristic of the NAC protein family. CsNAC protein was orthologous to Arabidopsis thaliana ATAF1. Phylogenetic analysis confirmed CsNAC belonged to the ATAF subfamily, which plays an important role in response to stress stimuli. RNA gel blot analysis showed that the expression of CsNAC gene was rapidly and strongly induced by stresses such as wounding and no oxygen. Low temperature (4°C) and exposure to ethylene also increased the expression level of CsNAC gene. However, its expression was suppressed by high temperature (40°C) but not affected by low oxygen (3%). Our results may provide the basis for future research of NAC-like gene’s role in stress-induced citrus peel pitting. Sequence data of CsNAC from this article have been deposited at GenBank under accession number EF596736.     

Cytoplasmic and Mitochondrial Creatine Kinases from the Skeletal Muscle of Sperm Whale (Physeter macrocephalus). Molecular Cloning and Enzyme Characterization

We have amplified two cDNAs, coding for creatine kinases (CKs), from the skeletal muscle of sperm whale Physeter macrocephalus by PCR, and cloned these cDNAs into pMAL plasmid. These are the first CK cDNA and deduced amino acid sequences from cetaceans to be reported. One of the two amino acid sequences is a cytoplasmic, muscle-type isoform (MCK), while the other was identified as a sarcomeric, mitochondrial isoform (sMiCK) that included a mitochondrial targeting peptide. The amino acid sequences of sperm whale MCK and sMiCK showed 94–96% sequence identity with corresponding isoforms of mammalian CKs, and all of the key residues necessary for CK function were conserved. The phylogenetic analyses of vertebrate CKs with three independent methods (neighbor-joining, maximum-likelihood and Bayes) supported the clustering of sperm whale MCK with Bos and Sus MCKs, in agreement with the contemporary view that these groups are closely related. Sperm whale MCK and sMiCK were expressed in Escherichia coli as a fusion protein with maltose-binding protein, and the kinetic constants (K _m, K _d and k _cat) were determined for the forward reaction. Comparison of kinetic constants with those of human and mouse CKs indicated that sperm whale MCK has a comparable affinity for creatine (K _m^Cr = 9.38 mM) to that of human MCK, and the sMiCK has two times higher affinity for creatine than the human enzyme. Both the MCK and sMiCK of sperm whale display a synergistic substrate binding (K _d /K _m = 3.1–7.8) like those of other mammalian CKs.     

水稻NAM基因家族的全基因组鉴定及表达分析

植物特异性转录因子NAM家族从属于NAC转录因子超家族,在植株生长发育、生理代谢以及应对各种胁迫反应中均发挥重要作用。该研究采用生物信息学方法鉴定水稻基因组中的NAM基因,分析其时空表达模式、亚细胞定位以及蛋白相互作用,并采用实时定量qRT PCR方法分析不同外源激素(如SA、ABA和MeJA)以及非生物胁迫(包括干旱、盐和冷)处理下各NAM基因的表达特征,为进一步探索NAM基因在非生物胁迫中的功能和应激机制以及激素调控途径奠定基础。结果显示：(1)从水稻基因组中共鉴定出48个NAM基因,进化分析将其分为5个亚家族;NAM基因在水稻基因组中存在9对片段复制事件。(2)组织表达分析显示,NAM基因在水稻不同组织及发育时期表现特异性表达,特别是叶鞘、茎和节的生长过程中高表达,且大多数是核定位,并存在多种蛋白互作。(3)实时定量qRT PCR表达分析显示,10个NAM基因在不同组织中均特异表达;大部分NAM基因在盐和干旱胁迫下表达上调,而在冷胁迫下表达降低;SA、ABA和MeJA处理均可显著改变各NAM基因的表达水平。研究表明,NAM基因在水稻生长发育、激素应答和非生物胁迫响应中具有重要作用。     

<Emphasis Type="Italic">Physcomitrella patens</Emphasis> is highly tolerant against drought,salt and osmotic stress

In order to determine the degree of tolerance of the moss Physcomitrella patens to different abiotic stress conditions, we examined its tolerance against salt, osmotic and dehydration stress. Compared to other plants like Arabidopsis thaliana, P. patens exhibits a high degree of abiotic stress tolerance, making it a valuable source for the identification of genes effecting the stress adaptation. Plants that had been treated with NaCl tolerated concentrations up to 350 mM. Treatments with sorbitol revealed that plants are able to survive concentrations up to 500 mM. Furthermore, plants that had lost 92% water on a fresh-weight basis were able to recover successfully. For molecular analyses, a P. patens expressed sequence tag (EST) database was searched for cDNA sequences showing homology to stress-associated genes of seed plants and bacteria. 45 novel P. patens genes were identified and subjected to cDNA macroarray analyses to define their expression pattern in response to water deficit. Among the selected cDNAs, we were able to identify a set of genes that is specifically up-regulated upon dehydration. These genes encode proteins exerting their function in maintaining the integrity of the plant cell as well as proteins that are known to be members of signaling networks. The identified genes will serve as molecular markers and potential targets for future functional analyses.     

酿酒葡萄‘赤霞珠’VvbHLH 93基因的克隆及分析北大核心CSCD

bHLH93转录因子参与调节植物的生长发育、应对各种胁迫等多种生理过程,该研究以酿酒葡萄‘赤霞珠’为试验材料,采用RT-PCR方法克隆葡萄VvbHLH 93基因全长,并进行生物信息学分析;用qRT-PCR法分析bHLH93在不同组织和果实不同发育时期的表达量,为进一步探索VvbHLH 93基因的功能及其机制奠定基础。结果表明:(1)成功克隆获得葡萄VvbHLH 93,该基因cDNA全长为1319 bp,开放阅读框长度为939 bp,编码312个氨基酸,相对分子量为35.18 kD,理论等电点为4.68,无信号肽和跨膜域,属于bHLH转录因子家族。(2)葡萄bHLH93蛋白与荷花的亲缘关系较近;VvbHLH93蛋白的C端为酸性结构区,富含丝氨酸、苏氨酸磷酸化位点;VvbHLH 93基因的启动子含有光响应元件和低温、干旱、赤霉素等应答元件。(3)qRT-PCR分析表明,VvbHLH 93在‘赤霞珠’中的表达具有组织特异性,在叶片中基本不表达,在茎中的表达量最高;随着赤霞珠葡萄果实的发育成熟,VvbHLH93相对表达量不断降低,到幼果期(直径>2 mm)后第5周开始相对表达量基本为0,总体呈现降低的变化趋势;与对照相比,在低温胁迫、盐胁迫、高温胁迫及充分灌溉胁迫下VvbHLH 93表达量显著降低。研究推测,VvbHLH 93基因可能是葡萄抗逆胁迫的负转录因子。     

<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>, a model system for functional validation of abiotic stress responsive genes

Stress tolerance is a multigenic character and there are many stress responsive genes, which are stress specific. Although many of these have been cloned, their functional significance remains fragmentary. Hence it is important to identify the relevant stress genes involved in altering the metabolism for adaptation. Overexpression is one of the several approaches and Chlamydomonas is a suitable system to study the functional relevance of stress genes. Stress responses can only be assessed on prior exposure to sublethal induction stress. In this study the acclimation response of Chlamydomonas was assessed for different abiotic stresses using physiological screens like chlorophyll stability, membrane damage, cell viability, accumulation of free radicals, survival and recovery growth. We demonstrate that Chlamydomonas responds to diverse stresses and is a potential system to study the relevance of stress genes. The relevance of choline oxidase A (codA), a key enzyme in glycinebetaine biosynthesis, was examined by developing transformants expressing codA gene from Arthrobacter globiformis. Southern positive transformants showed enhanced accumulation of glycinebetaine. The transformants also showed enhanced growth under salinity, high light coupled with methylviologen-induced oxidative stress, high temperature and cold stress. However the transgenics were not tolerant to PEG-mediated simulated osmotic stress, LiCl, menadione and UV stress. Increased cell survival and decreased chlorophyll degradation in transformants under acclimated conditions further confirmed the relevance of codA in imparting stress tolerance. Our results indicated that the relevance of stress responsive genes can be efficiently validated for diverse abiotic stresses using Chlamydomonas system. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. R. Hema and M. Senthil-Kumar contributed equally.     

羊肚菌分子鉴定及功能基因研究进展

羊肚菌作为珍贵的食药用真菌,具有很高的经济价值.就近年来羊肚菌分子生物学研究的进展从分子鉴定、系统学研究、功能基因等方面进行了分析总结.结果显示,目前利用分子标记已实现羊肚菌属快捷的分子鉴定,较全面揭示了属内种群的遗传多样性和亲缘关系;利用组学技术初步探索了羊肚菌菌核形成及子实体发育机制;此外还克隆表达了与羊肚菌抗逆响...     

橘小实蝇神经肽sulfakinin的分子生物学特性及其表达模式

【目的】分析橘小实蝇sulfakinin的时空表达模式及其对饥饿的响应,初步明确sulfakinin在调节橘小实蝇行为和生理方面的功能。【方法】利用RT-PCR技术克隆橘小实蝇神经肽sulfakinin的c DNA序列,采用实时定量PCR技术分析该基因在橘小实蝇不同发育阶段、成虫不同组织、幼虫不同组织及其在饥饿胁迫下的表达模式,并运用半定量RT-PCR技术进一步验证。【结果】实时定量PCR分析结果表明,sulfakinin在橘小实蝇的幼虫期和成虫期显著性高表达,卵期和蛹期几乎不表达。其中早期成虫表达量最高,约为晚期成虫的2倍,幼虫期表达量位于两者之间。在橘小实蝇幼虫、成虫不同组织中,sulfakinin在中枢神经系统的表达量显著高于其他组织,在触角的表达量仅次于中枢神经系统。此外,橘小实蝇在经饥饿处理24 h的过程中,sulfakinin的表达量均出现下调,其中从饥饿处理结果可知在6 h下降幅度最大,12 h和24 h表达量下降幅度减少。半定量RT-PCR技术进一步检测结果与实时定量PCR技术检测结果一致。【结论】sulfakinin在橘小实蝇中枢神经系统可能调节橘小实蝇成虫对饥饿胁迫的响应。其在触角中的高表达可能与橘小实蝇嗅觉的敏感性相关。本研究为进一步研究橘小实蝇sulfakinin的生理功能、评估其药靶潜力奠定了理论基础。     

Expressed sequence tags from the zhikong scallop (Chlamys farreri): Discovery and annotation of host-defense genes

A high-quality cDNA library was constructed from whole body tissues of the zhikong scallop, Chlamys farreri, challenged by Listonella anguillarum. A total of 5720 clones were sequenced, yielding 5123 expressed sequence tags (ESTs). Among the 3326 unique genes identified, 2289 (69%) genes had no significant (E-value < 1e?5) matches to known sequences in public databases and 194 (6%) matched proteins of unknown functions. The remaining 843 (25%) genes that exhibited homology with genes of known functions, showed broad involvement in metabolic processes (31%), cell structure and motility (20%), gene and protein expression (12%), cell signaling and cell communication (8%), cell division (4%), and notably, 25% of those genes were related to immune function. They included stress response genes, complement-like genes, proteinase and proteinase inhibitors, immune recognition receptors and immune effectors. The EST collection obtained in this study provides a useful resource for gene discovery and especially for the identification of host-defense genes and systems in scallops and other molluscs.     

A catalytic subunit of the sugar beet protein kinase CK2 is induced by salt stress and increases NaCl tolerance in Saccharomyces cerevisiae

Salinity is an important limiting factor in plant growth and development. We have cloned a catalytic subunit of the sugar beet protein kinase CK2 (BvCKA2) by functional expression in yeast of a NaCl-induced cDNA library. BvCKA2 was able to increase the yeast tolerance to NaCl and to functionally complement the cka1 cka2 yeast double mutant upon over-expression. Southern blot analysis indicated that, in sugar beet, the BCKA2 gene is a member of a multigene family. The mRNA levels of BvCKA2 were up-regulated in response to NaCl stress which suggests that protein kinase CK2 may be involved in the plant response to salt stress.     

Molecular cloning, in vitro expression and characterization of a plant squalene synthetase cDNA

Squalene synthetase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) catalyzes the first committed step for sterol biosynthesis and is thought to play an important role in the regulation of isoprenoid biosynthesis in eukaryotes. Using degenerate oligonucleotides based on a conserved region found in yeast and human squalene synthetase genes, a cDNA was cloned from the plant Nicotiana benthamiana. The cloned cDNA contained an open reading frame of 1234 bp encoding a polypeptide of 411 amino acids (M _r 47002). Northern blot analysis of, poly(A)⁺ mRNA from N. benthamiana and N. tabacum cv. MD609 revealed a single band of ca. 1.6 kb in both Nicotiana species. The identity and functionality of the cloned plant squalene synthetase cDNA was further confirmed by expression of the cDNA in Escherichia coli and in a squalene synthetase-deficient erg9 mutant of Saccharomyces cerevisiae. Antibodies raised against a truncated form of the protein recognized an endogenous plant protein of appropriate size as well as the full-length bacterially expressed protein as detected by western analysis. Comparison of the deduced primary amino acid sequences of plant, yeast, rat and human squalene synthetase revealed regions of conservation that may indicate similar functions within each polypeptide.