997 resultados para Pea lectin gene
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Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean. Knowledge on lectin at molecular level would help us to understand its regulation mechanism in crustacean immune system. A novel C-type lectin gene (Fclectin) was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1482 bp with an 861 bp open reading frame, encoding 287 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids. It also contains two carbohydrate recognition domains/C-type lectin-like domains (CRD1 and CRD2), which share 78% identity with each other. CRD1 and CRD2 showed 34% and 30% identity with that of mannose-binding lectin from Japanese lamprey (Lethenteron japonicum), respectively. Both CRD1 and CRD2 of Fclectin have I I amino acids residues, which are relatively invariant in animals' C-type lectin CRDs. Five residues at Ca2+ binding site I are conserved in Fclectin. The potential Ca2+/carbohydrate-binding (site 2) motif QPD, E, NP (Gln-Pro-Asp, Glu, Asn-Pro) presented in the two CRDs of Fclectin may support its ability to bind galactose-type sugars. It could be deduced that Fclectin is a member of C-type lectin superfamily. Transcripts of Fclectin were found only in hemocytes by Northern blotting and RNA in situ hybridization. The variation of mRNA transcription level in hemocytes during artificial infection with bacteria and white spot syndrome virus (WSSV) was quantitated by capillary electrophoresis after RT-PCR. An exploration of mRNA expression variation after LPS stimulation was carried out in primarily cultured hemocytes in vitro. Expression profiles of Fclectin gene were greatly modified after bacteria, LPS or WSSV challenge. The above-stated data can provide us clues to understand the probable role of C-type lectin in innate immunity of shrimp and would be helpful to shrimp disease control. (c) 2006 Elsevier Ltd. All rights reserved.
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本文通过根农杆菌(Agrobacterium tumfaciens)介导法分别将Signal和KDEL修饰的豇豆胰蛋白酶抑制剂(Cowpea trypsin inhibitor, CpTI)基因、豌豆外源凝集素(Pea lectin, P-Lec)和大豆Kunitz型胰蛋白酶抑制剂(Soybean Kunitz typsin inhibitor, SKTI)双价抗虫基因、雪花莲外源凝集素(Galanthus nivals agglutinin, GNA)基因以及高效复合启动子OM控制的苏云金杆菌(Bacillus thuringiensis, B.t.)杀虫毒蛋白基因导入了陆地棉(Gossypium hirsutum L.)栽培品种新陆早1号、新陆中2号、晋棉7号、冀合321、辽9和晋棉12号,并获得了大批转基因再生植株。 实验中对影响棉花转化和再生的一些条件进行了研究,从根农杆菌培养、棉花无菌苗的制备、转化操作和共培养等方面对转化条件进行了探讨;从激素配化、植物表达载体、外植体类型、基因型等方面对抗性愈伤组织的诱导进行了摸索;从激素、从碳源、培养容器、pH值、抗褐化剂及固化剂的选择等方面对影响植株再生的条件进行了优化。 本文开创性地采用嫁接代替移栽,从而极大地提高了转化植株定植成活率,缩短了缓苗时间并增加了转化植株当代的繁殖系数。 在建立了一套较为高效的陆地棉转化及再生系统基础上,本文还进行了其它转化方式和转化体系的初步探讨。利用棉花幼嫩种子无菌苗下胚轴作为外植体,通过改变愈伤组织诱导培养基配方面提高胚性愈伤组织的诱导频率,进而得到更多的体细胞胚状和再生植株,缩短再生周期;尝试用胚性愈伤组织作为外植体的根农杆菌介导法转化,确定了一些与转化有关的条件;建立了一套棉花茎尖培养程序,为运用基因枪法轰击棉花茎尖分生组织或用根农杆菌直接转化茎尖分生组织,以克服根农杆菌转化棉花时体胚发生的基因型局限开辟了一条新途径。 本文还建立了一种快速鉴定转化植株后代的方法。这一简便方法还有助于进行转基因棉纯合系的筛选以及外源基因的遗传稳定性研究。 转基因植株经Npt-II ELISA、PCR、PCR Southern 检测证明外源抗虫基因CpTI、SKTI、P-lec、GNA以及B.t.基因已存在于转化植株基因组内。修饰的CpTI转基因植株抗棉铃虫(Heliothis armigera Hubner)试验结果表明,其杀虫效果显著优于前期未修饰的CpTI转化植株。P-lec和SKTI双价转基因植株抗棉铃虫试验结果表明,转基因植株对棉铃虫幼虫具有较强的杀虫活力。 目前,已获得转以上抗虫基因棉花T1代植株。为今后进一步将植物基因工程技术应用于棉花遗传改良打下了基础。
Resumo:
C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, a novel multidomain C-type lectin gene from scallop Chlamys farreri (designated as Cflec-4) was cloned by RACE approach based on EST analysis. The full-length cDNA of Cflec-4 was of 2086 bp. The open reading frame was of 1830 bp and encoded a polypeptide of 609 amino acids, including a signal sequence and four dissimilar carbohydrate-recognition domains (CRDs). The deduced amino acid sequence of CflecA shared high similarities to other C-type lectin family members. The phylogenetic analysis revealed the divergence between the three N-terminal CRDs and the C-terminal one, suggesting that the four CRDs in Cflec-4 originated by repeated duplication of different primordial CRD. The potential tertiary structure of each CRD in Cflec-4 was typical double-loop structure with Ca2+-binding site 2 in the long loop region and two conserved disulfide bridges at the bases of the loops. The tissue distribution of Cflec-4 mRNA was examined by fluorescent quantitative real-time PCR. In the healthy scallops, the Cflec-4 transcripts could be only detected in gonad and hepatopancreas, whereas in the Listonella anguillarum challenged scallops, it could be also detected in hemocytes. These results collectively suggested that CflecA was involved in the immune defense of scallop against pathogen infection and provided new insight into the evolution of C-type lectin superfamily. (C) 2009 Elsevier Ltd. All rights reserved.
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植物血红蛋白在共生固氮根瘤中主要是对固氮酶进行嫌氧保护,确保固氮酶活性,在植物根中协助氧的运输或作为感觉氧压的信号分子.豆科植物凝集素功能之一是对相应专一的根瘤菌有识别作用,并有利于根瘤菌的聚集和侵染。本论文将非豆科结瘤植物Parasponia andersonii血红蛋白基因及豌豆凝集索基因转入水稻,目的是此二基因表达后,豌豆凝集素可聚集、识别豌豆根瘤菌,并有助于它们的侵染,血红蛋白则可对根瘤菌固氮酶进行嫌氧保护,保障其发挥固氮作用,从而为实现水稻结瘤和固氮打下初步基础。 本论文将带有Parasponia血红蛋白基因的pL305质粒,带有潮霉素磷酸转移酶基因及Parasponia血红蛋白基因的农杆菌双元载体质粒pLX412-Hb用花粉管通道法转化水稻l用pLX412-Hb和带有Bar基因、豌豆凝集素基因、Parasponia血红蛋白基因的质粒pLHB用基因枪法转化水稻幼胚及愈伤组织,带有pLX412-Hb的Agrobactmum tumefaciens LBA4404转化烟草,得到如下结果: 1.用pL305质粒花粉管通道法转化水稻,运作1512朵花,得到707粒种子.将230粒种子萌发,发芽220粒;白化苗4株,成苗206株;计发芽率96%,白化苗率1.7%,成苗率90%。取120株提取DNA以Parasponia血红蛋白基因为探针进行点杂交,有6株为阳性结果,阳性率5%.再取4株DNA点杂交阳性植株DNA,以Parasponia血红蛋白基因为探针进行Southem杂交,有3株有阳性结果,阳性率为75%,照此推算转化植株约占总植株的4%.目前,这些转基因植株已开花结实。 2.用pLX412-Hb质粒花粉管通道法转化水稻,运作743朵花,得到340粒种子。将120粒种子萌发,发芽117粒,白化苗3株,成苗111株;计发芽率97. 5%,白化苗率2. 5%,成苗率92. 5%.取30株提取DNA以Parasponia血红蛋白基因为探针进行点杂交,有4株为阳性结果,阳性率13%。再取4株DNA点杂交呈阳性的植株DNA,以Parasponia血红蛋白基因为探针进行Southern杂交,有2株有阳性结果,阳性率为50%,照此推算转化植株约占总植株的7%。目前部分转基因植株巳开花结实. 3.用pLX412 - Hb质粒基因枪法转化水稻幼胚及愈伤组织,抗性筛选得到15株再生植株,以Parasponia血红蛋白基因为探针进行点杂交,有14株为阳性结果,阳性率93%.取7株DNA点杂交呈阳性的植株DNA,以Parasponia血红蛋白基因为探针进行Southern杂交,都为阳性结果,阳性率为100%,照此推算转化植株约占总植株的93%.目前,部分转基因植株已开花结实. 4.用pLX412 -Hb质粒通过Agrobacterrum tumeFaciens LBA4404转化烟草,抗性筛选得到的再生植株以Parasponia血红蛋白基因为探针进行Southern杂交,结果为阳性. 5.用pLHB基因枪法转化的愈伤组织,抗性筛选得到已分化出绿芽点的愈伤组织,以Parasponia血红蛋白基因为探针或以豌豆凝集素基因为探针进行Southern杂交,杂交结果均为阳性. 6.最近国外巳发现大麦有血红蛋白基因.本论文以大麦血红蛋白cDNA为探针对未转基因的水稻进行Southern杂交,结果有阳性带。说明水稻有与大麦血红蛋白基因高度同源序列。以Parasponia血红蛋白基因为探针时杂交结果为阴性,说明水稻血红蛋白基因与Parasponiaa血红蛋白基因核苷酸序列相差较大. 7.提取Southern杂交证明有Parasponia血红蛋白基因整合的水稻根及叶总RNA,以Parasponia血红蛋白基因cDNA为探针进行RNA点杂交,结果根总RNA为阳性,叶总RNA为阴性。初步证明Parasponia血红蛋白基因在水稻根中表达。 8.提取未转基因的水稻根及叶RNA,以大麦血红蛋白基因cDNA为探针进行RNA点杂交,结果:根总RNA为阳性结果,叶总RNA为阴性结果.初步证明水稻血红蛋白基因在水稻根中表达。 总之,本论文证明已将Parasponia血红蛋白基因整合进水稻植株染色体,将豌豆凝集素基因、Parasponia血红蛋白基因整合进水稻愈伤组织染色体,初步证明水稻有血红蛋白基因,内外源血红蛋白基因都有组织特异性表达,从而为本研究的战略设想奠定初步基础。
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Lhcb2基因是LHCII基因家族中的一个重要成员。目前,对于其特性、结构和功能还不清楚。本文对豌豆Lhcb2基因的克隆、表达、功能及其在大肠杆菌中的表达产物与色素在体外的重组进行了比较系统的探讨,主要的结果如下: 1.采用RT-PCR技术,从豌豆幼叶中克隆了一个约800 bp的Lhcb2 cDNA。以特异探针进行Southern杂交的结果初步证明,Lhcb2基因以单拷贝形式存在于豌豆基因组中,这在文献中尚未见报道。 2.不同光照时间和温度对豌豆幼苗进行处理的RT-PCR,Northem blotting分析表明,Lhcb2基因转录水平上的表达受光照的控制,且明显地表现出对光照时间的依赖性。用400 μmolm-2.s-1的白光照射0~1.5小时Lhcb2基因未见表达,而在光照2小时以上则大量表达,推测该基因的表达可能要求某种(或某些)需光中间物的合成和积累。温度对Lhcb2基因的表达亦有显著的影响,相同的光照处理,4 ℃下Lhcb2基因的表达量比25℃下的表达量低一倍左右。 3.将豌豆Lhcb2基因反向插入植物表达载体pBIl21中,构建CaMV 35S启动子控制下反义Lhcb2基因的植物表达载体pBIantiLhcb2,通过农杆菌LBA4404介导,在Kan浓度为100 mg/L的筛选培养基上,获得120个抗Kan阳性植株。PCR检测表明至少有80个抗性植株为PCR阳性反应。Southern blotting分析表明,外源反义Lhcb2基因已整合到烟草基因组中。转基因植株在表型上主要表现为三大类型:叶色类似于未转基因的绿色植株、叶色发黄的植株、叶色发白甚至枯死的植株。这几类转基因植株光谱特性的分析表明,Lhcb2基因不仅影响光能的捕获,而且还可能参与激发能分配的调节作用。 4.将豌豆的Lhcb2基因亚克隆至原核表达载体pET-3d上,通过定点突变使其在大肠杆菌BL2l(DE3)中得到高效表达,其表达量约占大肠杆菌总蛋白的40%,并经纯化后获得了电泳纯的Lhcb2蛋白。应用改进的液氮/室温冻融重组方法将纯化的蛋白与色素进行体外重组,建立了高效的重组系统。实验结果表明重组后所获得的LHCB2单体与用生化方法从菠菜类囊体膜中分离纯化的天然LHC II单体相比较,其在部分变性胶的电泳行为,低温荧光发射光谱和激发光谱,以及室温吸收光谱和CD光谱的特征等方面都非常相似,说明大量表达的Lhcb2蛋白与色素已成功地重组,并具有与天然的LHCII单体相类似的组成和结构,这在国际上尚属首次。在此基础上又构建了N端和C端氨基酸残基缺失的突变体,并对这些缺失的氨基酸残基在LHCB2中的可能作用进行了初步的研究。结果表明,N端的前12个氨基酸残基、C端的前10个氨基酸残基和第11位的色氨基酸残基对LHCB2单体的形成不是必需的。 此外,从菠菜中纯化了LHCII,并对其多肽和色素组成及其光谱特性进行了比较系统的研究。同时对用不同浓度OGP和Mg2+处理所获得的不同聚集程度的LHCII的光谱特性进行了研究,并对Mg2+在其中的可能作用进行了初步的探讨。
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Invasive infection caused by Neisseria meningitidis is a worldwide public health problem. Previous reports have indicated that carriage of common ‘defective’ structural polymorphisms of the host mannose-binding lectin gene (MBL2) greatly increases an individual’s risk of developing the disease. We report the largest case–control study so far to investigate the effect of these polymorphisms in meningococcal disease (296 PCR-positive cases and 5196 population controls, all of European ancestry) and demonstrate that no change in risk is associated with the polymorphisms overall or in any age-defined subgroup. This finding contrasts with two smaller studies that reported an increase in risk. A systematic review of all studies of MBL2 polymorphisms in people of European ancestry published since 1999, including 24 693 individuals, revealed a population frequency of the combined ‘defective’MBL2 allele of 0.230 (95% confidence limits: 0.226–0.234). The past reported associations of increased risk of meningococcal disease were because of low ‘defective’ allele frequencies in their study control populations (0.13 and 0.04) that indicate systematic problems with the studies. The data from our study and all other available evidence indicate that MBL2 structural polymorphisms do not predispose children or adults to invasive meningococcal disease.
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AbstractInvasive infection caused by Neisseria meningitidis is a worldwide public health problem. Previous reports have indicated that carriage of common 'defective' structural polymorphisms of the host mannose-binding lectin gene (MBL2) greatly increases an individual's risk of developing the disease. We report the largest case-control study so far to investigate the effect of these polymorphisms in meningococcal disease (296 PCR-positive cases and 5196 population controls, all of European ancestry) and demonstrate that no change in risk is associated with the polymorphisms overall or in any age-defined subgroup. This finding contrasts with two smaller studies that reported an increase in risk. A systematic review of all studies of MBL2 polymorphisms in people of European ancestry published since 1999, including 24 693 individuals, revealed a population frequency of the combined 'defective'MBL2 allele of 0.230 (95% confidence limits: 0.226-0.234). The past reported associations of increased risk of meningococcal disease were because of low 'defective' allele frequencies in their study control populations (0.13 and 0.04) that indicate systematic problems with the studies. The data from our study and all other available evidence indicate that MBL2 structural polymorphisms do not predispose children or adults to invasive meningococcal disease.
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To provide tools for functional molecular genetics of the protozoan parasite Entamoeba histolytica, we investigated the use of the prokaryotic neomycin phosphotransferase (NEO) gene as a selectable marker for the transfection of the parasite. An Escherichia coli-derived plasmid vector was constructed (pA5'A3'NEO) containing the NEO coding region flanked by untranslated 5' and 3' sequences of an Ent. histolytica actin gene. Preceding experiments had revealed that amoebae are highly sensitive to the neomycin analogue G418 and do not survive in the presence of as little as 2 micrograms/ml. Transfection of circular pA5'A3'NEO via electroporation resulted in Ent. histolytica trophozoites resistant to G418 up to 100 micrograms/ml. DNA and RNA analyses of resistant cells indicated that (i) the transfected DNA was not integrated into the amoeba genome but was segregated episomally, (ii) in the amoebae, the plasmid replicated autonomously, (iii) the copy number of the plasmid and the expression of NEO-specific RNA were proportional to the amount of G418 used for selection, and (iv) under continuous selection, the plasmid was propagated over an observation period of 6 months. Moreover, the plasmid could be recloned into E. coli and was found to be unrearranged. To investigate the use of pA5'A3'NEO to coexpress other genes in Ent. histolytica, a second marker, the prokaryotic chloramphenicol acetyltransferase (CAT) gene under control of an Ent. histolytica lectin gene promoter was introduced into the plasmid. Transfection of the amoebae with this construct also conferred G418 resistance and, in addition, allowed continuous expression of CAT activity in quantities corresponding to the amount of G418 used for selection. When selection was discontinued, transfected plasmids were lost as indicated by an exponential decline of CAT activity in trophozoite extracts.
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Ecological processes are central to the formation of new species when barriers to gene flow (reproductive isolation) evolve between populations as a result of ecologically-based divergent selection. Although laboratory and field studies provide evidence that 'ecological speciation' can occur, our understanding of the details of the process is incomplete. Here we review ecological speciation by considering its constituent components: an ecological source of divergent selection, a form of reproductive isolation, and a genetic mechanism linking the two. Sources of divergent selection include differences in environment or niche, certain forms of sexual selection, and the ecological interaction of populations. We explore the evidence for the contribution of each to ecological speciation. Forms of reproductive isolation are diverse and we discuss the likelihood that each may be involved in ecological speciation. Divergent selection on genes affecting ecological traits can be transmitted directly (via pleiotropy) or indirectly (via linkage disequilibrium) to genes causing reproductive isolation and we explore the consequences of both. Along with these components, we also discuss the geography and the genetic basis of ecological speciation. Throughout, we provide examples from nature, critically evaluate their quality, and highlight areas where more work is required.
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A cDNA encoding the chloroplast/mitochondrial form of glutathione reductase (GR:EC 1,6,4,2) from pea (Pisum sativum L.) was used to map a single GR locus, named GORI. In two domesticated genotypes of pea (cv, Birte and JI 399) it is likely that the GORI locus contains a single gene. However, in a semi-domesticated land race of pea sequences were detected but closely related sets of GR gene sequences were in JI 281 represent either a second intact gene or a partial or pseudogene copy. A GR gene was cloned from ev. Birte, sequenced and its structure analysed. No features of the transcription or structure of the gene suggested a mechanism for generating any more than one form of . From these data plus previously published biochemical evidence was suggested a second, distinct gene encoding for the cytosolic form of GR should be present in peas. The GORI-encoded GR mRNA can be detected in all main organs of the plant and no alternative spliced species was present which could perhaps account for the generation of multiple isoforms of GR. The mismatch between the number of charge-separable isoforms in pea and the proposed number suggests that different GR isoforms arise by some form of post-transnational modification.
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A leishmaniose visceral (LV) ou calazar é uma doença endêmica, crônica, grave e de alta letalidade se não tratada. Os estudos apontam a proteína Lectina Ligante de Manose (MBL), codificada pelo gene MBL2, como uma peça-chave na imunidade inata, dada a sua função no reconhecimento microbiano, na eliminação, inflamação e morte celular. Neste trabalho realizamos um estudo do tipo caso-controle que teve como objetivo investigar a associação entre variantes no gene MBL2 e a suscetibilidade à LV em indivíduos residentes em áreas endêmicas da Ilha de São Luís-MA. A amostra foi constituída por 322 indivíduos, sendo 161 casos com LV, não aparentados, de ambos os sexos, residentes em áreas endêmicas da doença na Ilha de São Luís e 161 controles saudáveis, não infectados e não aparentados da mesma região. A identificação dos casos de LV se deu por meio do contato constante com os principais hospitais e ambulatórios de referência para a doença na cidade. Também foram feitas buscas de pacientes com LV em ambiente domiciliar, a partir de registros da FUNASA-MA. A análise molecular consistiu na genotipagem de 6 variantes localizadas na região promotora [posições -550 (C>G), -221(G>C), +4(C>T)] e codificadora [códons 52 (C>T), 54 (G>A) e 57 (G>A)] do gene MBL2, através da reação em cadeia da polimerase e sequenciamento automático. A dosagem da proteína MBL no soro foi realizada pelo teste de ELISA. Verificamos que os fenótipos MBL dependem do conjunto de alelos presentes no gene MBL2, sendo nítido o efeito que as variantes defectivas causam nos níveis da proteína. Não encontramos diferença significativa entre casos e controles em relação à distribuição dos genótipos MBL2 e dos níveis séricos de MBL. As frequências alélicas das variantes exônicas na amostra total mostram que o alelo A é o mais comum (74,8%) e que os alelos defectivos (B, C e D) se encontram principalmente em heterozigose (36,6%), o que reforça a ideia de que alelos MBL2 defectivos são mantidos na população por conferirem vantagem seletiva aos heterozigotos. Em relação aos 3 principais polimorfismos existentes na região promotora, verificamos ser a variante -221G (Y) a mais frequente (88%) seguida de +4C (P) (73%) e de -550C (L) (67%). Identificamos oito haplótipos em MBL2 num total de 644 cromossomos avaliados, em 30 combinações diferentes, sendo HYPA e LYQA os mais frequentes e HYPD e HYPB os mais raros. Todos os portadores de combinações de haplótipos homozigotos para alelos defectivos apresentaram níveis séricos de MBL indetectáveis. Os genótipos LYQA/LYQA e HYPA/HYPA apresentaram as maiores concentrações médias de MBL no soro. A combinação entre SNPs no éxon 1 e na região promotora do gene MBL2 resulta em grande variação nas concentrações de MBL em indivíduos saudáveis. Consideramos que o conjunto de dados gerados é uma contribuição valiosa que poderá ser expandida para outros cenários.
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Gaining insight into the mechanisms of chemoreception in aphids is of primary importance for both integrative studies on the evolution of host plant specialization and applied research in pest control management because aphids rely on their sense of smell
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A lectina ligante de manose (MBL) é uma proteína considerada de fase aguda com importante papel na primeira linha de defesa do sistema imune inato, cujos níveis séricos são determinados geneticamente. A MBL ativa a via da lectina do complemento, além de mediar a opsonização e fagocitose de microrganismos. Vários estudos associam os níveis séricos de MBL à suscetibilidade ou resistência a agentes infecciosos entre eles o Mycobacterium tuberculosis, agente causador da tuberculose humana. Neste estudo, com o objetivo de avaliar a ocorrência de uma possível associação entre os polimorfismos e a tuberculose, avaliamos as freqüências das mutações no éxon 1 do gene MBL em um grupo de 167 pacientes com tuberculose, subdivididos em 3 grupos: pacientes com tuberculose pulmonar, pacientes com tuberculose extrapulmonar, pacientes com tuberculose multirresistente a drogas, e grupo controle com 159 profissionais da saúde, negativos para tuberculose. A identificação dos alelos MBL *A, *B, *C e *D foi realizada por meio da reação em cadeia da polimerase, utilizando seqüências de iniciadores específicos e posterior digestão enzimática. As análises das freqüências alélicas e genotípicas do éxon 1 não mostraram qualquer diferença significativa entre pacientes com tuberculose e grupo controle (p>0,05). Não foram observadas associações significativas entre os grupos de tuberculose pulmonar, extrapulmonar e tuberculose multirresistente a drogas, quando relacionados entre si e ao grupo controle. Os dados obtidos em nosso estudo não demonstraram evidencias de qualquer influência das variações do éxon 1 do gene MBL na tuberculose ativa, sugerindo que os polimorfismos nessa região do gene não tem nenhuma influencia na susceptibilidade à tuberculose.