应用荧光光谱法研究植物LEA3蛋白11-氨基酸基序的保护功能

胚胎晚期富集蛋白(late embryogenesis abundant,LEA)是增强生物抵抗干旱、低温和盐渍等多种胁迫的重要功能蛋白,但其保护机理仍不清楚。本文利用紫外光谱法证实,含多拷贝11-氨基酸基序的多肽(如PM2D和PM2E)可较好的保护经冻融的乳酸脱氢酶(LDH)活性。进一步通过荧光光谱法证实,含多拷贝11-氨基酸基序的多肽可通过多位点协同结合模式,稳定LDH酶的结构。而含低拷贝11-氨基酸基序的多肽(如PM2F和PM2G)与LDH酶只有一种结合位点,二者的结合强度较弱,因而不能表现出对LDH酶活性的保护作用。此外,含多拷贝11-氨基酸的多肽与海藻糖在保护LDH酶活性上存在协同作用,且二者有着不同的保护机理。

植物抗逆蛋白(LEA3)22-氨基酸耐盐结构域在酵母细胞中的鉴定

大豆PM2蛋白属LEA3(late embryogenesis abundant3)蛋白。本文构建了编码PM2全长及含22-氨基酸结构域多肽(PM2、PM2A、PM2B和PM2C)的酵母表达载体。转化酵母得到四种重组菌。SDS-PAGE电泳和ESI-MS/MS或MALDI-TOF/TOF质谱结果表明,重组菌可表达目标多肽。测定对照菌及重组菌在无胁迫、高盐(1.5mol.L-1NaCl)和高渗透(2mol.L-1山梨糖)下的生长曲线。结果表明,在高盐胁迫下,四种重组菌胁迫后的恢复明显好于对照菌。多肽对高盐耐受能力的大小为:PM2C>PM2B≈PM2A≈PM2。证明大豆PM2蛋白的表达可提高酵母耐盐性,且22-氨基酸基序为PM2蛋白的耐盐结构域。结合前文在大肠杆菌中的结果,为"LEA蛋白可以类似机制参与原核和真核生物耐盐保护作用"的假说提供实验支持。然而,在高山梨糖胁迫下,对照菌和酵母重组菌的生长情况无明显差异。

动物多肽与钠通道选择性互作的微基序空间等电点

电压门控钠通道(Voltage-gated sodium channels,Nav,钠通道)是产生和传播神经冲动的重要离子通道,是动物多肽类毒素靶通道之一。动物多肽结合不同的钠通道受体位点,改变Nav的构象和通道动力学特性。本文聚焦钠通道与动物多肽的功能性结合,以经典动物多肽AaH2、Protoxin-II与Nav1.7互作的冷冻电镜颗粒结构为模板,探究多肽结合与钠通道激活、失活相关的电压感受器构象变化,提出多肽与钠通道互作的微结构域、氨基酸基序和空间等电点效价模型,以期为新型钠通道亚型特异性人工肽的设计和研制提供新思路。

植物胚胎发育晚期丰富蛋白1组的结构与功能

植物胚胎发育晚期丰富蛋白(late embryogenesis abundant proteins,LEA)是植物胚胎发生后期种子中大量积累的一类蛋白质。根据蛋白质的氨基酸基序和保守结构特点,LEA蛋白一般分为6组,其中第1组LEA蛋白(LEA1)含有高度保守的20氨基酸基序。LEA1蛋白在水溶液中主要呈无规则结构,具高亲水性和热稳定性,与植物抗逆功能密切相关。本文就LEA1蛋白的功能和结构等方面的研究做一综述。

基于宏基因组学方法挖掘新型α-L-鼠李糖苷酶资源

α-L-鼠李糖苷酶是特异性切割末端含α-L-鼠李糖的天然化合物的一类糖苷水解酶,该酶在食品、医药、化学等行业都有广泛的应用。本研究旨在利用保守氨基酸基序结合PCR驱动的宏基因组学方法,从提取的健康人体粪便宏基因组DNA中获得新型细菌源α-L-鼠李糖苷酶基因。通过对CAZy数据库GH78家族中193条细菌源α-L-鼠李糖苷酶氨基酸序列进行多重序列比对,首次将α-L-鼠李糖苷酶家族分为3个亚家族,并确定了其中两个亚家族的两对保守氨基酸基序。基于保守基序氨基酸序列设计简并引物,PCR扩增保守基序间基因片段,对PCR产物克隆测序,结果获得12条α-L-鼠李糖苷酶基因片段,对其编码的氨基酸序列在Gen Bank数据库进行Blast序列比对,其中两条基因片段的氨基酸序列一致性仅为52%,一条为73%,其余9条在94%以上。根据Gen Bank数据库中序列一致性94%以上片段对应全长基因序列设计上下游引物,以人体粪便宏基因组DNA为模板,扩增获得3条α-L-鼠李糖苷酶全长基因,并将其克隆于载体p ET-28a,在E.coli BL21(DE3)内进行异源表达,目的蛋白多以包涵体形式存在于沉淀中,少量以可溶性形式存在于上清中。人体肠道细菌宏基因组为新型α-L-鼠李糖苷酶基因发现提供了潜在的基因资源库,基于保守氨基酸基序驱动的宏基因组学方法,从人体肠道以及环境宏基因组中直接获取新酶基因是可行的。

Sequence Analysis of Type III Effector tccP and tccP2 Genes in Escherichia coli O157:H7 from Chinese Water-chestnut

[Objective] This study aimed to analyze the type III effector tccP and tccP2 genes in Escherichia coli O157：H7 from Chinese water-chestnut. [Method] Gene-specific and locus-specific primers were utilized to amplify tccP/tccP2 and their flanking regions for sequence analysis. [Result] E. coli O157：H7 CWN11 harbored intact tccP and tccP2 genes, however, the number of proline-rich repeats in tccP gene was only one that probably resulted in biological incapability, whereas, the tccP2 gene consisted of five and half proline-rich repeats and could encode functional protein. [Conclusion] Here, we reported the first sequence of tccP gene that consisted of only one proline-rich repeat and tccP2 was assumed to play a crucial role in colonization and subsequent signaling cascades.

科技

动物慢病毒Gag蛋白细胞内组装与释放机制破译1月6日,中国农业科学院哈尔滨兽医研究所王晓钧领衔的马传染病和慢病毒病研究团队,在国际著名病毒学专业期刊《病毒学杂志》（Journal of Virology）上在线发表文章,报道了马传染性贫血病毒（EIAV）Gag蛋白细胞内组装和释放机制研究的最新发现。研究发现,与人获得性免疫缺陷病毒（HIV-1）Gag主要定位于细胞膜内缘不同,

Molecular cloning of the heat shock protein 20 gene from Paphia textile and its expression in response to heat shock

P. textile is an important aquaculture species in China and is mainly distributed in Fujian, Guangdong, and Guangxi Provinces. In this study, an HSP20 c DNA designated Pt HSP20 was cloned from P. textile. The full-length c DNA of Pt HSP20 is 1 090 bp long and contains a 5′ untranslated region(UTR) of 93 bp, a 3′ UTR of 475 bp, and an open reading frame(ORF) of 522 bp. The Pt HSP20 c DNA encodes 173 amino acid residues and has a molecular mass of 20.22 k Da and an isoelectric point of 6.2. Its predicted amino acid sequence shows that Pt HSP20 contains a typical α-crystallin domain(residues 77–171) and three polyadenylation signal-sequences at the C-terminus. According to an amino acid sequence alignment, Pt HSP20 shows moderate homology to other mollusk s HSPs. Pt HSP20 m RNA was present in all of the test tissues including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, with the highest concentration found in the gonad. Under the stress of high temperature, the expression of Pt HSP20 m RNA was down-regulated in all of the tissues except the adductor muscle and gonad.

The Full-length Genome Analysis of a Street Rabies Virus Strain Isolated in Yunnan Province of China

The epidemic of rabies has rapidly increased and expanded in Yunnan province in recent years. In order to further analyze and understand the etiological reasons for the rapid expansion of rabies in Yunnan, a strain of rabies virus CYN1009D in Yunnan was isolated, and the complete genomic sequencing was carried out, and the bioimfomative analysis on genes/encoded proteins and phylogeny with reference to sequences in GenBank was performed. The complete genome of CYN1009D was 11923 nt in length and belonged to genotype I. The genes encoding different structural proteins were all conserved in their lengths, in comparison to other strains in China. The amino acid sequence was conserved at different antigen sites of NP, but the variation was detected at the secondary phosphorylation site of position 375; variations were also detected in the phosphorylation sites at positions 63-63 and 162 of PP; the sites playing important roles in virus synthesis, budding and viral morphology in MP were conserved; two glycosylation sites were detected at Asn37 and Ash319 in GP, the neutralizing antigen sites in GP were conserved; the initial amino acid of LP （ML） was different from that of most of the strains in China （MM）; the variations in G-L region in the intergenic region were significant. The phylogenic tree showed that CYN1009D has a closer genetic relationship to the strains in Southeast Asia, indicating that prevention and control on rabies in borderland areas should be reinforced meanwhile efforts are made to control rabies in China.

A new ALF from Litopenaeus vannamei and its SNPs related to WSSV resistance

Anti-lipopolysaccharide factors （ALFs） are basic components of the crustacean immune system that defend against a range of pathogens. The cDNA sequence of a new ALF, designated nLvALF2, with an open reading flame encoding 132 amino acids was cloned. Its deduced amino acid sequence contained the conserved functional domain of ALFs, the LPS binding domain （LBD）. its genotnic sequence consisted of three exons and four introns, nLvALF2 was mainly expressed in the Oka organ and gills of shrimps. The transcriptional level of nLw4LF2 increased significantly after white spot syndrome virus （WSSV） infection, suggesting its important roles in protecting shrimps from WSSV. Single nucleotide polymorphisms （SNPs） were found in the genomic sequence of nLvALF2, of which 38 were analyzed for associations with the susceptibility/resistance of shrimps to WSSV. The loci g.2422 A〉G, g.2466 T〉C, and g.2529 G〉A were significantly associated with the resistance to WSSV （P〈0.05）. These SNP loci could be developed as markers for selection of WSSV-resistant varieties ofLitopenaeus vannamei.

Cloning and Analysis of Calmodulin Gene from Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensis, cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exons and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca2+ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.

Phylogenetic Analysis of the Non-structural (NS) Gene of Influenza A Viruses Isolated in Kazakhstan in 2002-2009

Although the important role of the non-structural (NS1 and NEP) gene of influenza A in virulence of the virus is well established, our knowledge about the extent of variation in the NS gene pool of influenza A viruses in their natural reservoirs in Kazakhstan is incomplete. 17 influenza A viruses of different subtypes were studied in this paper. Seven types of haemagglutinin and five different neuraminidase subtypes in eight combinations were found among the isolated viruses. A comparison of nucleotide sequences of isolated viruses revealed a substantial number of silent mutations, which results in high degree of homology in amino acid sequences. By phylogenetic analysis it was shown that two distinct gene pools, corresponding to both NS allele A with 5 Clades and B, were present at the same time in Kazakhstan. The degree of variation within the alleles was very low. In our study allele A viruses had a maximum of 5% amino acid divergence in Clade while allele B viruses had only 4% amino acid divergence.

Sequencing and expression analysis of CD3γ/δ and CD3ε chains in mandarin fish, Siniperca chuatsi

The genomic and cDNA sequences of the CD3γ/δ and CD3ε homologues in the mandarin fish, Siniperca chuatsi, were determined. As in other vertebrate CD3 molecules, the deduced amino acid sequences of mandarin fish CD3γ/δ and CD3ε contained conserved residues and motifs, such as cysteine residues and CXXC and immunoreceptor tyrosine-based activation motifs. However, mandarin fish CD3γ/δ and CD3ε showed some differences to their mammalian counterparts, specifically the absence of a negatively charged residue in the transmembrane region of CD3γ/δ. Additionally, while an N-glycosylation site was present in CD3c, the site was not observed in CD3γ/δ. The CD3γ/δ and CD3ε subunit sequences contain six and five exons, respectively, consistent with homologues from Atlantic salmon, Salmo salar. Phylogenetic analysis also revealed that CD3γ/δ and CD3ε in mandarin fish are closely related to their counterparts in Acanthopterygian fish. Real-time PCR showed CD3γ/δ and CD3ε were expressed mainly in the thymus and spleen in normal healthy fish and, to a lesser extent, in mucosal-associated lymphoid tissues, such as the intestine and gills. When lymphocytes isolated from head kidney were treated with the mitogens phytohemagglutinin, concanavalin, and polyriboinosinic polyribocytidylic acid, mRNA expression levels of CD3γ/δ and CD3ε were significantly elevated within 12 h of treatment. This indicated the presence of T lymphocytes in the head kidney of teleost fish, and also the recognition of mitogens by the lymphocytes. Mandarin fish infected with the bacterial pathogen Flavobacterium columnare also showed an increase in the expression of CD3γ/δ and CD3ε mR_NA, indicating that CD3γ/δ and CD3ε lymphocytes are involved in the immune response of this species.

Molecular cloning and mRNA expression analysis of myosin heavy chain(MyHC)from fast skeletal muscle of grass carp,Ctenopharyngodon idella

The myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle.We have isolated the cDNA clone encoding MyHC of the grass carp,Ctenopharyngodon idella. The sequence comprises 5 934 bp,including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues.The deduced amino acid sequence showed 69%homology to rabbit fast skeletal MyHC and 73%–76%homology to the MyHCs from the mandarin fish,walleye pollack,white croaker,chum salmon,and carp.The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80%homology to the corresponding regions of other fish MyHCs.The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR.The MyHC gene showed the highest expression in the muscles compared with the kidney,spleen and intestine.Developmentally,there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage.The highest expression was detected in hatching larva.Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.

Molecular Cloning and Characteristics Analysis of ghrelin Gene in Asian Swamp Eel(Monopterus albus)

Ghrelin is an important signaling molecule linking reproductive and energy metabolism. In this study, ghrelin gene of Monopterus albus was cloned. Its structure and function were analysized preliminarily. By Rapid Amplification of cDNA Ends(RACE) technique, full-length cDNA and DNA sequences of ghrelin gene were obtained. The full-length ghrelin cDNA(GenBank accession no. JX122807) was 552 bp long, containing a 115 bp 5'-untranslated region, a 324 bp open reading frame and a 113 bp 3'-untranslated region. The full-length ghrelin DNA was 1 323 bp, consisting of three introns and four exons. The exon/intron junction sequences conformed to the GT/AG rule. Three introns were 594, 84 and 93 bp in length, respectively; four exons were229, 78, 112 and 133 bp in length, respectively. The results of amino acid sequence analysis showed that the deduced propreghrelin sequence of M. albus contained a signal peptide(SP) consisting of 22 amino acid residues, a mature peptide(MP)consisting of 19 amino acid residues and a C-terminal amino acid residue. Among them, the third amino acid of MP was serine(Ser^3) as the site for N-acylation and N-deacetylation reactions; the C-terminal amino acid residue sequence might contain a peptide hormone obestatin, which is a physiological antagonist of mature Ghrelin peptide. The homology and phylogenic relationships analyses of amino acid sequences suggested that propreghrelin of M. albus had high similarity to those of several Perciformes fishes; the propreghrelins of M. albus, Perciformes and Heterosomata fishes were clustered into a subgroup. The high conservatism of the gene structure and the amino acid sequences indicated that Ghrelin exerts important physiological functions and plays similar physiological mechanisms in vertebrates.

Molecular Characterization of the Duck Enteritis Virus UL4 Gene

Duck enteritis virus(DEV) is a herpesvirus that causes an acute,contagious and fatal disease. In the present article,the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction(PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame(ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups,and the duck enteritis virus branched separately,closely related to the Mardiviruses group comprising Gallid herpesvirus 2(GaHV-2) ,Gallid herpesvirus 3(GaHV-3) and Meleagrid herpesvirus 1(MeHV-1) . The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.

Expression and Analysis of Microtus fortis Against Schistosoma japonicum CD72

The total RNA was extracted from Microtus fortis liver tissue which before being infected and after being infected 10 d and 15 d by the Schistosoma japonicum cercariae. Using rattus norvegicus CD72 gene probes were used to hybridize analysis of CD72 difference expression in the Microtus fortis liver tissues which were infected with Schistosoma japonicum before and after being infected. At the same time, the cDNA sequence and encoded amino acid sequence of the rattus norvegicus CD72 gene and CD72 protein structural domains were analyzed by using bioinformatics. The results showed that the CD72 expression levels in the liver tissue of Microtus fortis after being infected was significantly higher than before being infected. The rattus norvegicus CD72 cDNA sequence of a total length is 1479 bp and encode 364 amino acid residues and rattus norvegicus CD72 protein containing a CD72 superfamily domain.

A cadmium metallothionein gene of ridgetail white prawn Exopalaemon carinicauda(Holthuis,1950) and its expression

Metallothioneins （MTs） are a group of low molecular weight cysteine-rich proteins capable of binding heavy metal ions. A cadmium metallothionein （EcMT-Cd） cDNA with a 189 bp open reading frame （ORF） that encoded a 62 amino acid protein was obtained from Exopalaemon carinicauda. Seventeen cysteines were in the deduced amino acid sequence, and the cysteine （Cys）-rich characteristic was revealed in different metallothioneins in other species. In addition, the deduced amino acid sequence did not contain any aromatic amino acid residues, such as tyrosine （Tyr）, tryptophan （Trp）, and phenylalanine （Phe）. EcMT- Cd mRNA was expressed in all tested tissues （the ovary, muscle, stomach, and hepatopancreas）, and its expression profiles in the hepatopancreas were very different when shrimps were exposed to seawater containing either 50 pmol/L CuSO4 or 2.5 gmol/L CdC12. The expression of EcMT-Cd was significantly up-regulated in shrimp exposed to CuSO4 for 12 h and down-regulated in shrimps exposed to CdC12 for 12 h. After 24 h exposure to both metals, its expression was down-regulated. By contrast, at 48 h the EcMT-Cd was up-regulated in test shrimps exposed to CdC12. The transcript of EcMT-Cd was very low or even absent before the zoea stage, and the expression of EcMT-Cd was detected from mysis larvae-I, then its expression began to rise. In conclusion, a cadmium MT exists in E. carinicauda that is expressed in different tissues and during different developmental stages, and responds to the challenge with heavy metal ions, which provides a clue to understanding the function of cadmium MT

Expression and Analysis of Microtus fortis against Schistosoma japonicum CD36 Gene

The total RNA was extracted from Microtusfortis liver tissue which before being infected and after being infected 10 d and 15 d by the Schistosoma japonicum cercariae. Using Rattus norvegicus CD36 gene probe to hybridize analysis of CD36 difference expression in the Microtus fortis liver tissues which were infected with Schistosorna japonicum before and after being infected. At the same time, the cDNA sequence and encoded amino acid sequence of the Rattus norvegicus CD36 gene and CD36 protein structural domains were analysized by using bioinformatics. The results showed that the CD36 expression levels in the liver tissue of Microtus fortis after being infected were significantly higher than before being infectied. The Rattus norvegicus CD36 cDNA sequence of a total length is 1625 bp and encoded 472 amino acid residues and Rattus norvegicus CD36 protein containing a CD36 superfamily domain.

Cloning and Characterization of a Lycium chinense Carotenoid Isomerase Gene Enhancing Carotenoid Accumulation in Transgenic Tobacco

Carotenoid isomerase （CRTISO）is a key enzyme that catalyzes the conversion of cis-lycopene to all- trans tycopene. In this study, we isolated and characterized the CRTISO gene from Lycium chinense （LcCRTISO） for the first time. The open reading flame of LcCRTISO was 1 815 bp encoding a protein of 604 amino acids with a molecular mass of 66.24 kDa. Amino acid sequence analysis revealed that the LcCRTISO had a high level of simi- larity to other CRTISO. Phylogenetic analysis displayed that LcCRTISO kept a closer relationship with the CRTISO of plants than with those of other species. Semi-quantitative PCR analysis indicated that LcCRTISO gene was expressed in all tissues tested with the highest expression in maturing fruits. The overexpression of LcCRTISO gene in transgenic tobacco resulted in an increase of total carotenoids in the leaves with [3-carotene and lutein being the predominants. The results obtained here clearly suggested that the LcCRTISO gene was a promising candidate for carotenoid production.