利用RACE技术扩增大豆抗病基因同源cDNA 5′末端序列

利用抗病基因保守序列筛选大豆cDNA文库,获得一抗病基因同源cDNA片段,命名为KR3-1。根据KR3-1设计两个基因特异引物（GSP 和 NGSP）,分别与通用引物(UPM)和巢式通用引物（NUP）共同扩增,成功地克隆到了该基因的5′末端序列。该扩增片段长447 bp,与已知序列重叠部分为129 bp。 Abstract:Based on part of a known partial cDNA sequence of a disease resistance gene homolog,KR3-1,obtained by screening a cDNA library from soybean,5′-RACE-PCR was carried out with gene specific primers and universal primers.After the nested PCR reaction,an amplified fragment of 447 bp in length which overlapped the known KR3-1 sequence by 129 bp was obtained subsequently.Thus,a 5′ cDNA end of KR3 was successfully cloned.     

一种新的cDNA末端快速扩增获取全长cDNA的方法

为克隆精子发生相关基因的全长cDNA,根据mRNA差异显示获得的ESTs设计引物。利用一种新的cDNA末端快速扩增方法（SMART RACE）扩增该EST的5′末端,并进行克隆测序,与cDNA差异显示获得ESTs拼接后,获得了三个新的全长cDNA。结果表明：SMAR RACE是一种简便、有效的克隆cDNA5′末端未知序列的技术。     

RLM-RACE法扩增獐茅液泡膜Na^＋/H^＋逆向转运蛋白基因5′-cDNA末端序列

根据已获得的盐生植物獐茅（Aeluropus littoralisvar.sinensisDebeaux）液泡膜Na^＋/H^＋逆向转运蛋白（Na＋/H＋antiporter）基因部分cDNA片段,设计2条特异引物（GSP1、GSP2）,采用RLM-RACE（RNA ligase-medi-ated rapid amplification of 5′and 3′cDNA ends）法获得了其5-′cDNA末端序列,并找出了转录起始位点。该片段长816 bp,与芦苇液泡膜Na^＋/H^＋逆向转运蛋白基因序列同源性最高达91%,与拟南芥、盐角草、水稻、大麦、小麦的同源性分别为81%、82%、86%、87%和81%,为该基因全长的克隆及启动子的研究奠定了基础。与其它测定基因转录起始位点的方法相比,RLM-RACE方法更快捷、简便、准确。     

一种新的甘露糖结合凝集素--朱顶兰凝集素基因的克隆及序列分析

运用同源克隆的方法设计简并引物,通过3′和5′RACE技术,从石蒜科植物朱顶兰(Amaryllis vittata Ait)总RNA中克隆了编码此凝集素(AVA)的全长cDNA序列.该基因全长686 bp,起始密码子位于第41～43 bp,终止密码子位于515～517 bp处,开放阅读框长474 bp,编码158个氨基酸,包含信号肽序列、成熟蛋白序列和C-末端剪切序列的前体蛋白.成熟蛋白由109个氨基酸残基组成,分子量为11.9kD.成熟蛋白在氨基酸水平上与雪花莲凝集素、水仙凝集素、石蒜凝集素和君子兰凝集素分别有73.4%、85.3%、80.7%和83.5%的同源性;朱顶兰凝集素的分子模式显示其与雪花莲凝集素有极其相似的三维结构;在Blocks数据库中检索AVA蛋白氨基酸序列的结构域,发现有3个凝集素功能结构域,并具有3个典型的甘露糖专一结合位点盒(QDNY).     

一种新的甘露糖结合凝集素——朱顶兰凝集素基因的克隆及序列分析

运用同源克隆的方法设计简并引物,通过3′和5′RACE技术,从石蒜科植物朱顶兰(Amaryllis vittata Ait)总RNA中克隆了编码此凝集素(AVA)的全长cDNA序列。该基因全长686 bp,起始密码子位于第41～43 bp,终止密码子位于515～517bp处,开放阅读框长474 bp,编码158个氨基酸,包含信号肽序列、成熟蛋白序列和C-末端剪切序列的前体蛋白。成熟蛋白由109个氨基酸残基组成,分子量为11.9kD。成熟蛋白在氨基酸水平上与雪花莲凝集素、水仙凝集素、石蒜凝集素和君子兰凝集素分别有73.4％、85.3％、80.7％和83.5％的同源性;朱顶兰凝集素的分子模式显示其与雪花莲凝集素有极其相似的三维结构;在Blocks数据库中检索AVA蛋白氨基酸序列的结构域,发现有3个凝集素功能结构域,并具有3个典型的甘露糖专一结合位点盒(QDNY)。     

天麻中一种抗真菌蛋白基因的克隆

依据天麻(GastrodiaelataBl.f.flavidaS.Chow)抗真菌蛋白GAFP_1的N端部分氨基酸序列设计简并引物,通过RACE(快速分离cDNA末端)的方法扩增得到GAFP_1全长cDNA。该cDNA包含一个编码171个氨基酸的ORF,推导的多肽序列与测得的蛋白质部分序列相同;在5′端有一个长为55bp的5′非编码区;终止密码子下游有一个141bp长的3′非编码区,其中含有两个加poly(A)信号及长度为26个腺苷酸的poly(A)。经检索发现该推导蛋白序列与火烧兰(Epipactishelloborine)和二叶兰(Listeraovata)的甘露糖结合蛋白以及雪花莲(Galanthusnivalis)中的甘露糖结合凝集素具有很高同源性。     

小反刍兽疫病毒基因组全长cDNA的构建及其序列的测定

根据小反刍兽疫Nigeria75/1株全基因组序列设计并合成PPRV特异引物,进而应用RT-PCR技术分5段扩增了PPRV全基因组cDNA。将扩增的各个cDNA重叠片段JF1、JF2、JF3、JF4和JF5分别克隆到载体上,建立了PPRV的初级cDNA克隆。在扩增5′末端时,引入AscI酶切位点和T7启动子序列;在基因组3′末段引入PacI酶切位点,后者供cDNA模板的线性化之用。将扩增片段再依次连接,最后亚克隆到质粒pok12中,获得了PPRV全长基因组cDNA克隆pok12-PPRV。通过序列同源性和进化树分析,结果表明,Nigeria75/1与MV和RPV的遗传关系最近。Nigeria75/1株全长cDNA的序列测定及构建,为拯救PPRV和在分子水平进一步深入研究PPRV打下坚实的基础。     

猪FGL2基因cDNA末端序列检测及结构分析

检测猪FGL2基因cDNA末端序列并对该基因结构初步分析。α-32P dCTP放射性同位素标记cDNA探针筛选猪基因组DNA文库;cDNA末端快速扩增(rapid amplification of cDNA end,RACE)。以猪正常小肠及心脏组织提取新鲜总RNA,反转录后作为模板,设计基因特异性引物,采用Advantage 2 聚合酶混合物进行PCR扩增;依据猪与人FGL2基因3′端已知同源序列设计PCR上游引物,以人FGL2基因3′末端序列设计下游引物,以猪基因组DNA为模板采用Advantage 2 聚合酶混合物进行PCR反应;PCR载体重组质粒DNA亚克隆扩增。同位素探针未能筛选到特异阳性克隆,RACE反应检测到特异性转录起始位置及第一个转录终止位置,但仍未检测到第二个转录终止位置。猪基因组DNA行PCR扩增成功检测到猪FGL2基因3′末端未知序列及第二个转录终止位置。     

绞股蓝法呢基焦磷酸合酶基因的克隆及其序列分析

目的:克隆并分析绞股蓝法呢基焦磷酸合酶(FPS)基因的全长序列。方法:参照罗汉果法呢基焦磷酸合酶基因,设计扩增绞股蓝FPS基因的3′RACE引物,采用3'RACE和5'RACE法克隆绞股蓝FPS基因全长cDNA。结果:获得绞股蓝FPS基因全长cDNA序列共1288个核苷酸,包含一个1026核苷酸的开放读框,编码342个氨基酸残基,推断该蛋白的相对分子质量为3.94×104。NCBI Blast结果显示绞股蓝FPS基因编码蛋白的氨基酸序列与已知的植物FPS氨基酸序列的同源性为91%~74%,核酸序列的同源性为88%~78%。结论:克隆了绞股蓝FPS基因全长cDNA序列,为进一步研究绞股蓝FPS基因的表达及三萜皂苷合成通路关键酶分子的进化奠定了基础。     

基于简并引物同步克隆两个红麻PDIL同源基因cDNA5'-末端序列

探索利用同一套简并引物结合通用引物同步扩增两个红麻PDIL同源基因的cDNA5’-末端序列,以期为同一转录组中两个旁系同源基因cDNA5' RACE的同步扩增提供借鉴.通过红麻HcPDIL5-2a和HcPDIL5-2b cDNA中间片段及3’-末端已知序列的比时,在其完全保守区段设计了一条引物用于两个基因5' RACE的共反转录;在其部分保守区段设计了两条简并引物,并利用其在两个基因的5'RACE扩增时退火温度的差异,结合通用引物巢式PCR同步扩增两个基因的cDNA 5 ’-末端未知序列.在两个基因全长cDNA拼接序列的基础上设计两对特异引物分别扩增它们的cDNA全长序列,测序结果进一步验证了序列拼接和cDNA 5' RACE同步扩增的可靠性.进化分析证实两个基因属于PDIL基因家族成员.     

Cloning and molecular characterization of a novel lectin gene from Pinellia ternata

The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.     

Molecular cloning and characterization of a mannose-binding lectin gene from <Emphasis Type="Italic">Pinellia cordata</Emphasis>

Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein was successfully expressed in Escherichia coli with the molecular weight expected.     

cDNA cloning and characterization of a mannose-binding lectin fromZingiber officinaleRoscoe (ginger) rhizomes

Using RNA extracted fromZingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA ofZ. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA ofzoa was 746 bp and contained a 510 bp open reading frame (ORF) encoding a lectin precursor of 169 amino acids with a signal peptide. ZOA was a mannose-binding lectin with three typical mannose-binding sites (QDNY). Semi-quantitative RT-PCR analysis revealed thatzoa expressed in all the tested tissues ofZ. officinale including leaf, root and rhizome, suggesting it to be a constitutively expressing form. ZOA protein was successfully expressed inEscherichia coli with the molecular weight expected. To our knowledge, this is the first mannose-binding lectin cDNA cloned from the family Zingiberaceae. Our results demonstrate that monocot mannose-binding lectins also occur within the family Zingiberaceae     

Purification, characterization, and molecular cloning of lectin from winter buds of Lysichiton camtschatcensis (L.) Schott

A novel lectin was purified to homogeneity from winter buds of Lysichiton camtschatcensis (L.) Schott of the Araceae family. It was a tetramer composed of two non-covalently associated polypeptides with small subunits (11 kDa) and large subunits (12 kDa). Sequencing of both subunits yielded unique N-terminal sequences. A cDNA encoding the lectin was cloned. The isolated cDNA contained an open reading frame that encoded 267 amino acids. It encoded both subunits, indicating that the lectin is synthesized as a single precursor protein that is post-translationally processed into two different subunits with 45% sequence identity. Each subunit contained a mannose-binding motif known to be conserved in monocot mannose-binding lectins, but its activity was not inhibited by monosaccharides, including methyl α-mannoside. Asialofetuin and yeast invertase were potent inhibitors. Lectin activity was detected in the buds formed during the winter season but not in the expanded leaves.     

Cloning and nucleotide sequence of a full-length cDNA for human 14 kDa beta-galactoside-binding lectin

A full-length cDNA for a 14K-type human lung beta-galactoside-binding lectin was cloned. The cDNA includes a 405 bp open reading frame coding 135 amino acids including the initiator methionine, and having a single internal EcoRI site and a polyadenylation signal. The deduced amino-acid sequence agreed completely with the sequence of a human placenta lectin determined by direct amino-acid sequence analysis (Hirabayashi, J. and Kasai, K. (1988) J. Biochem. 104, 1-4). It showed extensive sequence similarity with other vertebrate 14K-type lectins and a 35K-type lectin (carbohydrate-binding protein 35) of mouse 3T3 cell. Search of a Genbank sequence data base revealed significant sequence similarity between the beta-galactoside-binding lectins and the carboxyl-terminal half of an IgE-binding protein, the cDNA of which has been cloned from rat basophilic leukemia cells. Thus, 14K-type lectin, 35K-type lectin and IgE-binding protein appeared to form a superfamily of proteins. Almost all invariant residues are located in the central region of the 14K-type lectins, so this region may constitute an essential part of the lectins, such as the sugar-binding domain.     

A novel mannose-binding tuber lectin from Typhonium divaricatum (L.) Decne (family Araceae) with antiviral activity against HSV-II and anti-proliferative effect on human cancer cell lines

A novel mannose-binding tuber lectin with in vitro antiproliferative activity towards human cancer cell lines and antiviral activity against HSV-II was isolated from fresh tubers of a traditional Chinese medicinal herb, Typhonium divaricatum (L.) Decne by a combined procedure involving extraction, ammonium sulfate precipitation, ion exchange chromatography on DEAE-SEPHAROSE, CM-SEPHAROSE and gel-filtration on sephacryl S-200. The apparent molecular mass of the purified Typhonium divaricatum lectin (TDL) was 48 kDa. TDL exhibits hemagglutinating activity toward rabbit erythrocytes at 0.95 microg/ml, and its activity could be strongly inhibited by mannan, ovomucoid, asialofetuin and thyroglobulin. TDL showed antiproliferative activity towards some well established human cancer cell lines, e.g. Pro-01 (56.7 +/- 6.8), Bre-04 (41.5 +/- 4.8), and Lu-04 (11.4 +/- 0.3). The anti-HSV-II activity of TDL was elucidated by testing its HSV-II infection inhibitory activity in Vero cells with TC(50) and EC(50) of 5.176 mg/ml and 3.054 microg/ml respectively. The full-length cDNA sequence of TDL was 1145 bp and contained an 813-bp open reading frame (ORF) encoding a 271 amino acid precursor of 29-kDa. Homology analysis showed that TDL had high homology with many other mannose-binding lectins. Secondary and three-dimensional structures analyses showed that TDL is heterotetramer and similar with lectins from mannose-binding lectin superfamily, especially those from family Araceae.     

Purification,characterization, molecular cloning,and expression of novel members of jacalin-related lectins from rhizomes of the true fern Phlebodium aureum (L) J. Smith (Polypodiaceae)

A lectin was purified from rhizomes of the fern Phlebodium aureum by affinity chromatography on mannose-Sepharose. The lectin, designated P. aureum lectin (PAL), is composed of two identical subunits of approximately 15 kDa associated by noncovalent bonds. From a cDNA library and synthetic oligonucleotide probes based on a partial amino acid sequence, 5'- and 3'-rapid amplification of cDNA ends allowed the generation of two similar full-length cDNAs, termed PALa and PALb, each of which had an open reading frame of 438 bp encoding 146 amino acid residues. The two proteins share 88% sequence identity and showed structural similarity to jacalin-related lectins. PALa contained peptide sequences exactly matching those found in the isolated lectin. PALa and PALb were expressed in Escherichia coli using pET-22b(+) vector and purified by one-step affinity chromatography. Native and recombinant forms of PAL agglutinated rabbit erythrocytes and precipitated with yeast mannan, dextran, and the high mannose-containing glycoprotein invertase. The detailed carbohydrate-binding properties of the native and recombinant lectins were elucidated by agglutination inhibition assay, and native lectin was also studied by isothermal titration calorimetry. Based on the results of these assays, we conclude that this primitive vascular plant, like many higher plants, contains significant quantities of a mannose/glucose-binding protein in its storage tissue, whose binding specificity differs in detail from either legume mannose/glucose-binding lectins or monocot mannose-specific lectins. The identification of a jacalin-related lectin in a true fern reveals for the first time the widespread distribution and molecular evolution of this lectin family in the plant kingdom.     

Molecular characterization of a new lectin from the marine alga Ulva pertusa

A new lectin, named UPL1, was purified from a green alga Ulvapertusa by an affinitychromatography on the bovine-thyroglobulin-Sepharose 4B column. The molecular mass of the algal lectinwas about 23 kD by SDS-PAGE, and it specifically agglutinated rabbit erythrocytes. The hemagglutinatingactivity for rabbit erythrocytes could be inhibited by bovine thyroglobulin and N-acetyl-D-glucosamine. Thelectin UPL1 required divalent cations for maintenance of its biological activity, and was heat-stable, and hadhigher activity within pH 6-8. The N-terminal amino acid sequence of the purified lectin was determined(P83209) and a set of degenerate primers were designed. The full-length cDNA of the lectin was cloned byrapid amplification ofcDNA ends (RACE) method (AY433960). Sequence analysis of upll indicated it was! 084 bp long, and encoded a premature protein of 203 amino acids. The N-terminal sequence of the matureUPL1 polypeptide started at amino acid 54 of the deduced sequence from the cDNA, indicating 53 aminoacids lost due to posttranslational modification. The primary structure of the Ulva pertusa lectin did not showamino acid sequence similarity with known plant and animal lectins. Hence, this protein may be the paradigmof a novel lectin family.     

Molecular cloning and characterization of a mannose-binding lectin gene from Crinum asiaticum

Full-length cDNA of a mannose-binding lectin or agglutinin gene was cloned from a traditional Chinese medicinal herb Crinum asiaticum var. sinicum through RACE-PCR cloning. The full-length cDNA of C. asiaticum agglutinin (caa) was 820 bp and contained a 528 bp open reading frame encoding a lectin precursor (preproprotein) of 175 amino acid residues with a 22 aa signal peptide. The coding region of the caa gene was high in G/C content. The first 20 bp of the 5' UTR had a dC content of 50%, which was a typical feature of the leader sequence. By cutting away the signal peptide, the CAA proprotein was 15.79 kDa with a pl of 9.27 and contained 3 mannose-binding sites (QDNY). Random coil and extended strand constituted interlaced domination of the main part of the secondary structure. B-lectin conserved domain existed within N24 to G130. Predicted three-dimensional structure of CAA proprotein was very similar to that of GNA (Galanthus nivalis agglutinin). It is significant that besides certain homologies to known monocot mannose-binding lectins from Amaryllidaceae, Orchidaceae, Alliaceae and Liliaceae, caa also showed high similarity to gastrodianin type antifungal proteins. No intron was detected within the region of genomic sequence corresponding to the caa full-length cDNA. Southern blot analysis indicated that the caa gene belonged to a low-copy gene family. Northern blot analysis demonstrated that caa mRNA was constitutively expressed in all the tested tissue types including the root, bulb, leaf, rachise, flower and fruit tissues.     

Cloning, characterization, and structural analysis of a C-type lectin from Bothrops insularis (BiL) venom

Lectins are carbohydrate-binding molecules that mediate a variety of biological processes. In this work, we identify and characterize a lectin from Bothrops insularis venom, with respect to its biochemical properties and theoretical structure. Initially, from a venom gland cDNA library, we cloned and sequenced a cDNA encoding a protein with high identity to snake venom lectins. A lectin molecule was purified to homogeneity from the venom by affinity column and gel filtration. This protein named BiL displayed hemagglutinating activity that was inhibited by galactose, lactose, and EDTA. Mass spectrometry analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that BiL is a disulfide-linked dimeric protein consisting of monomers with 16,206 m/z. The amino acid sequence, deduced from its cDNA sequence, was confirmed by Edman sequencing and by peptide mass fingerprint analysis. BiL shows similarity to other C-type lectin family members. Modeling studies provide insights into BiL dimeric structure and its structural determinants for carbohydrate and calcium binding.