cDNA cloning and in vitro synthesis of the Dolichos biflorus seed lectin

The Dolichos biflorus seed lectin contains two structurally related subunits. A cDNA library was constructed using RNA isolated from D. biflorus seeds actively synthesizing the seed lectin. The library was expressed in Escherichia coli using a lambda Charon 16 vector, and lectin-specific antiserum was used to isolate a seed lectin cDNA. Hybridization of the D. biflorus seed lectin cDNA to RNA isolated from seeds actively producing both lectin subunits identifies a single-size RNA of 1100 bases. An oligodeoxyribonucleotide probe, constructed from an amino acid sequence common to both lectin subunits, detects the same size RNA. Translation of seed mRNA in vitro and immunoprecipitation of translation products using a lectin-specific antiserum yields a single polypeptide of slightly higher molecular mass than the largest seed lectin subunit. This seed lectin precursor is indistinguishable from a polypeptide synthesized from mRNA hybrid selected by the seed lectin cDNA. These data support the existence of a single polypeptide precursor for both subunit types of the D. biflorus seed lectin and suggest that differences between the subunit types arise by posttranslational processing.     

cDNA cloning and expression of Bauhinia purpurea lectin.

Bauhinia purpurea lectin (BPA) was purified from seeds of B. purpurea alba. The purified lectin was digested with an endoproteinase, Asp-N, or trypsin and then the amino acid sequences of the resultant fragments were analyzed. Furthermore, a cDNA library for BPA was constructed using RNA isolated from germinated Bauhinia purpurea seeds. By gene cloning, the nucleotide sequence of BPA cDNA and its deduced amino acid sequence were analyzed. The cloned BPA cDNA comprised 1,152 nucleotides and the open reading frame of the cDNA encodes a polypeptide of 290 amino acids including a signal peptide composed of 28 amino acids. BPA expressed in Escherichia coli showed a relative molecular mass of 29 kDa on sodium dodecyl sulfate-polyacrylamide gel. On comparison of its sequence with those of other leguminous seed lectins, BPA showed high homology to the others.     

Isolation and characterization of a dehydrin gene from Cicer pinnatifidum, a drought-resistant wild relative of chickpea

Chickpea is an important legume crop of the tropics and subtropics. As it is cultivated mostly during the dry season, drought is the major cause of poor chickpea yields. A wild relative of chickpea, Cicer pinnatifidum , is more tolerant than chickpea itself to various abiotic stresses, including drought. A cDNA clone encoding a dehydrin gene, cpdhn1 , was isolated from a cDNA bank prepared from ripening seeds of C. pinnatifidum . Dehydrins are proteins which accumulate in seeds during late embryogenesis and also during stress brought about by drought and other abiotic factors in many plants. The polypeptide deduced to correspond to this gene, cpdhn1 , consists of 195 amino acid residues with a molecular mass of 20.4 kDa. The polypeptide is a Y₂K dehydrin exhibiting two conserved 'Y'-segments in the amino-terminal region and one conserved 'K'-segment close to the carboxy terminus. Northern blot analyses showed that cpdhn1 expression was induced not only during seed development, but also in leaves in response to drought, chilling and salinity and also to treatment with ABA or methyl jasmonate. The induction of cpdhn1 expression by methyl jasmonate and ABA indicates that the gene may also be involved in the response to biotic stress. The CpDHN1 protein may thus improve the tolerance of chickpea to a variety of environmental stresses, both abiotic and biotic.     

Molecular cloning of the bark and seed lectins from the Japanese pagoda tree (Sophora japonica)

cDNA clones encoding the bark and seed lectins from Sophora japonica were isolated and their sequences analyzed. Screening of a cDNA library constructed from polyA RNA isolated from the bark resulted in the isolation of three different lectin cDNA clones. The first clone encodes the GalNAc-specific bark lectin which was originally described by Hankins et al. whereas the other clones encode the two isoforms of the mannose/glucose-specific lectin reported by Ueno et al.. Molecular cloning of the seed lectin genes revealed that Sophora seeds contain only a GalNAc-specific lectin which is highly homologous to though not identical with the GalNAc-specific lectin from the bark. All lectin polypeptides are translated from mRNAs of ca. 1.3 kb encoding a precursor carrying a signal peptide. In the case of the mannose/glucose-specific bark lectins this precursor is post-translationally processed in two smaller peptides. Alignment of the deduced amino acid sequences of the different clones revealed striking sequence similarities between the mannose/glucose-binding and the GalNAc-specific lectins. Furthermore, there was a high degree of sequence homology with other legume lectins which allowed molecular modelling of the Sophora lectins using the coordinates of the Pisum sativum, Lathyrus ochrus and Erythrina corallodendron lectins.     

Isolation of a low-sulfur tolerance gene from <Emphasis Type="Italic">Eichhornia crassipes</Emphasis> using a functional gene-mining approach

Genes enhancing nutrient utilization efficiency are needed for crop improvement. Here, we report the isolation of a gene conferring low-sulfur tolerance from water hyacinth (Eichhornia crassipes) using a functional gene-mining method. In doing this, an entry cDNA library was constructed from the roots of nutrient-starved water hyacinth using recombination cloning and subsequently shuttled into the plant transformation- and expression-ready vector. The plant transformation- and expression-ready library was transferred into Arabidopsis and a seed library of 50,000 independent transgenic lines was generated. Three transgenic lines with enhanced low-sulfur tolerance were isolated from the seed library. One of the transgenic lines, shl143-1, with improved tolerance to sulfate deficiency and an improved root system was further analyzed. It was found that a water hyacinth jacalin-related lectin gene (EcJRL-1) was overexpressed in shl143-1. Recapitulation analysis confirmed that the overexpression of the EcJRL-1 cDNA caused the phenotype. Therefore, this study demonstrates that a jacalin-related lectin is involved in root elongation under sulfur-deficient conditions.     

应用酵母单杂交系统分离水稻蜡质基因5''调控区结合蛋白的基因

在水稻蜡质基因５’上游区中一段３１ｂｐ 核苷酸序列能与水稻未成熟种子核蛋白特异结合。为了克隆这一核蛋白基因,以此３１ ｂｐ 序列构建成“鱼饵”质粒,从水稻ｃＤＮＡ文库中筛选到１３ 个阳性克隆。根据这些阳性克隆中插入ｃＤＮＡ 片段的相互杂交结果,对这些克隆进行了分组。     

Cloning, mapping and genomic organization of a fish C-type lectin gene from homozygous clones of rainbow trout (Oncorhynchus mykiss)

Utilizing a splenic cDNA library and rapid amplification of cDNA 5' ends (5'-RACE), a C-type lectin gene was cloned from a homozygous cloned rainbow trout. The 1176 bp cDNA contains a 714 bp open reading frame from which a 238-amino-acid (aa) (27 kDa) protein was deduced. It was confirmed that this protein belongs to the C-type animal lectins, and is a type II membrane receptor. The predicted protein from this sequence contains a 48 aa cytoplasmic domain, a 20 aa transmembrane domain (TM), a 46 aa stalk region and a 124 aa carbohydrate-recognition domain (CRD). The stalk region contains a leucine-zipper, and an N-glycosylation site was also found in the CRD. Sequence alignment and phylogenetic analysis of the CRD indicate that the protein has similarity with human dendritic cell immunoreceptor (DCIR), gp120 binding C-type lectin (gp120BCL) and mammalian hepatic lectins. The N-terminus (aa 4-183) has similarity with NKG2, a group of C-type lectin receptors important in human natural killer cell function. The genomic DNA (gDNA) containing this gene was amplified and sequenced. The 4569 bp gDNA contains five exons and four introns. The first three exons encode the cytoplasmic domain, the TM and stalk region, respectively. Unlike the other type II C-type lectin receptors in which the CRD was encoded by three exons, the CRD of this lectin was encoded by two exons. A transposon Tc1-like fragment was found in intron III. Intron IV is composed of a simple repeat. Tissue-specific expression of the gene was studied by RT-PCR, and it was mainly expressed in spleen and peripheral blood leukocyte (PBL). Using AluI to digest the fragment containing exon I, intron I and exon II, an RFLP was produced between the sequences of this gene in two cloned fish, OSU 142 and Arlee (AR). Seventy-one doubled haploids (DH) of OSU X AR were screened, and the gene was mapped to linkage group XIV on the published map (Young et al., Genetics 148 (1998) 839).     

Expression of soybean lectin gene deletions in tobacco

A series of constructs containing the developmentally regulated soybean lectin gene (Le1) were used to transform tobacco plants in order to assess developmental and quantitative regulation conferred by flanking sequences. The largest of the lectin constructs contained approximately 3,000 base pairs (bp) of Le1 5 flanking region and 1,500 bp of the 3 flanking region. The smallest construct contained no 5 flanking region and 194 bp of the 3 flanking region. ELISA assays of lectin in individual tobacco seeds and Southern blot analyses confirmed that most constructs were inherited as unique insertion events. Maximal expression of Le1 required more than 338 bp of 5 sequence, indicating that far upstream factors are involved in quantitative control of lectin expression. Lectin expression declined more than 80% between deletions with 1,700 versus 338 bp of 5 flanking sequence. In contrast, developmental control of lectin expression was maintained by Le1 inserts with only 190 bp of 5 sequence. The lectin promoter offers a potential means to target high levels of gene expression to the developing seeds of soybean or other dicotyledonous plants.     

A gibberellin-regulated xyloglucan endotransglycosylase gene is expressed in the endosperm cap during tomato seed germination.

Xyloglucan endotransglycosylases (XETs) modify xyloglucans, major components of primary cell walls in dicots. A cDNA encoding an XET (LeXET4) was isolated from a germinating tomato (Lycopersicon esculentum Mill.) seed cDNA library. DNA gel blot analysis showed that LeXET4 is a single-copy gene in the tomato genome. LeXET4 mRNA was strongly expressed in germinating seeds, was much less abundant in stems, and was not detected in roots, leaves or flower tissues. During germination, LeXET4 mRNA was detected in seeds within 12 h of imbibition with maximum mRNA abundance at 24 h. Tissue prints showed that LeXET4 mRNA was localized exclusively to the endosperm cap region. Expression of LeXET4 was dependent on exogenous gibberellin (GA) in GA-deficient (gib-1 mutant) tomato seeds, while abscisic acid, a seed germination inhibitor, had no effect on LeXET4 mRNA expression in wild-type seeds. LeXET4 mRNA disappeared after radicle emergence, even though degradation of the lateral endosperm cell walls continued. The temporal, spatial and hormonal regulation pattern of LeXET4 gene expression suggests that XET has a role in endosperm cap weakening, a key process regulating tomato seed germination.     

Isolation and characterization of cDNA clones encoding ADP-glucose pyrophosphorylase (AGPase) large and small subunits from chickpea (Cicer arietinum L.)

Four cDNA clones encoding two large subunits and two small subunits of the starch regulatory enzyme ADP-glucose pyrophosphorylase (AGPase) were isolated from a chickpea (Cicer arietinum L.) stem cDNA library. DNA sequence and Southern blot analyses of these clones, designated CagpL1, CagpL2 (large subunits) and CagpS1 and CagpS2 (small subunits), revealed that these isoforms represented different AGPase large and small subunits. RNA expression analysis indicated that CagpL1 was expressed strongly in leaves with reduced expression in the stem. No detectable expression was observed in seeds and roots. CagpL2 was expressed moderately in seeds followed by weak expression in leaves, stems and roots. Similar analysis showed that CagpS1 and CagpS2 displayed a spatial expression pattern similar to that observed for CagpL2 with the exception that CagpS1 showed a much higher expression in seeds than CagpS2. The spatial expression patterns of these different AGPase subunit sequences indicate that different AGPase isoforms are used to control starch biosynthesis in different organs during chickpea development.     

孔石莼凝集素蛋白基因的克隆与表达

根据孔石莼(Ulva pertusa)凝集素(Lectin)蛋白cDNA全长序列(GenBank登录号:AY433960)设计引物,以其总DNA为模板,采用PCR技术扩增蛋白DNA序列,经克隆、测序获得基因序列。结果表明,孔石莼凝集素蛋白(UPL)基因序列长约为670 bp,含有一个大小为56 bp的内含子。此外,设计带酶切位点的引物,以UPL-cDNA为模板,扩增其开放阅读框,并与表达载体pGEX-2T连接,构建原核表达载体pG2T-UPL,并在大肠杆菌BL21(DE3)中成功表达大小约为47 kD的目的蛋白。