Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.     

Molecular cloning and characterization of Scotch pine defensin 2

A 252 bp cDNA fragment that corresponds to defensin 2 (PsDef2) was amplified from a cDNA library from seven-day plantlets of Pinus sylvestris L. This fragment encodes a protein that consists of 83 amino acid residues. The protein contains an N-terminal signal peptide, which includes 33 amino acid residues. A mature form of defensin 2 of Scotch pine contains a gamma-thionine domain and it is also characterized by specific conservative residues that are common to all plant defensins.     

一个水稻OsWNK基因的分离及表达分析

在褐飞虱取食后的水稻cDNA差减文库中筛选到与拟南芥AtWNK1激酶基因高度同源的EST(GenBank登录号:BU572310),以该EST为探针,从褐飞虱取食后的水稻cDNA文库中分离到OsWNK基因的全长cDNA,该基因编码一个含677个氨基酸残基的蛋白激酶,与以前克隆出的一种拟南芥蛋白激酶基因(GenBank登录号:DQ837532)只有3个氨基酸残基的差异。Northern杂交结果显示,在褐飞虱取食后,该基因的表达上升。表明该激酶基因参与褐飞虱取食的应答反应,可能与水稻抗褐飞虱有关.     

Isolation of a cotton CAP gene: a homologue of adenylyl cyclase-associated protein highly expressed during fiber elongation

The cDNA encoding CAP (adenylyl cyclase-associated protein) was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. The cDNA (GhCAP) contained an open reading frame that encoded 471 amino acid residues. RNA blot analysis showed that the cotton CAP gene was expressed mainly in young fibers.     

Medicago truncatula Mt-ZFP1 encoding a root enhanced zinc finger protein is regulated by cytokinin, abscisic acid and jasmonate, but not cold.

A Medicago truncatula zinc finger protein cDNA (Mt-ZFP1) was isolated from a M.truncatula seedling cDNA library using RT-PCR product as a probe. The predicted amino acid sequence of Mt-ZFP1 is over 79% similar to S-SCOF-1 from soybean, a novel cold-inducible zinc finger protein involved in cold stress signal transduction mediated by abscisic acid (ABA). The secondary structure of Mt-ZFP1 protein was almost identical to that of S-SCOF-1. Mt-ZFP1 also contained a typical C2H2-type zinc finger domain and a putative nuclear located signal. RNA gel blot hybridization demonstrated that the Mt-ZFP1 gene was actively expressed in roots, with a lower abundance in leaf and stem tissues. Cold treatment did not induce the expression of Mt-ZFP1 in either leaves and stems or roots. Exogenous application of cytokinins marginally increased the accumulation of Mt-ZFP1 mRNA, while ABA and jasmonate treatments decreased the levels of Mt-ZFP1 mRNA. DNA gel-blot analysis demonstrated that Mt-ZFP1 is present as a single copy gene in the M. truncatula genome. These data suggest that Mt-ZFP1 is a novel zinc finger protein with different physiological functions to that of S-SCOF-1. The similar cold-inducible factor like S-SCOF-1 might not exist in M. truncatula.     

The expression of tgas118, encoding a defensin in Lycopersicon esculentum, is regulated by gibberellin.

A flower specific cDNA, tgas118, has been isolated after differential screening of a gib-1 anther cDNA library of Lycopersicon esculentum. The corresponding mRNA was present in all tissues analysed. Northern blot analysis revealed that in wild-type tomato the gene was predominantly expressed throughout flower development, while in the gibberellin (GA)-deficient mutant of tomato (gib-1) the abundance declined. Treatment of the mutant with GA resulted in an accumulation of the tgas118 mRNA within hours in leaf and bud tissues. In the leaf, GA1, GA3 and GA9 were effective in enhancing the expression while GA4 was not. In addition to GA, wounding and dehydration also increased the accumulation of tgas118 mRNA in leaf tissue. In situ hybridization showed that application of 50 ng GA3 bud(-1) induced a similar spatial expression of the tgas118 mRNA in gib-1 buds 24 h post treatment to that found in wild-type flower buds. The deduced TGAS118 protein displays up to 77% similarity with defensins and as its expression is up-regulated by stimuli such as wounding it is proposed that it may play a role in protection against pathogens.     

A novel gene of tomato preferentially expressed in fruit encodes a protein with a Ca2+-dependent lipid-binding domain

A cDNA clone, called CLB1, was isolated from a cDNA library from tomato (Lycopersicon esculentum) and characterized. The CLB1 cDNA contains an open reading frame of 1518 bp, and encodes a putative protein of 506 amino acids with a predicted molecular mass of 54 633 Da. The deduced CLB1 amino acid sequence contains a domain that exhibits from 26% to 37% identity with the Ca²⁺-dependent lipid-binding domains of cytosolic phospholipase A₂, protein kinase C, Rabphilin-3A, and Synaptotagmin I of animals. Southern blot analysis indicates that the CLB1 gene belongs to a small gene family in the tomato genome. The CLB1 mRNA is preferentially expressed in fruit tissues.     

Cloning and characterization of a novel human leptin receptor overlapping transcript-like 1 gene (LEPROTL1)

A new full-length cDNA encoding a novel protein was isolated from our human fetal brain cDNA library. The cDNA consists of 2701 bp and has a putative open reading frame encoding 131 amino acids which possesses a JAK binding site (Pro(46)-Ile-Pro(48) which is preceded by a cluster of hydrophobic residues) and is highly homologous to the leptin receptor gene-related protein (OB-RGRP). Northern blot analysis showed that this new gene is widely expressed in human tissues and radiation hybrid mapping placed the gene to human chromosome 8p21.1-8p21.2.     

A novel defensin from the lentil Lens culinaris seeds

A novel 47-residue plant defensin was purified from germinated seeds of the lentil Lens culinaris by ammonium sulfate precipitation, gel filtration, chromatography, and RP-HPLC. The molecular mass (5440.41 Da) and complete amino acid sequence (KTCENLSDSFKGPCIPDGNCNKHCKEKEHLLSGRCRDDFRCWCTRNC)¹ of defensin, termed Lc-def, were determined. Lc-def has eight cysteines forming four disulfide bonds. The total RNA was isolated from lentil germinated seeds, RT-PCR and subsequent cloning were performed, and cDNA was sequenced. A 74-residue predefensin contains a putative signal peptide (27 amino acid) and a mature protein. Lc-def shows high sequence homology with legumes defensins, exhibits an activity against Aspergillus niger, but does not inhibit proteolytic enzymes.     

氮磷饥饿诱导的水稻糖转运体基因的cDNA克隆和鉴定

运用快速扣除杂交 (RaSH)方法构建了水稻氮饥饿诱导的cDNA文库。从该文库获得了一个cDNA克隆OsNSI1 (Oryzasativanitrogenstarva tion inducible 1 )。该全长cDNA编码 5 77个氨基酸 ,蛋白分子量为 6 1 .2kD。推测得出的氨基酸序列与其他物种的糖转运体有很高的同源性。水合性分析表明OsNSI1包含有 1 2个跨膜区域和一个中心亲水环。这些数据提示OsNSI1是一个糖转运体蛋白。Southern印迹分析表明OsNSI1是一个单拷贝基因。Northern印迹分析表明OsNSI1主要在叶及根中表达 ,氮、磷饥饿能强烈诱导其表达增强     

Identification of a glialblastoma cell differentiation factor-related gene mRNA in human microvascular endothelial cells

Vascular endothelial cells (VEC) transduce mitogenic and chemoattractant signals in response to erythropoietin (Epo). An analysis of changes in gene expression in VEC would be helpful to understanding the molecular nature of mitogenic signals. An effective method for analysis of gene expression is through differential display. Using this approach, we obtained from Epo-treated human microvascular endothelial cells (HMVEC) a cDNA fragment with characteristics of the 3'end of mRNA. Using the cDNA fragment, we then isolated a full-length clone from a HMVEC cDNA library. The cDNA of interest encodes a protein consisting of 404 amino acids with a carboxy-terminal end sequence identical to glialblastoma cell differentiation factor-related protein (GBDR1). Northern blot analysis showed that GBDR1 mRNA was ubiquitously expressed in human tissues. In Southern blot analysis, GBDR1 cDNA identified a single gene on chromosome 9. Since analysis of the amino acid sequence revealed several putative phosphorylation sites for different protein kinases, the GBDR1 protein was expressed and purified from bacterial extracts and, as predicted, casein kinase II phosphorylated GBDR1 in vitro. Immunofluorescence and biochemical data revealed that the GBDR1 protein is not entirely localized in the cytosolic fraction, suggesting that it may interact with another protein(s). These findings demonstrate that GBDR1 is an intracellular signaling molecule that may play a role in the regulation of endothelial cell growth.     

Cloning and functions analysis of a pyruvate dehydrogenase kinase in Brassica napus

Pyruvate dehydrogenase kinase (PDK) is a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), which plays a key role in intermediary metabolism. In this study, a 1,490-bp PDK in Brassica napus (BnPDK1) was isolated and cloned from Brassica cDNA library. BnPDK1 has an 1,104 open reading frame encoding 367 amino acids. Genomic DNA gel blot analysis result indicated that BnPDK1 is a multi-copy gene. RNA gel blot analysis and RNA in situ hybridization were used to determine the expression of BnPDK1 in different organs. BnPDK1 gene was ubiquitously expressed in almost all the tissues tested, having the highest expression in the stamen and the young silique. Over-expression of BnPDK1 in transgenic Arabidopsis lines would repress the PDC activity, and resulted in the decrease of seed oil content and leaf photosynthesis. These results implied that BnPDK1 was involved in the regulation of fatty acid biosynthesis in developing seeds.     

外来入侵植物紫茎泽兰不同组织RNA提取方法

从植物组织中提取高质量的RNA是进行cDNA文库构建等分子生物学研究的前提。在苯酚法的基础上,改进并得到了一种适合紫茎泽兰根、茎、叶总RNA快速提取的方法,消除了蛋白质、DNA、多糖等的污染。该方法提取的紫茎泽兰不同组织总RNA纯度高、完整性好,可用于RT-PCR、cDNA文库构建、Northern杂交等分子生物学实验,而且简单、经济、重复性好,适合于多种植物组织总RNA的提取。Northern杂交表明F3’H基因在紫茎泽兰的根、茎、叶等组织中广泛存在,但在叶中的表达量最高,在根中的表达量最低。     

细胞内物质转运调节分子ARFGAP3的克隆表达及生化活性

ADP核糖基化因子-GTP酶活化蛋白(ARF GAP)是重要的细胞内物质转运调节分子.在22周孕龄人胎肝cDNA文库中发现一种新基因,其编码的氨基酸序列与大鼠ARF1 GAP有32%同源性.将这种新基因命名为“ARFGAP3”,对其进行功能研究,利用逆转录-聚合酶链式反应(RT-PCR),从人胎盘总RNA中扩增ARFGAP3全长cDNA序列,并将其亚克隆到pGEM-T载体;采用RNA印迹法和斑点杂交法,检测其组织表达谱,发现在多种腺体和睾丸中有很高水平ARFGAP3基因转录,并且只有一种约2.7 kb的转录本.利用基因重组技术,构建表达质粒pBAD/Thio-ARFGAP3,在大肠杆菌中表达,采用亲和层析法纯化表达产物,利用肠激酶切除重组融合蛋白N端引导序列.检测重组ARFGAP3的生化活性,证实ARFGAP3对ARF1具有GAP活性,促进ARF1结合的GTP水解为GDP,磷脂酰肌醇二磷酸(PIP2)增强其GAP活性,而磷脂酰胆碱(PC)抑制其GAP活性.     

Molecular characterization of a stigma-specific gene encoding an arabinogalactan-protein (AGP) from Nicotiana alata

Arabinogalactan-proteins (AGPs) were isolated from the pistils of Nicotiana alata , deglycosylated, and the protein backbones fractionated by reversed-phase HPLC as previously reported. A major fraction, RT35 was isolated and peptide sequences were obtained after protease digestion. A gene-specific degenerate oligonucleotide was designed according to the amino acid sequences and a 380 bp PCR fragment was amplified in vitro from pistil RNA. The PCR fragment was used to screen a pistil cDNA library and a 762 bp cDNA clone (AGP Na 3) was isolated and sequenced. The AGP Na 3 cDNA encodes a 169 amino acid protein which consists of three domains: an N-terminal secretion signal, a Pro-rich domain and a C-terminal Cys-rich domain. The mature protein has 145 amino acid residues (16.7 kDa) and a predicted pl of 7.5. Northern blot analyses showed that the AGP Na 3 gene was only expressed in the pistils of N. alata and of closely related Nicotiana species but not in other plants or suspension-cultured cells. Further Northern blot analysis and in situ hybridization showed that within the pistil, it was primarily expressed in the stigmatic tissues of mature flowers.