Cloning and expression of a PR5-like protein from Arabidopsis: inhibition of fungal growth by bacterially expressed protein

Pathogenesis-related (PR)-5 proteins are a family of proteins that are induced by different phytopathogens in many plants and share significant sequence similarity with thaumatin. We isolated a complementary DNA (ATLP-3) encoding a PR5-like protein from Arabidopsis which is distinct from two other previously reported PR5 cDNAs from the same plant species. The predicted ATLP-3 protein with its amino-terminal signal sequence is 245 amino acids in length and is acidic with a pI of 4.8. The deduced amino acid sequence of ATLP-3 shows significant sequence similarity with PR5 and thaumatin-like proteins from Arabidopsis and other plants and contains a putative signal sequence at the amino-terminus. The expression of ATLP-3 and a related gene (ATLP-1) that we previously isolated from Arabidopsis was induced by pathogen infection and salicylic acid, a known inducer of pathogenesis-related genes. Southern blot analysis indicates that the ATLP-1 and ATLP-3 are coded by single-copy genes. To study the effect of ATLP-1 and ATLP-3 proteins on fungal growth, the cDNA regions corresponding to putative mature protein were expressed in Escherichia coli and the cDNA encoded proteins were purified. ATLP-1 and ATLP-3 proteins cross-reacted with anti-osmotin and anti-zeamatin antibodies. ATLP-3 protein showed antifungal activity against several fungal pathogens suggesting that ATLP-3 may be involved in plant defense against fungal pathogens.     

Isolation and analysis of a genomic clone encoding a pokeweed antiviral protein

Partial cDNAs encoding a pokeweed antiviral protein were obtained by polymerase chain reaction from the poly(A)⁺ RNA of seeds, leaves, and roots using two specific primers based on the amino acid sequence of a pokeweed antiviral protein from the seeds (PAP-S). Using the cDNAs as a radioactive probe, 17 and 39 positive plaques were isolated from libraries containing the genomic DNA of Phytolacca americana digested with Bam HI partially and completely, respectively. The plaques were grouped into nine types by Southern hybridization. The type genomic clone encodes a protein of 294 amino acids. Its amino acid sequence is similar but not identical to that of PAP-S. A comparison of the two amino acid sequences suggested that the deduced protein contains extrapeptides of 24 and 9 amino acids at the NH₂ and the COOH terminals, respectively. The putative protein was expressed in Escherichia coli and shown to depurinate the specific adenine of wheat 25S rRNA, indicating that the protein encoded by a type genomic clone is a functional protein exhibiting RNA N-glycosidase activity.     

Cloning and Expression of a New Gene Encoding an Antifungal Protein in Phytolacca americana

protein (Pa-AFP) with molecular weight about 4 kD was purified from the seeds of Phytolacca americana L. , which obviously inhibits the growth of Rhizoctonia solani Kiihn in vitro. The authors isolated mRNA from the seeds of pokeberry and designed a degenerate PCR primer according to the N-terminal sequence of the purified protein. The full-length cDNA encoding Pa-AFP was cloned by RT-PCR and 5'-RACE and sequenced. The deduced amino acid sequence indicates that a preprotein with 65 amino acid residues is firstly translated and then processed to a mature protein with 38 amino acids. The DNA encoding the mature protein was subcloned into expression vector pGEX-4T1, and expressed efficiently in E. coli BL21 as a GST- Pa-AFP fusion protein. The fusion protein was purified by glutathione-Sepharose 4B affinity colmnn chromatography. The purified fusion protein was specifically digested by thrombin and the Pa-AFP was further purified by filtration column chromatography.     

Ha-DEF1, a sunflower defensin,induces cell death in <Emphasis Type="Italic">Orobanche</Emphasis> parasitic plants

Plant defensins are small basic peptides of 5–10 kDa and most of them exhibit antifungal activity. In a sunflower resistant to broomrape, among the three defensin encoding cDNA identified, SF18, SD2 and HaDef1, only HaDef1 presented a preferential root expression pattern and was induced upon infection by the root parasitic plant Orobanche cumana. The amino acid sequence deduced from HaDef1 coding sequence was composed of an endoplasmic reticulum signal sequence of 28 amino acids, a standard defensin domain of 50 amino-acid residues and an unusual C-terminal domain of 30 amino acids with a net positive charge. A 5.8 kDa recombinant mature Ha-DEF1 corresponding to the defensin domain was produced in Escherichia coli and was purified by means of a two-step chromatography procedure, Immobilized Metal Affinity Chromatography (IMAC) and Ion Exchange Chromatography. Investigation of in vitro antifungal activity of Ha-DEF1 showed a strong inhibition on Saccharomyces cerevisiae growth linked to a membrane permeabilization, and a morphogenetic activity on Alternaria brassicicola germ tube development, as already reported for some other plant defensins. Bioassays also revealed that Ha-DEF1 rapidly induced browning symptoms at the radicle apex of Orobanche seedlings but not of another parasitic plant, Striga hermonthica, nor of Arabidopsis thaliana. FDA vital staining showed that these browning areas corresponded to dead cells. These results demonstrate for the first time a lethal effect of defensins on plant cells. The potent mode of action of defensin in Orobanche cell death and the possible involvement in sunflower resistance are discussed.     

油葵HaGPAT1基因的克隆及表达分析

该研究利用RT-PCR技术,从油葵(Helianthus annuus L.)种子中克隆了甘油-3-磷酸酰基转移酶(GPAT)基因(HaGPAT1),对其进行生物信息学分析,并通过实时荧光定量PCR技术(qRT-PCR)检测该基因在不同组织、种子不同发育时期以及不同胁迫条件下的表达特征。结果表明:HaGPAT1基因全长为1 656bp,编码551个氨基酸,相对分子量为62.132kD,等电点为8.84。系统进化树分析表明,HaGPAT1蛋白与高等植物莴苣的GPAT1亲缘关系最近。qRT-PCR分析表明,HaGPAT1基因在油葵花蕊中的表达水平最高,开花后17d的种子中次之;在干旱和盐胁迫条件下,HaGPAT1基因的表达水平均显著上调。研究推测,HaGAT1基因可能在油葵花器官发育中发挥重要作用,并且参与了油葵对干旱和高盐的抗性调节。     

Cloning and expression of antiviral/ribosome-inactivating protein from <Emphasis Type="Italic">Bougainvillea</Emphasis> x<Emphasis Type="Italic">buttiana</Emphasis>

A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP)from the leaves of Bougainvillea x buttiana was isolated.The cDNA consisted of 1364 nucleotides with an open reading frame (ORF)of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids.The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species.The deduced protein has been designated BBAP1 (Bougainvillea x buttiana antiviral protein1).The ORF was cloned into an expression vector and expressed in E.coli as a fusion protein of approximately 78 kDa.The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity,and imparted a high level of resistance against the tobacco mosaic virus (TMV).     

Tissue-specific and developmental regulation of a gene encoding a low molecular weight sulfur-rich protein in soybean seeds

A gene corresponding to a cDNA clone, SE60, encoding a low molecular weight sulfur-rich protein in soybean seeds was isolated from a soybean genomic library and characterized at the nucleotide level. The SE60 gene is interrupted by an intervening sequence of 694 by in size. The 5 flanking region of the gene contained various regulatory sequences such as the RY repeat and CACA elements found in other seed protein genes of legumes. The SE60 gene encoded a pre-protein of 75 amino acids, having a signal sequence of 28 amino acids at the N-terminus. The mature protein of 47 amino acids was basic and cysteine-rich. Northern blot analysis suggested that the SE60 gene is expressed in a tissue-specific and developmentally regulated manner during soybean seed development. The SE60 genes form a small multigene family composed of about four members in the soybean genome.     

Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

Cloning and characterization of a cDNA encoding type 1 diacylglycerol acyltransferase from sunflower (Helianthus annuus L.)

A full-length cDNA encoding a putative diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) was obtained from sunflower (Helianthus annuus L.) seeds. The 1524-bp open reading frame of this cDNA, designated as HaDGAT1, encodes a protein of 507 amino acids with a molecular mass of 58.5 kDa showing high homology to DGAT1 enzymes of other plants. The protein characters, such as a predicted structure with a long N-terminal hydrophilic domain followed by 9 transmembrane domains, acyl-CoA-binding signature, diacylglycerol (DAG)-binding and putative endoplasmic reticulum retrieval motifs (ER-DIR), also indicated that HaDGAT belongs to the DGAT1 family. HaDGAT1 is expressed in all plant tissues especially in developing seeds. Expression of recombinant HaDGAT1 in yeast showed an 1.76-fold increase of total fatty acids, especially unsaturated fatty acids such as palmitoleic acid (enhanced by 86.6%) and oleic acid (enhanced by 81.6%).     

The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.     

Molecular cloning of a cDNA fromBrassica napus L. for a homologue of acyl-CoA-binding protein

A cDNA encoding an acyl-CoA-binding protein (ACBP) homologue has been cloned from a gt11 library made from mRNA isolated from developing seeds of oilseed rape (Brassica napus L.). The derived amino acid sequence reveals a protein 92 amino acids in length which is highly conserved when compared with ACBP sequences from yeast, cow, man and fruit fly. Southern blot analysis ofBrassica napus genomic DNA revealed the presence of 6 genes, 3 derived from theBrassica rapa parent and 3 fromBrassica oleracea. Northern blot analysis showed that ACBP genes are expressed strongly in developing embryo, flowers and cotyledons of seedlings and to a lesser extent in leaves and roots.     

Sulphur and nitrogen nutrition influence the response of chickpea seeds to an added, transgenic sink for organic sulphur

In order to increase the concentration of the nutritionally essential sulphur amino acids in seed protein, a transgene encoding a methionine- and cysteine-rich protein, sunflower seed albumin (SSA), was transferred to chickpeas (Cicer arietinum L). Transgenic seeds that accumulated SSA contained more methionine and less oxidized sulphur than the controls, suggesting that additional demand for sulphur amino acids from the expression of the transgene stimulated sulphur assimilation. In addition, the activity of trypsin inhibitors, a known family of endogenous, sulphur-rich chickpea seed proteins, was diminished in transgenic, SSA-containing seeds compared with the non-transgenic controls. Together, these results indicate that the reduced sulphur sequestered into SSA was supplied partly by additional sulphur assimilation in the developing transgenic seeds, and partly by some diversion of sulphur amino acids from endogenous seed proteins. Growth of chickpeas on nutrient with a high sulphur-to-nitrogen ratio increased the total seed sulphur content and the accumulation of sulphur amino acids in the seeds, and partly mitigated the effect of SSA accumulation on the trypsin inhibitor amount. The results suggest that free methionine and O-acetylserine (OAS) acted as signals that modulated chickpea seed protein composition in response to the variation in sulphur demand, as well as in response to variation in the nitrogen and sulphur status of the plant.     

麻疯树核糖体失活蛋白基因的克隆和表达

麻疯树(Jatropha curcas L.)核糖体失活蛋白(curcin)是存在于麻疯树种子中的一种毒性较强的蛋白,它与蓖麻毒蛋白和相思子毒蛋白的性质相似,属Ⅰ型核糖体失活蛋白.从麻疯树种子中分离得到一种分子量为28.2 kD的蛋白质,其对无细胞系统中蛋白质合成的抑制活性较强,IC50为(0.19±0.01)nmol/L,具有RNA N-糖苷酶活性.依据curcin的N端部分氨基酸设计简并引物,通过RT-PCR和5'-RACE技术从未成熟种子总RNA中克隆到curcin全长cDNA序列.该cDNA全长由1 173个碱基组成,包含一个编码293个氨基酸的前体蛋白,前42个氨基酸为信号肽.推测的多肽序列与测定的蛋白质N端序列相同,与多种己发表的Ⅰ型核糖体失活蛋白和Ⅱ型核糖体失活蛋白的A链有一定的同源性.将curcin的编码区与表达载体pQE-30相连后,转入大肠杆菌(Escherichia coil)M15菌株中得到了有效的表达.将表达的融合蛋白纯化后发现,它具有抑制无细胞系统蛋白质合成的能力.