Cloning and characterization of a pollen-specific cDNA encoding a glutamic-acid-rich protein (GARP) from potato Solanum berthaultii

A pollen-specific cDNA was isolated from a cDNA library of in vitro germinated pollen of the diploid potato species Solanum berthaultii. The cDNA clone, designated SB401, hybridizes to a messenger RNA of 1.2 kb length in mature and germinated pollen. SB401 messenger RNA is absent from other parts of the plant, including other flower tissues. SB401 cDNA, which possesses a long stretch of AT-rich 5-untranslated leader sequence, encodes a glutamic acid-rich protein (GARP) which is hydrophilic throughout and contains six imperfect repeated motifs of the sequence V-V-E-K-K-N/E-E with the di-basic amino acid residue pair (K-K) as the core within the repeats. These repeats are spaced at irregular intervals and predicted to form an -helical structure. The SB401 protein was over-expressed in Escherichia coli and the purified protein was used for raising antiserum. Both E. coli-expressed and the endogenous SB401 proteins in pollen and pollen tubes appear much larger on SDS-polyacrylamide gels than their calculated molecular masses. Immunoblotting revealed the protein to be most abundant in germinated pollen. The structural features of SB401 protein and a possible role for the protein in pollen development, pollen germination, and pollen tube growth are discussed.     

Isolation and partial characterization of a novel pollen-specific cDNA with multiple polyadenylation sites from wheat

Wheat is the foremost staple food crop that offers bothcalories and proteins to a large global population. Wheat(hexaploid AABBDD genome, 16 billion bp) is a geneti-cally complex, self-pollinating plant with bisexualflowers that produce short-lived pollen. Very little is knownabout the molecular biology of its gametophyte develop-ment despite a longstanding interest in hybrid seeds. Mostof our information is from the studies on a few model andcrop plants such as Arabidopsis, tobacco, vegeta…     

Characterization of a pollen-expressed gene encoding a putative pectin esterase ofPetunia inflata

From a pollen tube cDNA library ofPetunia inflata, we isolated cDNA clones encoding a protein, PPE1, which exhibits sequence similarity with plant, bacterial, and fungal pectin esterases. Genomic clones containing thePPE1 gene were isolated using cDNA for PPE1 as a probe, and comparison of the cDNA and genomic sequences revealed the presence of a single intron in thePPE1 gene. During pollen development,PPE1 mRNA was first detected in anthers containing uninucleate microspores; it reached the highest level in mature pollen and persisted at a high level inin vitro germinated pollen tubes. The observed expression pattern of thePPE1 gene suggests that its product may play a role in pollen germination and/or tube growth.     

The pollen-specific gene Ntp303 encodes a 69-kDa glycoprotein associated with the vegetative membranes and the cell wall

The pollen-specific gene Ntp303 belongs to the class of late pollen specific genes. It is first transcribed directly after pollen mitosis. Biochemical properties, appearance and precise location of the NTP303 protein during pollen development and pollen tube growth were studied by amino-acid micro-sequencing, protein gel blotting and immuno-localization. Antisera were raised against recombinant proteins, encoded by sequences of the pollen-specific Ntp303 gene. The antibodies specifically recognized a 69-kDa glycoprotein. Electron-microscopic immuno-localization of the protein revealed the presence of high concentrations of the NTP303 protein at the vegetative plasma membranes that surround the vegetative cell, the generative cell and the sperm cells of pollen and pollen tubes. The generative plasma membranes of the generative cell and the sperm cells were negative. NTP303 protein was also present in the cell walls and in callose plugs. With this method it was shown that the NTP303 protein was already present in mid-bicellular pollen, after the first, asymmetrical pollen mitosis. Possible functions for the NTP303 protein are discussed in relation to its properties and its association with the vegetative plasma membranes. Received: 9 September 1999 / Revision accepted: 4 November 1999     

Isolation and characterization of a polygalacturonase gene highly expressed in Brassica napus pollen

A cDNA clone, Sta 44-4, corresponding to a mRNA highly expressed in Brassica napus cv. Westar stamens, was isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 44-4 is synthesized in pollen beginning at the late uninucleate stage and reaches a maximum in trinucleate microspores. Sta 44-4 displayed significant sequence similarity to known pollen polygalacturonase genes. The B. napus pollen polygalacturonase gene was shown to be part of a small gene family and to display some polymorphism among different cultivars.     

小麦花粉特异性表达的cDNA的分离及表达特性

应用抑制差示杂交和5′/3′RACE PCR方法分离了小麦(Triticum aestivum L.)花粉特异性表达的全长cDNA(TaPSG719,GenBank:AY451238)),该基因全长1172 bp,5′非编码区序列长达329 bp,包含多个上游可译框架(uORF);该基因编码188个氨基酸的蛋白质,大小约20 kD,等电点为12.1。Northern杂交和RT-PCR分析表明该基因在成熟花粉特异表达,而在小孢子、叶片、根和未成熟的种子、幼茎和子房等组织几乎检测不到。进一步研究小麦花粉发育过程的表达水平表明,TaPSG719在单核和双核小孢子阶段不表达,在开花前5d(已完成有丝分裂)开始表达并迅速增强达到高峰,但随着花粉的成熟表达水平逐渐下降。表明TaPSG719是一个花粉中晚期特异性表达基因。经BLAST同源性分析表明,与目前已登录的基因没有显著的同源性。Southern杂交表明TaPSG719可能为一个多拷贝基因。为研究TaPSG719 cDNA 5′非编码区序列的uORF对可译框架的翻译的影响,构建不同缺失或突变的表达载体,采用麦胚体外翻译系统,结果显示含uORF的5′非编码区序列能显著抑制蛋白质的翻译水平,表明TaPSG719基因表达至少部分是在翻译水平上调控。     

Isolation and characterization of pollen-specific maize genes with sequence homology to ragweed allergens and pectate lyases

A cDNA clone (Zm58.1) was isolated by differential screening from a cDNA library made to mature Zea mays pollen, and shown to be pollen-specific by RNA blot analysis. When this partial-length clone was used to probe a genomic library, a similar but distinct pollen-specific genomic clone (68% sequence identity) was isolated (Zm58.2). The putative proteins coded for by these two clones show sequence homology to several flower-expressed gene products from various plant species, including known pollen allergens from short ragweed (Ambrosia artemisiifolia), and to pectate lyases from the plant pathogenic bacteria Erwinia spp. The two genes map to different chromosomes.     

小麦花粉特异性表达的cDNA的分离及表达特性

应用抑制差示杂交和5′/3′RACE PCR方法分离了小麦(Triticum aestivum L.)花粉特异性表达的全长cDNA(TaPSG719,GenBank:AY451238)),该基因全长1 172 bp,5′非编码区序列长达329 bp,包含多个上游可译框架(uORF);该基因编码1 88个氨基酸的蛋白质,大小约20 kD,等电点为12.1.Northern杂交和RT-PCR分析表明该基因在成熟花粉特异表达,而在小孢子、叶片、根和未成熟的种子、幼茎和子房等组织几乎检测不到.进一步研究小麦花粉发育过程的表达水平表明,TaPSG719在单核和双核小孢子阶段不表达,在开花前5 d(已完成有丝分裂)开始表达并迅速增强达到高峰,但随着花粉的成熟表达水平逐渐下降.表明TaPSG719是一个花粉中晚期特异性表达基因.经BLAST同源性分析表明,与目前已登录的基因没有显著的同源性.Southern杂交表明TaPSG719可能为一个多拷贝基因.为研究TaPSG719 cDNA 5′非编码区序列的uORF对可译框架的翻译的影响,构建不同缺失或突变的表达载体,采用麦胚体外翻译系统,结果显示含uORF的5′非编码区序列能显著抑制蛋白质的翻译水平,表明TaPSG719基因表达至少部分是在翻译水平上调控.     

The identification of a pollen-specific LEA-like protein in Lilium longiflorum

A novel pollen‐specific LEA‐like protein, LP28, was detected in Lilium longiflorum using two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). Immunoblot analysis using antiserum raised against LP28 revealed that the protein was not found in somatic tissues or uninucleate microspores, but accumulated gradually in developing pollen following microspore mitosis. Furthermore, LP28 was abundant in germinated pollen after hydration. The cDNA clone corresponding to LP28 encoded a putative protein of 238 amino acids with a calculated molecular mass of 24·2 kDa and a pI of 4·7. The amino acid sequence is highly hydrophilic except for the N‐terminal hydrophobic signal peptide. The sequence has similarities with group 3 LEA (late embryogenesis abundant) proteins. Immunocytochemical analyses demonstrated that LP28 was mainly found in cytoplasmic granules of the vegetative cell until pollen maturation, but after hydration it appeared in the elongating pollen tube wall. LP28 might be a unique pollen‐specific protein that is transported to the pollen tube wall after germination. Therefore, it is assumed that LP28 plays a role not only in pollen maturation, but also in the growth of the pollen tube, which penetrates the stylar matrix.