Rice bicoid－related cDNA sequence and its expression during early embryogenesis

INTRODUCTIONThe establishment of embryonic polarity is oneof the critical events in embryogenesis. The polar distribution of maternal mRNA can be tracedto the unfertilized egg cell. After fertilization, itstranslational products trigger the zygotic targetgenes which define the embryonic region and fUIther direct the pattern formation and body planduring embryogenesis. In Drosophila, Bicoid is oneof the important maternal genes involved in thecontrol of embryo polarity and larvae segmen…     

C-terminal extension of rice glutamate decarboxylase (OsGAD2) functions as an autoinhibitory domain and overexpression of a truncated mutant results in the accumulation of extremely high levels of GABA in plant cells

Glutamate decarboxylase (GAD) converts L-glutamate to gamma-aminobutyric acid (GABA), which is a non-protein amino acid present in all organisms. Plant GADs carry a C-terminal extension that binds to Ca(2+)/calmodulin (CaM) to modulate enzyme activity. However, rice possesses two distinct types of GAD, OsGAD1 and OsGAD2. Although they both have a C-terminal extension, the former peptide contains an authentic CaM-binding domain (CaMBD), which is common to dicotyledonous plants, while the latter does not. Therefore, the role of the C-terminal extension in functional expression of OsGAD2 was investigated. An in vitro enzyme assay using recombinant OsGAD2 proteins revealed low activity in the presence or absence of Ca(2+)/CaM. However, a truncated version of GAD2 (OsGAD2DeltaC) had over 40-fold higher activity than wild-type GAD at physiological pH. These two DNA constructs were introduced simultaneously into rice calli via Agrobacterium to establish transgenic cell lines. Free amino acids were isolated from several lines for each construct to determine GABA content. Calli overexpressing OsGAD2 and OsGAD2DeltaC had about 6-fold and 100-fold the GABA content of wild-type calli, respectively. Regenerated OsGAD2DeltaC rice plants had aberrant phenotypes such as dwarfism, etiolated leaves, and sterility. These data suggest that the C-terminal extension of OsGAD2 plays a role as a strong autoinhibitory domain, and that truncation of this domain causes the enzyme to act constitutively, with higher activity both in vitro and in vivo.     

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     

Cloning and characterization of the chloroplast elongation factor EF-Tu cDNA of Oryza sativa L

From the rice leaf cDNA library, we have cloned a cDNA encoding rice chloroplast translational elongation factor EF-Tu (tufA). The rice tufA cDNA clone contains 1678 nucleotides and codes for a 467 amino acid protein including a putative chloroplast transit peptide of 59 amino acid residues. The predicted molecular mass of the mature protein is approximately 45 kDa. This cDNA clone contains the 61 nucleotides of the 5' untranslated region (UTR) and the 213 nucleotides of 3' UTR. Amino acid sequence identity of the rice tufA with the mature chloroplast EF-Tu proteins of tobacco, pea, arabidopsis, and soybean ranges from 83% to 86%. The deduced polypeptide of the rice tufA cDNA contains GTP binding domains in its N-terminal region and chloroplast EF-Tu signature regions in the C-terminal region. The rice tufA appears to exist as a single copy gene, although its homologues of maize and oat exist as multiple copy genes. The rice tufA gene is located in chromosome 1 and is more highly expressed in the leaf than in root tissue.     

An Arabidopsis thaliana lipoxygenase gene can be induced by pathogens, abscisic acid, and methyl jasmonate.

We isolated and characterized a 2.8-kb, full-length, Arabidopsis thaliana cDNA clone encoding a lipoxygenase. DNA sequence analysis showed that the deduced amino acid sequence of the Arabidopsis protein is 72 to 78% similar to that of legume seed lipoxygenases. DNA blot analysis indicated that Arabidopsis contains a single gene, LOX1, with appreciable homology to the cDNA clone. RNA blot analysis showed that the LOX1 gene is expressed in Arabidopsis leaves, roots, inflorescences, and young seedlings. LOX1 expression levels were highest in roots and young seedlings. In mature plants, LOX1 mRNA levels increased upon treatment with the stress-related hormones abscisic acid and methyl jasmonate and remained high for at least 96 h. Expression of the LOX1 gene was examined following infiltration of leaves with virulent (Psm ES4326) and avirulent (Pst MM1065) strains of Pseudomonas syringae. LOX1 mRNA levels were induced approximately 6-fold by both virulent and avirulent strains; however, the response to avirulent strains was much more rapid. Infiltration of leaves with Pst MM1065 resulted in maximal induction within 12 h, whereas maximal induction by Psm ES4326 did not occur until 48 h. When a cloned avr gene, avrRpt2, was transferred to Psm ES4326, LOX1 mRNA accumulated in a pattern similar to that observed for the avirulent strain Pst MM1065.     

Molecular cloning and functional expression of cDNA encoding a cysteine proteinase inhibitor,cystatin, from Job's tears (Coix lacryma-jobi L. var. Ma-yuen Stapf)

A lambdaZAP II cDNA library was constructed from mRNA in immature seeds of the grass Job's tears. A cDNA clone for a cysteine proteinase inhibitor, cystatin, was isolated from the library. The cDNA clone spanned 757 base pairs and encoded 135 amino acid residues. The deduced amino acid sequence was similar to that of cystatins from the gramineous plants rice, sorghum, and corn. The central Gln-Val-Val-Ala-Gly sequence thought to be one of the binding sites of cystatins was found. A remarkable characteristic of the peptide sequence of Job's-tears cystatin was the putative signal peptide that has been found in sorghum and corn but not in rice. The cystatin cDNA was expressed in Escherichia coli as a His-tagged recombinant protein. The purified recombinant protein inhibited papain.     

Isolation and characterization of a cDNA-clone coding for potato type A phytochrome

We have isolated and sequenced a cDNA clone encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 78% amino acid identity to the Arabidopsis phyA and 50% identity to the Arabidopsis phyB open reading frame, we have classified this cDNA clone as potato phyA phytochrome. The amino acid immediately preceding cysteine 323, which is the homologue of oat cystein 321, to which the chromophore has been shown to be attached, is a tyrosine residue. This contrasts with six other type A phytochrome sequences from both monocots and dicots that encode serine in this position. As already observed in three other cDNAs isolated from dicot species, the potato phyA clone encodes a short open reading frame (13 amino acids) preceding the phyA open reading frame (1123 amino acids), supporting the idea that this type of leader sequence might be involved in the regulated expression of the phytochrome apoprotein. Southern blot analysis revealed a single phyA gene as well as other related phytochrome sequences in the potato genome. phyA mRNA levels varied in different organs and were modulated by white light; in seedlings and sprouts, highest levels of mRNA were detected in the etiolated stage. Upon illumination with white light, mRNA levels decreased to the amount found in leaves of re-etiolated plants. Lowest expression was observed in leaves of plants grown in the light, in tubers irrespective of light treatment, and in roots of plants grown in the dark. In roots of plants grown in the light, elevated levels of phyA mRNA were detected. Using a monoclonal antibody generated against pea phytochrome as an immunochemical probe, the protein was only detectable in protein extracts from etiolated seedlings and sprouts.     

Identification and expression of a cDNA clone encoding aspartate aminotransferase in carrot

A full-length cDNA clone encoding aspartate aminotransferase (AAT) has been identified from a carrot root cDNA library. Degenerate oligo primers were synthesized from the known amino acid sequence of AAT form I from carrot (Daucus carota L. cv Danvers). These primers were utilized in a polymerase chain reaction to amplify a portion of a carrot AAT gene from first strand cDNA synthesized from poly(A)⁺ RNA isolated from 5-d-old cell suspension cultures. The resulting 750-bp fragment was cloned, mapped, and sequenced. The cloned fragment, mpAAT1, was used as a probe to identify a full-length cDNA clone in a library constructed from poly(A)⁺ RNA isolated from carrot roots. A 1.52-kb full-length clone, AAT7, was isolated and sequenced. AAT7 has 54% nucleotide identity with both the mouse cytoplasmic and mitochondrial AAT genes. The deduced amino acid sequence has 52 and 53% identity with the deduced amino acid sequences of mouse cytoplasmic and mitochondrial AAT genes, respectively. Further analysis of the sequence data suggests that AAT7 encodes a cytoplasmic form of carrot AAT; the evidence includes the (a) absence of a transit or signal sequence, (b) lack of “m-residues,” or invariant mitochondrial residues, in the carrot AAT sequence, and (c) high degree of sequence similarity with the amino acid sequence previously obtained for form I of carrot, a cytoplasmic isoenzyme. High- and low-stringency hybridizations to Southern blots of carrot nuclear DNA with AAT7 show that AAT is part of a small multigene family. Northern blot analysis of AAT7 suggests that AAT is expressed throughout cell culture up to 7 d and is highly expressed in roots but not in leaves.     

cDNA cloning of a novel cdc2+/CDC28-related protein kinase from rice

A cDNA clone, named R2, has been isolated by screening a rice cell cDNA library with a redundant oligonucleotide probe derived from the conserved ATP binding site of cdc2+/CDC28 protein kinases. The cDNA contained the entire coding sequence for a 424 amino acid polypeptide with a molecular mass of 47.6 kDa. The R2 mRNA, 2.1 kb in size, was expressed in both cultured rice cells and rice seedlings at similar levels. The predicted R2 protein has canonical motifs for ATP binding and catalysis, and is significantly homologous (up to 47%) to members of the cdc2+/CDC28 subfamily of serine/threonine protein kinase. The R2 protein is a novel member of the subfamily.     

Molecular cloning of cDNA for rat liver general acyl CoA dehydrogenase and homology between the rat liver and pig kidney enzymes

cDNA clone for general acyl CoA dehydrogenase (GAD) was isolated from a rat liver cDNA expression library in lambda gt11 using anti-pig kidney GAD antibody. Size of the isolated cDNA was estimated to be 1.5-1.6 kb. By immunological analysis of fusion protein and epitope selection, the cDNA clone was identified as that containing the GAD gene. Partial amino acid sequence deduced from nucleotide sequence of the cDNA coincided with that of the pig kidney enzyme. The antibody cross-reacted with rat liver enzyme and molecular weights of these enzyme proteins were shown to be almost the same. All these results indicate that rat liver GAD shares a common structure with pig kidney enzyme.     

Molecular cloning and characterization of anther-preferential cDNA encoding a putative actin-depolymerizing factor

A cDNA clone, LMP131A, which is preferentially expressed in mature anther was isolated from a lily cDNA library. Northern blot analysis and plaque hybridization expriments showed that the LMP131A mRNA is present at ca. 0.3% of the mRNA in mature pollen and is not detectable in carpel, petal, floral bud, leaf, or root. The clone contains an open reading frame of 139 amino acid residues which shows greater than 40% sequence identity in a 91 amino acid overlap to animal actin-depolymerizing factors (ADF), cofilin and destrin. The sequences at and near the actin-binding site are most conserved. Using the lily clone as a probe, a cDNA clone, BMP1, was isolated from a mature anther library of Brassica napus. The expression pattern of the BMP1 clone was the same as that of the lily clone. The Brassica anther-preferential clone contains an open reading frame which is 79% identical to the lily LMP131A protein. Southern blot analysis showed that there are one or a few copies of the putative ADF genes in B. napus and Arabidopsis thaliana.