Cloning and characterization of a novel chalcone synthase gene from Phalaenopsis hybrida orchid flowers

Chalcone synthase (CHS, EC 2.3.1.74) is the key enzyme involved in flavonoid and anthocyanin biosynthesis. A complete DNA sequence of chalcone synthase gene designated Pchs1 was isolated by means of usual and then inverse polymerase chain reactions from genomic DNA of an orchid, Phalaenopsis hybrida, cv. Formosa rose. Nucleotide sequence analysis based on alignment with published Phalaenopsis chs cDNA revealed that Pchs1 contained an intact open reading frame of 1173-bp with one 109-bp intron at the conserved site. The deduced polypeptide (PCHS1) from Pchs1 comprised 390 amino acids with a predicted mol wt of 42.5 kD. PCHS1 showed 61–65% identities with CHS from other plants and retained most of the conserved residues. Some putative cis-regulatory elements were present at the 5′ and 3′ flanking regions of Pchs1. Southern blot analysis predicted at least four chs-like genes, thus indicating the presence of a small multigene chs family in P. hybrida. Relative quantitative RT-PCR showed that Pchs1 is expressed in petals at early flower development as well as in lip tissue when the flower has just opened. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 250–258. The text was submitted by the authors in English.     

Cloning, characterization and expression analysis of a sucrose synthase gene from tropical epiphytic orchid Oncidium Goldiana

A full-length cDNA encoding sucrose synthase was isolated from the tropical epiphytic orchid Oncidium Goldiana. The cDNA is 2829 bp in length containing an open reading frame of 2447 bp encoding 816 amino acids with a predicted molecular mass of 93.1 kDa. The deduced amino acid sequence of O . Goldiana sucrose synthase ( Osus ) shares more than 80% identity with those from other monocotyledonous plants. The sucrose synthase gene was demonstrated to encode a functional sucrose synthase protein by expression as recombinant protein in Escherichia coli . The Osus mRNA is present in all the tissues analysed, with the highest levels in strong sinks such as developing inflorescence and root tips. Incubation with sucrose or glucose resulted in a significant increase in the steady-state Osus mRNA levels in root tips and mature leaves in a similar pattern to maize Sus1 . Expression of the Osus mRNA in mature leaves was markedly enhanced by anaerobic conditions and elevated CO₂. The expression pattern and regulation of the gene suggest that the sucrose synthase plays an important role in the growth and development of the tropical epiphytic orchid O . Goldiana.     

A characterization of the MADS-box gene family in maize

Studies on distantly related dicot plant species have identified homeotic genes that specify floral meristem identity and determine the fate of floral organ primordia. Most of these genes belong to a family characterized by the presence of a structural motif, the MADS-box, which encodes a protein domain with DNA-binding properties. As part of an effort to understand how such genes may have been recruited during the evolution of flowers with different organ types such as those found in maize, two members of this gene family in maize, ZAG1 and ZAG2, have been characterized previously. Here, the isolation and characterization of four new members of this gene family, designated ZAP1, ZAG3, ZAG4 and ZAG5, are described and the genetic map position of these and 28 additional maize MADS-box genes is determined. The first new member of this family appears to be the Zea mays ortholog of the floral homeotic gene APETALA1 (AP1) and has been designated ZAP1. One of these genes, ZAG4, is unusual in that its deduced protein sequence includes the MADS domain but lacks the K-domain characteristically present in this family of genes. In addition, its copy number and expression varies among different inbreds. A large number of maize MADS-box genes map to duplicated regions of the genome, including one pair characterized here, ZAG3 and ZAG5. These data underscore the complexity of this gene family in maize, and provide the basis for further studies into the regulation of floral organ morphogenesis among the grasses.     

Cloning, characterization and tissue specific expression of a sucrose synthase gene from tropical epiphytic CAM orchid Mokara Yellow

A full-length cDNA encoding sucrose synthase was isolated from the tropical epiphytic CAM orchid Mokara Yellow. The cDNA is 2748bp in length containing an open reading frame of 2447bp encoding 816 amino acids with a predicted molecular mass of 93.1 kDa. The deduced amino acid sequence of M. Yellow sucrose synthase (Msus1) shares more than 80% identity with those from other monocotyledonous plants. The sucrose synthase gene was demonstrated to encode a functional sucrose synthase protein by expression as recombinant protein in Escherichia coli. Northern blot analysis showed that the expression pattern of Msus1 mRNA is tissue specific with highest levels in strong sinks such as expanding leaves and root tips, but not detectable in mature leaves and flowers. Incubation with sugars resulted in a significant increase in the steady-state Msus1 mRNA levels in shoots of seedlings.     

Cloning and characterization of PhPI9 involved in floral development from Phalaenopsis Orchid

In the attempt to discover new genes involved in the floral development in monoeotyledonousin species,we have cloned and characterized the homologous PISTALLATA-like (PI-like) gone from Phalaenopsis hybrid cultivar named PhPI9 (Phalaenopsis PI STILLATA # 9).The eDNA of PhPI9 has a fragment of 834 bp and has 60% identity with the PISTILATA from Arabidopsis.The deduced amino acid sequence of PhPI9 had the typical PI-motif.It also formed a subelade with other monoeot PI-type genes in phylogenetie analysis.Southern analysis showed that PhPI9 was present in the Phalaenopsis orchid genome as a single copy.Furthermore,it was expressed only in the lip of the Phalaenopsis flower and no expression was detected in vegetative organs.Thus,as a B-function MADS-box gone,PhP19 specifies floral organ identity in orchids.     

一个新的水稻MADS—box基因的克隆及表达分析

根据ＭＡＤＳ－ｂｏｘ基因保守区结构,设计简并性引物,利用３＇ＲＡＣＥ从水稻（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）中克隆了１个新的水稻ＭＡＤＳ－ｂｏｘ基因的ｃＤＮＡ片段,同时利用５＆ＲＡＣＥ获得了全长ｃＤｎＡ命名为ＦＤＲＭＡＤＳＳ。序列分析表明,该ｃＤＮＡ全长１４０６ｂｐ,开放阅读框共编码２３３个到,具有典型的植物ＭＡＤＳ－ｂｏｘ基因的结构。推测的氨基酸序列与拟南芥的ＭＡＤＳ－ｂｏｘ基因,ＡＧＬ１４同     

Cloning and characterization of a cold-and ABA-inducible Arabidopsis gene

We have identified by differential screening a novel Arabidopsis thaliana gene, called kin1, which is induced at +44 °C. The nucleotide sequences of both the genomic clone and the corresponding cDNA were determined. The deduced 6.5 kDa polypeptide has an unusual amino acid composition being rich in alanine, glycine and lysine. The gene belongs to a family of at least two genes. Northern blot analysis revealed that the level of kin1 mRNA is increased 20-fold in cold-treated plants. In addition to being expressed in cold, kin1 mRNAlso induced by water stress and the plant hormone abscisic acid (ABA) which has been suggested to be a common mediator for osmotic stress responses and cold acclimation in plants. Sequence comparisons showed that the kin1 gene product has similarities to fish antifreeze proteins (AFPs).     

人端粒酶RNA基因的克隆与鉴定

以人血基因组ＤＮＡ为模板,合成两段２０个寡聚核苷酸为引物,经过ＰＣＲ扩增,得到４８０ｂｐ的片段,克隆到ｐＭＤ１８－Ｔ载体中,经电泳、酶切、ＰＣＲ鉴定后测定序列。序列分析表明氙克隆的人端粒酶ＲＮＡ（ｈｕｍａｎ ｔｅｌｏｍｅａｓｅ ＲＮＡ,ｈＴＲ）基因含有４８０ｂｐ,包括约４５０ｂｐ的编码模板区主序列和约３０ｂｐ的上游调控区序列,其中模板区的１１个核苷酸（５’－ＣＵＡＡＣＣＣＵＡＡＣ－３’）合成端粒亚     

Interactions of B-class complex proteins involved in tepal development in Phalaenopsis orchid

In our previous studies, we identified four DEFICIENS (DEF)-like genes and one GLOBOSA (GLO)-like gene involved in floral organ development in Phalaenopsis equestris. Revealing the DNA binding properties and protein-protein interactions of these floral homeotic MADS-box protein complexes (PeMADS) in orchids is crucial for the elucidation of the unique orchid floral morphogenesis. In this study, the interactome of B-class PeMADS proteins was assayed by the yeast two-hybrid system (Y2H) and glutathione S-transferase (GST) pull-down assays. Furthermore, the DNA binding activities of these proteins were assessed by using electrophoretic mobility shift assay (EMSA). All four DEF-like PeMADS proteins interacted individually with the GLO-like PeMADS6 in Y2H assay, yet with different strengths of interaction. Generally, the PeMADS3/PeMADS4 lineage interacted more strongly with PeMADS6 than the PeMADS2/PeMADS5 lineage did. In addition, independent homodimer formation for both PeMADS4 (DEF-like) and PeMADS6 (GLO-like) was detected. The protein-protein interactions between pairs of PeMADS proteins were further confirmed by using a GST pull-down assay. Furthermore, both the PeMADS4 homodimer and the PeMADS6 homodimer/homomultimer per se were able to bind to the MADS-box protein-binding motif CArG. The heterodimeric complexes PeMADS2-PeMADS6, PeMADS4-PeMADS6 and PeMADS5-PeMADS6 showed CArG binding activity. Taken together, these results suggest that various complexes formed among different combinations of the five B-class PeMADS proteins may increase the complexity of their regulatory functions and thus specify the molecular basis of whorl morphogenesis and combinatorial interactions of floral organ identity genes in orchids.