Isolation and characterization of a Brassica napus cDNA corresponding to a B-class floral development gene

B-class floral homeotic genes are required for the proper formation and identity of petals and stamens in dicot flowers. A partial cDNA clone encoding a B-class gene, BnAP3 (Brassica napus APETALA3), was isolated from a B. napus cDNA library derived from young inflorescence meristems. The 5' region of the cDNA was retrieved by RACE. The deduced amino acid sequence of the full-length clone exhibited high similarity to APETALA3 of Arabidopsis thaliana and functionally homologous proteins from other species. 5' RACE and Southern analysis suggests that BnAP3 has multiple alleles in B. napus. Expression analysis assayed by RT-PCR shows that BnAP3 is expressed in floral tissues, as well as non-floral tissues such as root and bract. Transformation of wild-type A. thaliana and B. napus plants with BnAP3 under the control of a promoter specific to reproductive organs converts carpels to stamens, while the expression of this construct in A. thaliana plants mutant for AP3 restores the development of third-whorl stamens in addition to directing a carpel to stamen conversion in the fourth whorl.     

Structure and evolutionary origin of the gene encoding a human serum mannose-binding protein.

The N-terminal sequence of the major human serum mannose-binding protein (MBP1) was shown to be identical at all positions determined with the amino acid sequence predicted from a cDNA clone of a human liver MBP mRNA. An oligonucleotide corresponding to part of the sequence of this cDNA clone was used to isolate a cosmid genomic clone containing a homologous gene. The intron/exon structure of this gene was found to closely resemble that of the gene encoding a rat liver MBP (MBP A). The nucleotide sequence of the exons differed in several places from that of the human cDNA clone published by Ezekowitz, Day & Herman [(1988) J. Exp. Med. 167, 1034-1046]. The MBP molecule comprises a signal peptide, a cysteine-rich domain, a collagen-like domain, a 'neck' region and a carbohydrate-binding domain. Each domain is encoded by a separate exon. This genomic organization lends support to the hypothesis that the gene arose during evolution by a process of exon shuffling. Several consensus sequences that may be involved in controlling the expression of human serum MBP have been identified in the promoter region of the gene. The consensus sequences are consistent with the suggestion that this mammalian serum lectin is regulated as an acute-phase protein synthesized by the liver.     

Isolation and characterization of cDNA clones corresponding to the genes expressed preferentially in floral organs of Arabidopsis thaliana

Seventeen cDNA clones of genes corresponding to mRNAs expressed preferentially in floral organs of Arabidopsis thaliana were obtained by differential screening of a flower bud cDNA library, and classified into five groups (1A, 17A, 1B, 4B and 5B) by cross-hybridization and restriction analysis. Sequence analysis revealed that the 1A-1 and 17A-1 clones encode vegetative storage proteins (VSPs). The VSP mRNAs were detected in a small amount in leaves and increased to a limited level by wounding. Both 1B-1 and 5B-1 clones were homologous to transmembrane protein cDNAs. The protein encoded by 4B-1 clone contained a proline-rich region, but no homologous proteins were found in databases.     

Characterization of a new myrosinase in Brassica napus

A full-length cDNA clone defining the new myrosinase gene family MC in Brassica napus was isolated and sequenced. Southern hybridization showed that the MC family probably consists of 3 or 4 genes in B. napus. MC genes are expressed in the developing seed, but not in the vegetative tissues investigated. In situ hybridizations to developing seeds showed that the MC genes are expressed in the myrosin cells of the embryo axis and the cotyledons. Complexes with myrosinase and myrosinase-binding protein (MBP) were purified and characterized. Sequencing of peptides from myrosinases occurring in the complexes showed that the 70 kDa myrosinase is encoded by the MC genes, whereas the 65 kDa myrosinase is encoded by the MB genes. This is in contrast to the 75 kDa myrosinase which occurs in free form and is encoded by the MA genes. Deglycosylations of the myrosinase complexes and the free myrosinase showed that the molecular sizes of the myrosinases could be reduced significantly by this treatment, and that the size differences between the different myrosinases are mainly due to differences in glycosylation.     

草原龙胆MADS-box基因的克隆及表达分析

植物MADS-box基因家族编码高度保守的转录因子,参与了包括花发育在内的多种发育进程。为阐释双子叶植物草原龙胆（Eustoma grandiflorum）花器官发育的分子调控机制,根据MADS-box基因保守序列设计简并引物,用3＇-RACE方法从草原龙胆中克隆了4个花器官特异表达的MADS-box家族基因。序列和系统进化树分析表明,这4个基因分别与金鱼草DEF基因、矮牵牛FBP3基因和FBP6基因以及拟南芥SEP3基因具有很高的同源性,分别属DEF/GLO、AG-like和SEP-like亚家族。从而将这4个基因分别命名为EgDEF1、EgGLO1、EgPLE1和EgSEP3-1。推导的氨基酸序列显示,这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域,每个基因均有其亚家族特异的C-末端功能域。基因特异性RT-PCR检测结果显示：EgDEF1在萼片、花瓣、雄蕊及胚珠中高丰度表达,在心皮中微量表达;而EgGLO1在花瓣和雄蕊中高丰度表达,在萼片中微量表达;在根、茎、叶等营养器官中均未检测到上述2个基因的表达。EgPLE1在雌蕊、心皮和胚珠中特异表达,但表达的丰度存在差异,在雄蕊中的表达有所减弱。SEP-like亚家族基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达丰度相对一致。     

Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.     

Cloning and Characterization of the cDNA Encoding a Novel Brain-Specific 14-kDa Protein

A new acidic protein with a molecular weight of 14,000 was purified from rat brain, in which it was specifically expressed, and partially sequenced by protein sequencing. On the basis of results obtained from the amino acid sequences, mixed oligonucleotides were synthesized and used as probes to clone a cDNA from a rat brain cDNA library. The cloned cDNA provided the full-length sequence of the 14-kDa protein. Northern blot hybridization using total RNA from several tissues of the rat provided evidence that the 14-kDa protein was expressed specifically in rat brain. Transfection of this cDNA into mammalian cells resulted in expression of the 14-kDa protein. The amino acid sequence predicted from the cDNA of the rat brain 14-kDa protein contained 137 amino acid residues. A hydropathy profile revealed a hydrophobic domain (amino acids 60-80) flanked by highly hydrophilic stretches on both sides. Whereas the N-terminal region of the 14-kDa protein contained four repeating motifs, EKTKEGV, the C-terminal domain was rich in glutamic acid and proline. A computer search of the amino acid sequence of the 14-kDa protein indicated no homology to any other protein reported so far.     

An RNA-binding protein, AtRBP1, is expressed in actively proliferative regions in Arabidopsis thaliana

A cDNA clone, named XF41, that encodes an RNA-binding protein was isolated from Arabidopsis thaliana. The deduced protein, named AtRBP1, contains two conserved consensus sequence-type RNA-binding domains (CS-RBDs) in the N-terminal half, a putative PY motif (a target of a WW domain) in the center, and uncharacterized C-terminal domain. A binding assay demonstrated that the AtRBP1 can bind to single-stranded nucleic acids in vitro. Analysis of localization of the GUS activity of transgenic Arabidopsis thaliana plants that have the chimeric gene containing the upstream sequence of the AtRBP1 gene and GUS gene demonstrated that the AtRBP1 gene is expressed in meristematic tissues such as the vegetative shoot apex and root tips, developing organs such as floral buds and pistils of young flowers, abscission layers of immature siliques and junctions of pedicels. Considering the specificity of the expression, AtRBP1 may be required in the progress of cell proliferation.     

Cloning and characterization of a bamboo LEAFY HULL STERILE1 homologous gene.

A cDNA named DlMADS8 was isolated from the young spikelets of the sweet bamboo, Dendrocalamus latiflorus by rapid amplification of cDNA end (RACE). DNA sequence analysis showed that DlMADS8 was composed of full ORF and 3'UTR, but without 5'UTR. The cDNA contained 1059 nucleotides and encoded a putative protein of 244 amino acid residues. The gene displayed the structure of a typical plant MADS-box gene, which consisted of a MADS domain, K domain, a short I region, and the C-terminal region. Phylogenetic analysis of plant MADS-box genes based on amino acid sequences revealed that DlMADS8 was grouped into the AGAMOUS-LIKE 2 (AGL2)-like subfamily. It was homologous to the LEAFY HULL STERILE1 (LHS1) genes of grasses. To study the functions of it, DlMADS8 cDNA clone driven by the CaMV 35S promoter was transformed into Arabidopsis thaliana. Transgenic plants of DlMADS8 exhibited the phenotypes of curled leaves and early flowering. After bolting, three novel phenotypes related to inflorescence development were observed in different transgenic plants. No obvious homeotic conversions of floral organs were observed in all of the 35S::DllMADS8 transgenic Arabidopsis plants. These results indicated that DlMADS8 probably plays a role in floral meristem determinacy and is involved in controlling the flowering time of D. latiflorus.     

棉花MADS框蛋白基因(GhMADS1)的克隆

作为转录因子,MADS框蛋白基因在植物花器官发育中有着重要的功能。为研究棉花花器官发育的分子机理,以棉花花器官突变体CHV1(cotton homeotic variant)和徐州142正常植株为材料,利用棉花EST数据库资料,通过EST序列整合,从陆地棉徐州142花蕾中克隆出一个MADS框蛋白的编码区段,GenBank登录号为AF538965。该片段(GhMADS1)长713bp,包含一个711bp的开放阅读框,推导的氨基酸序列(236个氨基酸)与葡萄、烟草、矮牵牛、拟南芥和金鱼草等的AGL2组MADS框蛋白有很高的序列相似性。系统进化分析同样将GhMADS1基因归人AGt2组MADS框蛋白。RT-PCR分析显示,该基因在陆地棉的花瓣、雄蕊、胚珠和纤维中表达,特别是在花瓣中表达量最高,而在根、茎、叶等营养器官和棉花同源异型突变体CHV1(所有花器官均变为苞叶状叶性器官)的变异花蕾中不表达。这些结果说明GhMADS1基因可能在棉花花器官发育中有着重要的功能。     

Nucleotide sequence of cDNA coding for dianthin 30, a ribosome inactivating protein from Dianthus caryophyllus.

Rabbit antibodies raised against dianthin 30, a ribosome inactivating protein from carnation (Dianthus caryophyllus) leaves, were used to identify a full length dianthin precursor cDNA clone from a lambda gt11 expression library. N-terminal amino acid sequencing of purified dianthin 30 and dianthin 32 confirmed that the clone encoded dianthin 30. The cDNA was 1153 basepairs in length and encoded a precursor protein of 293 amino acid residues. The first 23 N-terminal amino acids of the precursor represented the signal sequence. The protein contained a carboxy-terminal region which, by analogy with barley lectin, may contain a vacuolar targeting signal.     

草原龙胆MADS-box基因的克隆及表达分析

植物MADS-box 基因家族编码高度保守的转录因子, 参与了包括花发育在内的多种发育进程。为阐释双子叶植物草原龙胆(Eustoma grandiflorum)花器官发育的分子调控机制, 根据MADS-box基因保守序列设计简并引物, 用3'-RACE方法从 草原龙胆中克隆了4个花器官特异表达的MADS-box家族基因。序列和系统进化树分析表明, 这4个基因分别与金鱼草DEF基因、矮牵牛FBP3基因和FBP6基因以及拟南芥SEP3基因具有很高的同源性, 分别属DEF/GLO、AG-like和SEP-l ike亚家族。从而将这4个基因分别命名为EgDEF1、EgGLO1、EgPLE1和EgSEP3-1。推导的氨基酸序列显示, 这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域, 每个基因均有其亚家族特异的C-末端功能域。基因特异性RT-PCR检测结果显示: EgDEF1 在萼片、花瓣、雄蕊及胚珠中高丰度表达, 在心皮中微量表达; 而EgGLO1在花瓣和雄蕊中高丰度表达, 在萼片中微量表达; 在根、茎、叶等营养器官中均未检测到上述2个基因的表达。EgPLE1在雌蕊、心皮和胚珠中特异表达, 但表达的丰度存在差异, 在雄蕊中的表达有所减弱。SEP-like亚家族基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达丰度相对一致。