Characterization of JBURE-IIb isoform of Canavalia ensiformis (L.) DC urease

Ureases, nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and bicarbonate, are widespread in plants, bacteria, and fungi. Previously, we cloned a cDNA encoding a Canavalia ensiformis urease isoform named JBURE-II, corresponding to a putative smaller urease protein (78kDa) when compared to other plant ureases. Aiming to produce the recombinant protein, we obtained jbure-IIb, with different 3' and 5' ends, encoding a 90kDa urease. Three peptides unique to the JBURE-II/-IIb protein were detected by mass spectrometry in seed extracts, indicating that jbure-II/-IIb is a functional gene. Comparative modeling indicates that JBURE-IIb urease has an overall shape almost identical to C. ensiformis major urease JBURE-I with all residues critical for urease activity. The cDNA was cloned into the pET101 vector and the recombinant protein was produced in Escherichia coli. The JBURE-IIb protein, although enzymatically inactive presumably due to the absence of Ni atoms in its active site, impaired the growth of a phytopathogenic fungus and showed entomotoxic properties, inhibiting diuresis of Rhodnius prolixus isolated Malpighian tubules, in concentrations similar to those reported for JBURE-I and canatoxin. The antifungal and entomotoxic properties of the recombinant JBURE-IIb apourease are consistent with a protective role of ureases in plants.     

Isolation and characterization of a lectin gene from seeds of chickpea (Cicer arietinum L.).

A cDNA library was constructed in lambda TriplEx2 vector using poly (A(+)) RNA from immature seeds of Cicer arietinum. The lectin gene was isolated from seeds of chickpea through library screening and RACE-PCR. The full-length cDNA of Chichpea seed lectin(CpGL)is 972 bp and contains a 807 bp open reading frame encoding a 268 amino acid protein. Analysis shows that CpSL gene has strong homology with other legume lectin genes. Phylogenetic analysis showed the existence of two main clusters and clearly indicated that CpSL belonged to mannose-specific family of lectins. RT-PCR revealed that CAA gene expressed constitutively in various plant tissues including flower, leaf, root and stem. When chickpea lectin mRNA level was checked in developing seeds, it was higher in 10 DAF seeds and decreased throughout seed development.     

Jaburetox-2Ec: an insecticidal peptide derived from an isoform of urease from the plant Canavalia ensiformis

Canatoxin, a urease isoform from Canavalia ensiformis seeds, shows insecticidal activity against different insect species. Its toxicity relies on an internal 10 kDa peptide (pepcanatox), released by hydrolysis of Canatoxin by cathepsins in the digestive system of susceptible insects. In the present work, based on the N-terminal sequence of pepcanatox, we have designed primers to amplify by PCR a 270-bp fragment corresponding to pepcanatox using JBURE-II cDNA (one of the urease isoforms cloned from C. ensiformis, with high identity to JBURE-I, the classical urease) as a template. This amplicon named jaburetox-2 was cloned into pET 101 vector to obtain heterologous expression in Escherichia coli of the recombinant protein in C-terminal fusion with V-5 epitope and 6-His tag. Jaburetox-2Ec was purified on Nickel-NTA resin and bioassayed in insect models. Dysdercus peruvianus larvae were fed on cotton seed meal diets containing 0.01% (w/w) Jaburetox-2Ec and, after 11 days, all individuals were dead. Jaburetox-2Ec was also tested against Spodoptera frugiperda larvae and caused 100% mortality. In contrast, high doses of Jaburetox-2Ec were innocuous when injected or ingested by mice and neonate rats. Modeling of Jaburetox-2Ec, in comparison with other peptide structures, revealed a prominent beta-hairpin motif consistent with an insecticidal activity based on either neurotoxicity or cell permeation.     

The gene for the major cuticular wax-associated protein and three homologous genes from broccoli (Brassica oleracea) and their expression patterns

The gene for the major protein (WAX9) found in surface wax of broccoli, designated wax9D , and three homologous genes ( wax9A, B and C ) were isolated from a genomic library using the previously isolated cDNA encoding the WAX9 protein as the probe; all four genes were sequenced. Genomic Southern blot analysis using the WAX9 cDNA as a probe showed the presence of at least four homologous genes in broccoli genome. The sequence of the originally isolated WAX9 cDNA matched with that of gene D . All four genes have an intron two codons before the stop codon. The putative promoter regions of the four genes, beyond the first 200 bp immediately 5' to the translation start sites, are quite different. Essential elements such as TATA and CAAT boxes and several regions homologous to the promoter regions of other plant ltp genes were identified. The expression patterns of the genes were determined by RT-PCR with gene-specific primers and sequencing of the PCR products. All the genes were expressed in leaves and flower buds. While genes A, B and D also were expressed in stems and open flowers, expression of gene C was not detected in these organs. None of them were expressed in roots. The 972 bp 5'-flanking region of wax9D when fused to β-glucuronidase (GUS) gene, directed tissue-specific GUS expression in transgenic tobacco plants; GUS expression was found in the epidermis of leaves, stems and flower petals, sepals, trichomes, and ovules but not in roots.     

Isolation and expression analysis of LEA genes in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Late embryogenesis abundant (LEA) protein family is a large protein family that includes proteins accumulated at late stages of seed development or in vegetative tissues in response to drought, salinity, cold stress and exogenous application of abscisic acid. In order to isolate peanut genes, an expressed sequence tag (EST) sequencing project was carried out using a peanut seed cDNA library. From 6258 ESTs, 19 LEA-encoding genes were identified and could be classified into eight distinct groups. Expression of these genes in seeds at different developmental stages and in various peanut tissues was analysed by semi-quantitative RT-PCR. The results showed that expression levels of LEA genes were generally high in seeds. Some LEA protein genes were expressed at a high level in non-seed tissues such as root, stem, leaf, flower and gynophore. These results provided valuable information for the functional and regulatory studies on peanut LEA genes.     

麝香百合LLGLO1基因的克隆和表达

用RACE方法克隆的麝香百合花发育的GLOBOSA（GLO）类B功能基因LLGLO1,与其他多种单子叶植物的GLO类基因高度同源,且C区具有典型的PI结构基序。通过RT—PCR检测,百合不同组织中的LLGLO1基因表达模式与郁金香的GLO类基因相似。即主要集中在百合第一、二、三轮花器官中表达,心皮和茎中有微量表达,而且随着心皮的成熟,其在心皮中的表达量逐渐增加,但在百合叶片中则未检测到LLGLO1的表达,因此认为LLGLO1在百合花器官中呈特异性表达。LLGLO1在百合第一轮花器官中的表达支持了van Tunen对ABC模型的修正。     

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.     

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。