Molecular characterisation and expression of two venom allergen-like protein genes in Heterodera glycines.

Secretory proteins encoded by genes expressed in the oesophageal gland cells of plant-parasitic nematodes have key roles in nematode parasitism of plants. Two venom allergen-like protein cDNAs (designated hg-vap-1 and hg-vap-2)were isolated from Heterodera glycines gland cell cDNA libraries. Both cDNAs hybridised to genomic DNA of H. glycines in Southern blots. The hg-vap-1 cDNA contained an open reading frame encoding 215 amino acids with the first 25 amino acids being a putative secretion signal. The hg-vap-2 cDNA contained an open reading frame encoding 212 amino acids with the first 19 amino acids being a putative secretion signal. Genes of hg-vap-1 and hg-vap-2 contained four introns, which ranged in size from 44 to 574 bp, and five exons ranging in size from 43 to 279 bp. In situ hybridisation analyses showed that mRNAs of both vap genes accumulated specifically in the subventral gland cells of H. glycines during parasitism. The gland cell-specific expression and presence of predicted secretion signal peptides in both VAPs suggest that these proteins are secreted from the nematode and may play a role in the infection of host plants by this parasite.     

Inter- and intra-specific cuticle variation between amphimictic and parthenogenetic species of root-knot nematode (Meloidogyne spp.) as revealed by a bacterial parasite (Pasteuria penetrans)

Specific host–parasite interactions exist between species and strains of plant parasitic root-knot nematodes and the Gram-positive bacterial hyperparasite Pasteuria penetrans. This bacterium produces endospores that adhere to the cuticle of migrating juveniles, germinate and colonise the developing female within roots. Endospore attachment of P. penetrans populations to second-stage juveniles of the root-knot nematode species Meloidogyne incognita and Meloidogyne hapla showed there were interactive differences between bacterial populations and nematode species. Infected females of M. incognita produced a few progeny which were used to establish two nematode lines from single infective juveniles encumbered with either three or 26 endospores. Single juvenile descent lines of each nematode species were produced to test whether cuticle variation was greater within M. hapla lines that reproduce by facultative meiotic parthenogenesis than within lines of M. incognita, which reproduces by obligate parthenogenesis. Assays revealed variability between broods of individual females derived from single second-stage juvenile descent lines of both M. incognita and M. hapla suggesting that progeny derived from a single individual can differ in spore adhesion in both sexual and asexual nematode species. These results suggest that special mechanisms that produced these functional differences in the cuticle surface may have evolved in both sexually and asexually reproducing nematodes as a strategy to circumvent infection by this specialised hyperparasite.     

Latent infection: a low temperature survival strategy in steinernematid nematodes

1. Entomopathogenic nematodes penetrate and kill Galleria mellonella within 48 h at optimal temperatures.

2. Low temperature induces infection latency, preventing host death until optimal conditions resume.

3. Infected Galleria survived 25 days at 5°C. On transfer to 25°C, 100% and 12.5% of Steinernema carpocapsae and Steinernema riobravis infected larvae died within 72 h.

4. Infective juvenile penetration decreased with decreasing temperature; declining from 49.7 and 49.3 nematodes/host at 25°C to 6.3 and 0.25 nematodes/host at 5°C for S. carpocapsae and S. riobravis, respectively.

5. Latent infection occurs, albeit infrequently, due to low host penetration at low temperature.

Molecular cloning and characterization of soybean peroxidase gene families

Plant peroxidases play major roles in many physiological processes. A soybean seedbud (21 days after flowering) Uni-ZAP XR cDNA library was screened with a peroxidase-specific probe. The probe was generated by 3′ rapid amplification of cDNA ends with soybean seedbud total RNA and a degenerate primer derived from a plant peroxidase conserved amino acid region (distal heme ligand). Positive clones were recovered by PCR using the degenerate peroxidase-specific primer and the vector primer T₇ flanking the cloning site. Four cDNAs, designated GmEpa1, GmEpa2, GmEpb1, and GmEpb2, contained 1298, 1326, 1171, and 1145 nucleotides, excluding poly(A) tail, and encoded mature proteins of 303, 303, 292, and 292 amino acids, respectively. The four predicted amino acid sequences showed homology to other peroxidases. GmEpa1 and GmEpa2 exhibited 97% amino acid identity, GmEpb1 and GmEpb2 exhibited 93% amino acid identity, and GmEpa1 and GmEpb1 exhibited 47% amino acid identity. GmEPa1 and GmEPb1 were expressed as fusion proteins in Escherichia coli. The recombinant fusion proteins were sequestered in inclusion bodies and active forms of the two denatured proteins were recovered after in vitro folding in a medium containing hemin, urea and Ca²⁺. GmEpa1 and GmEpa2 messages were detected in developing seed and root, while GmEpb1 and GmEpb2 messages were present in root, leaf, stem and seed pod. These cDNAs and cDNA-specific primers will allow investigations into peroxidase’s role in development, stress response and in other physiological processes.     

大豆向热带地区发展的遗传基础

大豆(Glycine max)是光周期敏感的植物,该特性是决定其生育期及其生态适应区的关键因素。温带的大豆品种引种到热带地区(短日照)时,开花期和成熟期提前、产量降低,限制了大豆在热带地区的种植。长童期(LJ)大豆品种的发现是解决该问题的重要突破。在短日照条件下,LJ品种比温带品种开花晚、体量大、成熟晚且产量提高。前期研究发现,J位点是控制LJ性状的关键位点。近期,我国科学家通过精细定位克隆了J基因,发现其与拟南芥(Arabidopsis thaliana)早花基因(ELF3)同源。他们通过功能互补和近等基因系等方法验证了J基因的功能,在短日照条件下,等位基因j比J开花晚、成熟晚且产量提高。进一步研究发现,J蛋白与E1基因(豆科植物开花抑制因子)的启动子结合抑制E1基因的表达,从而解除E1对大豆开花基因(FT)的抑制,促进大豆在短日照下开花。研究还发现在大豆种质资源中存在多种j等位变异。该研究引领了大豆生育期遗传研究的新方向,揭示了大豆向热带地区发展的遗传基础。     

Use of differential display for the identification of touch-induced genes from an ethylene-insensitive Arabidopsis mutant and partial characterization of these genes

Touch has been shown to affect plant growth and development and ethylene has been shown to have similar effects. However, the mechanisms responsible for touch-induced responses remain unclear. Differential display PCR was used to identify touch-regulated genes from 3-week-light-grown ethylene-insensitive etr1-3 Arabidopsis (Columbia ecotype) mutant plants. The differential display PCR screening process yielded 32 cDNA fragments. Subsequent screening of the 32 fragments using northern analysis yielded three touch-inducible clones (A8A, G5A and G7F). These three cDNA were then used to screen a cDNA library. A 1.2 kb fragment for OPR3 was obtained from A8A screenings. This cDNA fragment encodes 12-oxophytodienoate-10, 11-reductase (OPR), an enzyme in the jasmonic acid biosynthetic pathway. OPR3 was found to be induced by touch, wounding, methyl jasmonate (MeJA), NaCl and CaCl₂ while ethylene and darkness had no effect. A 2 kb cDNA encoding a calcium-dependent protein kinase (CDPK32) was obtained with G5A screenings. CDPK32 was shown to be induced by touch, wounding, NaCl and darkness while ethylene and MeJA had little or no effect. A 1.4 kb cDNA encoding a novel protein was recovered from the cDNA library screenings with a G7F fragment. This cDNA had some sequence similarity to GDA1 and was designated GDL for GDA1-like cDNA. GDL was activated by touch, wounding, MeJA, NaCl and CaCl₂ while there was no induction with ethylene and darkness. Using differential display PCR we have successfully been able to identify three clones that are inducible by touch and not by ethylene.     

棉铃虫c-Jun氨基末端激酶基因的克隆、表达谱及对UV-A胁迫的响应

【目的】本研究旨在克隆棉铃虫Helicoverpa armigera c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)基因,并对其进行序列和表达模式分析,探讨该基因在棉铃虫生长发育及响应UV胁迫方面的作用。【方法】利用RT-PCR与RACE技术克隆棉铃虫JNK基因,并利用生物信息学方法对其编码的氨基酸序列进行分析;采用实时荧光定量PCR技术检测其在棉铃虫不同发育阶段(卵、1-6龄幼虫、蛹、雌雄成虫)、成虫不同组织(去除触角和复眼的头、胸、腹、触角、复眼、足、翅、中肠、卵巢)中及雌成虫在UV-A照射不同时间(0, 30, 60, 90, 120和150 min)下的相对表达量变化。【结果】克隆获得一个棉铃虫JNK基因并命名为HaJNK(GenBank登录号:MH719009),其cDNA序列全长为2 431 bp,开放阅读框(ORF)长1 191 bp,编码396个氨基酸,编码蛋白质的相对分子量为45.01 kD,等电点为6.35,无跨膜结构,无信号肽。系统进化分析显示,棉铃虫HaJNK与其他昆虫JNK具有很高的同源性。发育阶段表达分析表明,HaJNK在棉铃虫卵期表达量最高;组织特异性分析显示该基因在成虫复眼、胸部及卵巢部位特异性表达。UV-A照射能诱导棉铃虫雌成虫体内HaJNK的表达,随着照射时间的延长,其表达量呈现先升高后降低的趋势,在照射60 min时表达量达到峰值。【结论】HaJNK在棉铃虫不同龄期、成虫不同组织和UV-A照射不同时间的雌成虫中差异表达,提示其在响应UV-A胁迫的分子机制中具有重要意义。     

Factors Affecting Disease Development by Rhynchosporium alismatis in Starfruit (Damasonium minus), a Weed of Rice

The fungal pathogen Rhynchosporium alismatis is being developed for biological control of starfruit (Damasonium minus), an important aquatic weed in Australian rice fields. The development of R. alismatis in starfruit differs between juvenile and adult plants. Juvenile starfruit plants are stunted as a result of fungal infection, while in adult plants, the main effect is necrosis and chlorosis of floating leaves. A conidial concentration of 1×10⁴ conidia mL^-1 was adequate to cause disease symptoms on floating leaves, but the stunting effect on juveniles was caused by concentrations of at least 1×10⁵ conidia mL^-1. To successfully inoculate juvenile plants, the water must be drained before inoculation to expose plants to the inoculum. The artificial addition of dew periods did not enhance disease development in plants. The stunting of juvenile starfruit plants caused by the infection of R. alismatis may give rice plants a competitive advantage over the weed at the seedling stage