Cloning and characterization of HbMT2a, a metallothionein gene from Hevea brasiliensis Muell. Arg differently responds to abiotic stress and heavy metals.

Metallothioneins (MTs) are of low molecular mass, cysteine-rich proteins. They play an important role in the detoxification of heavy metals and homeostasis of intracellular metal ions, and protecting against intracellular oxidative damages. In this study a full-length cDNA of type 2 plant metallothioneins, HbMT2a, was isolated from 25 mM Polyethyleneglycol (PEG) stressed leaves of Hevea brasiliensis by RACE. The HbMT2a was 372bp in length and had a 237bp open reading frame (ORF) encoding for a protein of 78 amino acid residues with molecular mass of 7.772 kDa. The expression of HbMT2a in the detached leaves of rubber tree clone RY7-33-97 was up-regulated by Me-JA, ABA, PEG, H2O2, Cu(2+) and Zn(2+), but down-regulated by water. The role of HbMT2a protein in protecting against metal toxicity was demonstrated in vitro. PET-28a-HbMT2-beared Escherichia coli. Differential expression of HbMT2a upon treatment with 10 °C was observed in the detached leaves of rubber tree clone 93-114 which is cold-resistant and Reken501 which is cold-sensitive. The expression patterns of HbMT2a in the two rubber tree clones may be ascribed to a change in the level of endogenous H2O2.

Molecular and biochemical characterization of a cyanogenic ß-glucosidase in the inner bark tissues of rubber tree (Hevea brasiliensis Muell. Arg.).

Tapping causes the loss of large amounts of latex from laticifers and subsequently enhances latex regeneration, a high carbon- and nitrogen-cost activity in rubber tree. It is suggested that a 67 kDa protein associated with protein-storing cells in the inner bark tissues of rubber tree plays an important role in meeting the nitrogen demand for latex regeneration. Here, the 67 kDa protein was further characterized by a combination of cell biological, molecular biological and biochemical techniques. Immunogold labeling showed that the 67 kDa protein was specifically localized in the central vacuole of protein-storing cells. A full-length cDNA, referred to as HbVSP1, was cloned. The HbVSP1 contained a 1584 bp open reading frame encoding a protein of 527 amino acids. The putative protein HbVSP1 shared high identity with the P66 protein from rubber tree and proteins of the linamarase, and bg1A from cassava (Manihot esculenta). HbVSP1 contained the active site sequences of ß-glucosidase, TFNEP and I/VTENG. In vitro analysis showed that the 67 kDa protein exhibited the activity of both ß-glucosidase and linamarase and was thus characterized as a cyanogenic ß-glucosidase. Proteins immuno-related to the 67 kDa protein were present in leaves and lutoids of laticifers. Tapping down-regulated the expression of HbVSP1, but up-regulated the expression of genes encoding the key enzymes for rubber biosynthesis, while the effect of resting from tapping was the reverse. Taken together, the results suggest that the 67 kDa protein is a vacuole-localized cyanogenic ß-glucosidase encoded by HbVSP1 and may have a role in nitrogen storage in inner bark tissues of trunk during the leafless periods when rubber tree is rested from tapping.

Isolation and analysis of differentially expressed genes from peanut in response to challenge with Ralstonia solanacearum

Background: Bacterial wilt caused by Ralstonia solanacearum is the most devastating disease in peanut. Planting resistant peanut cultivars is deemed as the sole economically viable means for effective control of the disease. To understand the molecular mechanism underlying resistance and facilitate breeding process, differences in gene expression between seeds of Rihua 1 (a Virginia type peanut variety resistant to bacterial wilt) inoculated with the bacterial pathogen suspension (10(9) cfu ml-1) and seeds of the same cultivar treated with water (control), were studied using the GenefishingTM technology. Results: A total of 25 differentially expressed genes were isolated. Expression of genes encoding cyclophilin and ADP-ribosylation factor, respectively, were further studied by real time RT-PCR, and full length cDNAs of both genes were obtained by rapid amplification of cDNA ends. Conclusions: The study provided candidate genes potentially useful for breeding peanut cultivars with both high yield and bacterial wilt resistance, although confirmation of their functions through transgenic studies is still needed.

Characterization of HbEREBP1, a wound-responsive transcription factor gene in laticifers of Hevea brasiliensis Muell. Arg.

AP2/ERF transcription factors play an important role in regulation of the cross-talk between ethylene and jasmonate signaling pathways mediating defense responses of plants to biotic and abiotic stresses. In this study, an AP2/ERF transcription factor gene was isolated and characterized from laticifers of rubber tree by using RACE and real time PCR. The full length cDNA, referred to as HbEREBP1, was 1,095 bp in length and contained a 732 bp open reading frame encoding a putative protein of 243 amino acid residues. The molecular mass of the putative protein is 26.4 kDa with a pI of 9.46. The deduced amino acid sequence had a specific domain of AP2 superfamily and an ethylene-responsive element binding factor-associated amphiphilic repression motif, sharing 42.4, 39.1, and 38.0% identity with that of AtERF11, AtERF4, and AtERF8 in Arabidopsis, respectively. HbEREBP1 expression was down-regulated by tapping and mechanical wounding in the laticifers of adult trees. It was also down-regulated at early stage while up-regulated at late stage upon treatment with exogenous ethephon or methyl jasmonate, which was reverse to the case of defense genes in laticifers of epicormic shoots of rubber tree. Our results suggest that HbEREBP1 may be a negative regulator of defense genes in laticifers.

Identification of chilling-responsive transcripts in peanut (Arachis hypogaea L. )

To isolate differentially expressed peanut genes responsive to chilling, a suppression subtractive hybridization (SSH) cDNA library was constructed for a chilling tolerant peanut cultivar A4 with mRNAs extracted from the seeds imbibed at 2ºC and 15ºC, respectively, for 24 hrs. A total of 466 cDNA clones were sequenced, from which 193 unique transcripts (73 contigs and 120 singlets) were assembled. Of these unique transcripts, 132 (68.4 percent) were significantly similar to the sequences in GenBank non-redundant (nr) protein database, which belonged to diverse functional categories including metabolism, signal transduction, stress response, cell defense and transcriptional regulation. The remaining 61 (31.6 percent) showed no similarity to either hypothetical or known proteins. Six differentially expressed transcripts were further confirmed with real-time quantitative PCR (RT-qPCR).

MYC genes with differential responses to tapping, mechanical wounding, ethrel and methyl jasmonate in laticifers of rubber tree (Hevea brasiliensis Muell. Arg.).

MYC2 transcription factor is a key component of the core module COI1-JAZ-MYC2 of jasmonate signaling in Arabidopsis, but the MYC transcription factor (s) associated with jasmonate signaling in jasmonate-responsive laticifer cells remains to be identified. Two full-length cDNAs, designated HblMYC1 and HblMYC2, were isolated from laticifer cells in Hevea brasiliensis by the method of RACE. HblMYC1 contained 1431bp ORF encoding a putative protein of 476 amino acids while HblMYC2 contained 1428bp ORF encoding a putative protein of 475 amino acids. Bioinformatic analysis showed that the putative proteins, HblMYC1 and HblMYC2, possessed a bHLH domain and were most related to the MYC2 among the selected 27 MYC members with identified functions in Arabidopsis. In addition to the presence of cis-regulatory elements involving jasmonate responsiveness in the promoter regions of HblMYC1 and HblMYC2, the abscisic acid-, salicylic acid- and gibberellin-responsive elements were found in the promoter region of HblMYC1. Transcripts of HblMYC1 and HblMYC2 were most abundant in latex, relatively low in male flowers and nearly undetected in bark tissues and roots by real-time RT-PCR analysis. Regular tapping, mechanical wounding, and ethrel remarkably up-regulated HblMYC1 expression, but had little effect on the expression of HblMYC2 in laticifer cells. Successive tapping, however, significantly down-regulated the expression of HblMYC2 while up-regulating the expression of HblMYC1. The HblMYC2 expression took a mutual ebb and flow relationship with the HblMYC1 expression upon treatment with methyl jasmonate. Characterization of HblMYC1 and HblMYC2 will contribute to the understanding of jasmonate signaling in laticifiers, a kind of specialized tissue for natural rubber biosynthesis in Hevea brasiliensis.

Sodium azide mutagenesis resulted in a peanut plant with elevated oleate content

Screening of peanut seeds resulting from 0.39 percent sodium azide treatment with NIRS calibration equation for bulk seed samples identified a plant with more than 60 percent oleate. Oleate content in individual seeds of the plant, as predicted by NIRS calibration equation for intact single peanut seeds, ranged from 50.05 percent ~ 68.69 percent. Three seeds with >60 percent oleate thus identified were further confirmed by gas chromatography. Multiple sequence alignments of the FAD2B gene from Huayu 22 (wild type) and peanut seeds with elevated oleate (mutant type) revealed a C281T transition in the coding region causing an I94T substitution in the oleoyl-PC desaturase, which may be responsible for reduction in the enzyme activity.

Novel protocol to identify true hybrids in normal oleate x high oleate crosses in peanut

A novel hybrid identification protocol was developed for F0:1 peanut seeds resulting from crosses between normal oleate cultivars with wild type FAD2B gene and high oleate genotypes with an A insertion in FAD2B gene. Presence of a series of overlapped peaks in trace file of the PCR product amplified with bF19/R1 primers was an indication of hybridity. This protocol may facilitate high oleate breeding and genetic studies in peanut.

Simple method to prepare DNA templates from a slice of peanut cotyledonary tissue for polymerase chain reaction

An efficient DNA extraction method was developed for peanut seed, where only 3-5 mg cotyledonary tissue was enough for more than 50 PCR reactions with a reaction volume of 15 ul. Both low copy number and high copy number DNA sequences were successfully amplified. Processing one seed sample only took about half an hour. Sampling had no significant effects on germination and development. The DNA extraction method makes it possible to identify transformants and conduct molecular marker studies prior to sowing, and thus may greatly hasten research progress.

A rapid and cheap protocol for preparation of PCR templates in peanut

This paper describes a simple, low cost and reliable DNA template preparation protocol for polymerase chain reaction (PCR) using immature leaves from peanut seeds or leaves from field-grown plants. The technique may find wide utility in studies involving PCR-based molecular markers, rapid screening for transformants and gene cloning.