Purification and characterization of potato ribonuclease P

Ribonuclease P (RNase P), a ribonucleo-protein endoribonuclease, responsible for 5′ maturation of precursor tDNA, is well characterized in bacteria, yeast and human, but not in plant. Attempt has been made to partially purify and characterize nuclear RNase P from potato. cDNAs encoding two putative protein subunits of potato ribonuclease P (RNase P), StPop5 and StRpp25, were picked from potato EST library based on homology with respective human RNase P protein subunits. Both the cDNAs, 435 bp long StPop5 and 765 bp long StRpp25, were RT-PCR amplified, cloned and sequenced. StPop5 exhibited 46 % nucleotide sequence similarity to the hPop5 sequence. The deduced amino acid sequence of StPop5 had 23 % identity and 35 % similarity to hPop5. Both hRpp25 and StRpp25 had 46 % nucleotide sequence homology, and 17 % identity and 27 % similarity at a length of 271 amino acids. The molecular masses of purified 6× His-tagged recombinant StPop5 and StRpp25 proteins were 18 kDa and 33 kDa respectively. Potato nuclear RNase P was partially purified from leaves employing DEAE-Sephacel anion-exchange chromatography, and from floral buds employing DEAE-Sephacel and HiTrap Q anion-exchange chromatography, ammonium sulfate precipitation and gel filtration chromatography. Immunoprecipitation with polyclonal antisera, raised against recombinant StPop5 and StRpp25, demonstrated association of these two proteins with floral bud RNase P activity but not with leaf RNase P activity.     

Isolation and Characterization of the PKAr Gene From a Plant Pathogen,Curvularia lunata

By using EST database from a full-length cDNA library of Curvularia lunata, we have isolated a 2.9 kb cDNA, termed PKAr. An ORF of 1,383 bp encoding a polypeptide of 460 amino acids with molecular weight 50.1 kDa, (GeneBank Acc. No. KF675744) was cloned. The deduced amino acid sequence of the PKAr shows 90 and 88 % identity with cAMP-dependent protein kinase A regulatory subunit from Alternaria alternate and Pyrenophora tritici-repentis Pt-1C-BFP, respectively. Database analysis revealed that the deduced amino acid sequence of PKAr shares considerable similarity with that of PKA regulatory subunits in other organisms, particularly in the conserved regions. No introns were identified within the 1,383 bp of ORF compared with PKAr genomic DNA sequence. Southern blot indicated that PKAr existed as a single copy per genome. The mRNA expression level of PKAr in different development stages were demonstrated using real-time quantitative PCR. The results showed that the level of PKAr expression was highest in vegetative growth mycelium, which indicated it might play an important role in the vegetative growth of C. lunata. These results provided a fundamental supporting research on the function of PKAr in plant pathogen, C. lunata.     

A novel Glycine soja tonoplast intrinsic protein gene responds to abiotic stress and depresses salt and dehydration tolerance in transgenic Arabidopsis thaliana

Tonoplast intrinsic protein (TIP) is a subfamily of the aquaporin (AQP), also known as major intrinsic protein (MIP) family, and regulates water movement across vacuolar membranes. Some reports have implied that TIP genes are associated with plant tolerance to some abiotic stresses that cause water loss, such as drought and high salinity. In our previous work, we found that an expressed sequence tag (EST) representing a TIP gene in our Glycine soja EST library was inducible by abiotic stresses. This TIP was subsequently isolated from G. soja with cDNA library screening, EST assembly and PCR, and named as GsTIP2;1. The expression patterns of GsTIP2;1 in G. soja under low temperature, salt and dehydration stress were different in leaves and roots. Though GsTIP2;1 is a stress-induced gene, overexpression of GsTIP2;1 in Arabidopsis thaliana depressed tolerance to salt and dehydration stress, but did not affect seedling growth under cold or favorable conditions. Higher dehydration speed was detected in Arabidopsis plants overexpressing GsTIP2;1, implying GsTIP2;1 might mediate stress sensitivity by enhancing water loss in the plant. Such a result is not identical to previous reports, providing some new information about the relationship between TIP and plant abiotic stress tolerance.     

2-Cys peroxiredoxin responds to low temperature and other cues in Caragana jubata,a plant species of cold desert of Himalaya

A 2-Cys peroxiredoxin cDNA (CjPrx) was isolated and characterized from Caragana jubata, a temperate/alpine plant species of high altitude cold desert of Himalaya and Eurasia. The cDNA obtained was 1,064 bp long consisting of an open reading frame of 789 bp encoding 262 amino acids. The calculated molecular mass of the mature protein was 28.88 kDa and pI was 5.84. Deduced amino acid sequence of CjPrx shared a high degree homology with 2-CysPrx proteins from other plants. CjPrx had both the PRX_type 2-Cys domain and thioredoxin-like superfamily domains. CjPrx contained 26.72 % α-helices, 6.87 % β-turns, 20.61 % extended strands and 45.80 % random coils, and was a hydrophilic protein. Expression of CjPrx was modulated by low temperature, methyl jasmonate (MJ), salicylic acid and drought stress, but no significant change was observed in response to abscisic acid treatment. Among all the treatments, a strong up-regulation of CjPrx was observed in response to MJ treatment.     

Over-expression of tobacco <Emphasis Type="Italic">UBC1</Emphasis> encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>)

Ubiquitin (Ub)-conjugating enzyme (UBC, E2) receives Ub from Ub-activating enzyme (E1) and transfers it to target proteins, thereby playing a key role in Ub/26S proteasome-dependent proteolysis. UBC has been reported to be involved in tolerating abiotic stress in plants, including drought, salt, osmotic and water stresses. To isolate the genes involved in Cd tolerance, we transformed WT (wild-type) yeast Y800 with a tobacco cDNA expression library and isolated a tobacco cDNA, NtUBC1 (Ub-conjugating enzyme), that enhances cadmium tolerance. When NtUBC1 was over-expressed in tobacco, cadmium tolerance was enhanced, but the Cd level was decreased. Interestingly, 20S proteasome activity was increased and ubiquitinated protein levels were diminished in response to cadmium in NtUBC1 tobacco. By contrast, proteasome activity was decreased and ubiquitinated protein levels were slightly enhanced by Cd treatment in control tobacco, which is sensitive to Cd. Moreover, the oxidative stress level was induced to a lesser extent by Cd in NtUBC1 tobacco compared with control plants, which is ascribed to the higher activity of antioxidant enzymes in NtUBC1 tobacco. In addition, NtUBC1 tobacco displayed a reduced accumulation of Cd compared with the control, likely due to the higher expression of CAX3 (Ca²⁺/H⁺ exchanger) and the lower expression of IRT1 (iron-responsive transporter 1) and HMA-A and -B (heavy metal ATPase). In contrast, atubc1 and atubc1atubc2 Arabidopsis exhibited lower Cd tolerance and proteasome activity than WT. In conclusion, NtUBC1 expression promotes cadmium tolerance likely by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or the Ub-independent 20S proteasome and by diminishing oxidative stress through the activation of antioxidant enzymes and decreasing Cd accumulation due to higher CAX3 and lower IRT1 and HMA-A/B expression in response to 50 µM Cd challenge for 3 weeks.     

Isolation and characterization of a wheat IF2 homolog required for innate immunity to stripe rust

Key message

The wheat eIF2 homolog, TaIF2, is induced by the stripe rust pathogen CYR 32 at an early stage of inoculation and is related to the innate immunity resistance level in wheat.

Abstract

The initiation of translation represents a critical control point in the regulation of gene expression in all organisms. We previously identified an upregulated EST S186 (EL773056) from an SSH-cDNA library of the Shaanmai 139 strain of wheat (Triticum aestivum) infected with Puccinia striiformis (Pst). In the present work, we isolated a cDNA clone and identified it as a wheat IF2 homolog. This cDNA consisted of 1,314 nucleotides and contained an open reading frame of 795 nucleotides encoding a polypeptide of 254 amino acids. The amino acids represent a conserved domain in EF-Tu, mtIF2-II, and mtIF2-Ivc. The alignment result showed that it maybe a partial cDNA of the initiation factor 2/eukaryotic initiation factor 5B (IF2/eIF5B) superfamily gene. Paradoxically, results of a Swiss-model analysis suggesting a low QMEAN Z-score implied that it was a membrane protein. Quantitative RT-PCR studies confirmed that the wheat eIF2 (TaIF2) homolog was differentially expressed in three near-isogenic lines. Critical time points for the induction of resistance by inoculation with Pst CYR32 in YrSM139-1B + YrSM139-2D immune resistance genotype occurred at 1 and 3 dpi (days post-infection). RNAi test showed that the inoculated BSMV-IF2 leaves of Shaanmai 139 showed obvious cell death after 15 days of inoculation with CYR 32. qRT-PCR analysis of the target gene in cDNA samples isolated from BSMV-IF2-Pst, BSMV-0-Pst and Pst infected leaves confirmed that the expression of TaIF2 is suppressed by BSMV-IF2 at 3 dpi. This suggested that TaIF2/eIF5B plays an important role in the mechanism of innate immunity to stripe rust pathogen.     

Identification of candidate SNPs for drug induced toxicity from differentially expressed genes in associated tissues

The full-length cDNA sequence (1158 bp) encoding a ribosomal L5 protein, designated as TaL5, was firstly isolated from common wheat (Triticum aestivum L.) using the rapid amplification of cDNA ends method (RACE). The open reading frame (ORF) of TaL5 gene was 906 bp, and its deduced amino acid sequence (301 residues) shared high similarity to those of other higher plant L5 proteins. TaL5 protein contained a putative 5S binding region (74 amino acids). TaL5 DNA sequence was further cloned, and sequence analysis showed that it contained 7 introns and 8 exons. Predicated using TargetP software, TaL5 protein was putatively located in mitochondria and contains a transit peptide of 12 amino acids. During grain filling period, temporal expression pattern of TaL5 gene was approximately consistent with the rates of starch accumulation in grains. Additionally, TaL5 gene was dramatically induced by salt, drought and freezing stresses, exogenous abscisic acid (ABA) and salicylic acid (SA) in wheat seedlings. These implied that TaL5 gene could function in growth, development and abiotic stresses in wheat plants.     

Characterization of Squalene synthase Gene from Chlorophytum borivilianum (Sant. and Fernand.)

Saponins are important group of secondary metabolites known for their pharmacological properties. Chlorophytum borivilianum contains high amount of saponins and is thus, recognized as an important medicinal plant with aphrodisiac properties. Though the plant is well known for its pharmaceutical properties, there is meager information available about the genes and enzymes responsible for biosynthesis of saponins from this plant. Squalene synthase (SqS) is the key enzyme of saponin biosynthesis pathway and here, we report cloning and characterization of SqS gene from C. borivilianum. A full-length CbSqS cDNA consisting of 1,760 bp was cloned which contained an open reading frame (ORF) of 1,233 bp, encoding a protein of 411 amino acids. Analysis of deduced amino acid sequence of CbSqS predicted the presence of conserved isoprenoid family domain and catalytic sites. Phylogenetic analysis revealed that CbSqS is closer to Glycine max and monocotyledonous plants. 3D structure prediction using various programs showed CbSqS structure to be similar to SqS from other species. C-terminus truncated recombinant squalene synthase (TruncCbSqS) was expressed in E. coli M15 cells with optimum expression induced with 1 mM IPTG at 37 °C. The gene expression level was analyzed through semi-quantitative RT-PCR and was found to be higher in leaves as compared to the roots.     

Cloning and characterisation of a phenylalanine ammonia-lyase gene from Rhus chinensis

Key message

The gene and cDNA sequence encoding PAL from Chinese medicinal plant Rhus chinensis were cloned and analyzed, furthermore the biochemical properties, kinetic parameters, differential expression and key sites were studied.

Abstract

Rhus chinensis is a well-known Chinese medicinal plant. Phenylalanine ammonia-lyase (PAL) is the first enzyme of phenylpropanoid pathway. Several recent studies suggested that PAL also play an important role in plant–aphid interaction. In this study, both the cDNA and the genomic sequence encoding PAL from Rhus chinensis (designated as RcPAL) were cloned and analyzed. The 3,833 bp gene contained a 1,342 bp intron and two extrons. The ORF was 2,124 bp and predicted to encode a 707-amino acid polypeptide. The results of real-time PCR showed that RcPAL expressed in all tested tissues and followed the order: stems > young leaves > petioles > roots > seeds > mature leaves. RcPAL was successfully expressed in E. coli with the pET-28a-RcPAL recombinant vector. The recombinant protein exhibited a high level of PAL activity. Biochemical properties and kinetic parameters of recombinant RcPAL were further studied. The results showed that the optimal temperature and pH for RcPAL activity were 45 °C and 9.0, and the K _m and K _cat values were 7.90 mM and 52.31 s^?1, respectively. The active sites and substrate selectivity site were also investigated with site-directed mutagenesis methods, suggesting that Phe¹²⁶ is responsible for the substrate selectivity. To our knowledge, this was the first full-length PAL gene cloned and characterized from the family Anacardiaceae so far.     

Molecular cloning and expression analysis of a squalene synthase gene from a medicinal plant, Euphorbia pekinensis Rupr.

Euphorbia pekinensis Rupr., which is also known as a medicinal plant, produces a large amount of alkaloids, phytosterols and triterpenes. In this study, we reported on the cDNA cloning and characterization of a novel squalene synthase (SQS) from E. pekinensis. Squalene synthase catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to produce squalene (SQ), the first committed precursor for sterol and triterpene biosynthesis. The full length cDNA named EpSQS (Genbank Accession Number JX509735) contained 1,614 bp with an open reading frame of 1,236 bp encoding a polypeptide of 411 amino acids. The deduced amino acid sequence of the EpSQS named EpSQS exhibited a high homology with other plant SQSs, and contained a single domain surrounded by helices. Phylogenetic analysis showed that EpSQS belonged to the plant SQS kingdom. Tissue expression analysis revealed that EpSQS expressed strongly in roots, weakly in stems and leaves, implying that EpSQS was a constitutive expression gene. The recombinant protein was expressed in Escherichia coli and detected by SDS-PAGE and western blot. The high performance liquid chromatography (HPLC) analysis showed that EpSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene.