Subtractive Library Research Articles

Identification and characterization of novel NF-kB dependent genes involved in HTLV-I pathogenesis

The NF-kB transcription factor plays pivotal roles in the pathogenesis and therapy-resistance of human cancers, including adult T-cell leukemia (ATL) induced by the oncoretrovirus HTLV-I. However, the downstream target genes of NF-kB involved in cancer biology and therapy remain largely unknown. To address this important issue, we have developed a novel approach called subtraction-based complementary gene expression cloning strategy. Given the characteristic anti-apoptosis activity of cancer cells, we used this approach to identify NF-kB-dependent anti-apoptotic genes involved in HTLV-I oncogenesis. The principle of this strategy is that expression of anti-apoptotic genes induced by HTLV-I-activated NF-kB should protect normal T cells from apoptosis induced by death inducers such as FasL. Briefly, a subtractive cDNA retroviral library enriched in genes induced by HTLV-I-NF-kB was generated and used to infect FasL-sensitive T cells. The infected T cells were treated with FasL and G418 (selective marker of cDNA expression). The FasL-and G418-resistant clones were isolated by limiting dilution, and the functional genes involved in FasL-resistance were fished out by RT-PCR and DNA sequencing. Using this strategy, several known NF-kB-dependent apoptotic genes have been identified, such as IAP1, Bcl-xL, c-FLIP and DcR2, indicating the reliability of our approach. Notably, numerous novel NF-kB-dependent anti-apoptotic genes were also identified. One of these novel genes has been confirmed to be expressed highly in HTLV-I-transformed T cells and primary ATL cells, and can be induced in normal T cells by HTLV-I in an NF-kB-dependent manner. Our mechanistic studies further indicate that this novel protein binds to mitochondria and prevents FasL activation of Bid, Caspase 9 and Caspase 3 but not Caspase 8. Currently, we are actively investigating the pathophysiological role of this novel gene in the biology and therapy of ATL and other cancers associated with deregulated NF-kB.

Identification of hydrogen peroxide responsive ESTs involved in phenylethanoid glycoside biosynthesis in Cistanche salsa cell culture

Hydrogen peroxide is an effective abiotic elicitor that can induce secondary metabolite biosynthesis in plants. We show that in cell suspension culture of a salt-tolerant medicinal plant Cistanche salsa, the production of bioactive components phenylethanoid glycosides (PeGs) was increased after an H2O2 treatment. To identify genes related to PeGs biosynthesis affected by H2O2, we constructed a suppression subtractive hybridization library of H2O2 responsive genes using a C. salsa cell line and identified 105 expressed sequence tags (ESTs) and 85 genes. EST library functional annotation and gene ontology analyses showed genes related to various stress responses, biosynthesis of secondary metabolites, and transcriptional regulation. Among them we identified two genes related to the PeGs biosynthesis pathway (4-coumarate coenzyme A ligase and cinnamate 4-hydroxylase), and two WRKY type transcription factors. The expressions of selected genes after the H2O2 treatment were analyzed by RT-qPCR. An early increased transcription of PeG biosynthesis pathway genes after the treatment revealed that H2O2 induced PeGs biosynthesis via up-regulation of its key genes.

Identification of Theobroma cacao genes differentially expressed during Phytophthora megakarya infection

Cocoa black pod caused by Phytophthora is one of the most serious diseases affecting cocoa production. This project set out was to improve our knowledge of the molecular mechanisms involved in Theobroma cacao resistance. We performed a transcriptional screening using macroarrays containing 89 ESTs from subtractive libraries enriched with genes differentially expressed in response to T. cacao/Phytophthora interactions. A gene expression analysis revealed the induction of genes involved in stress signal transduction, general and specific defence genes and genes involved in hormone signalling pathways. RT-qPCR showed that the level expression of two genes in the resistant plant was higher during the first eight hours after pathogen inoculation. Three genes from a family encoding a protease inhibitor displayed different levels of expression depending on resistant and susceptible T. cacao clones. A comparison of promoter sequences revealed a motif present only in genes that were up-expressed in the resistant plant.

Production and secretion of naphthoquinones is mediated by the MFS transporter MFS1 in the entomopathogenic fungus Ophiocordyceps sp. BCC1869.

Naphthoquinones are deep red polyketide pigments produced by the ant-pathogenic fungus Ophiocordyceps sp. BCC1869. In culture, biosynthesis of these naphthoquinones remains at a low level during the first 20 days and reaches its maximum production level at approximately 50 days. The MFS transporter gene MFS1 was previously identified in Ophiocordyceps sp. BCC1869 from a subtractive EST library between the fungus grown under naphthoquinone-inductive and naphthoquinone-repressive conditions. We cloned and sequenced this transporter gene, which has an open reading frame of 1505 bp and three introns (48, 52, and 58 bp). Phylogenetic analysis showed this MFS transporter was tightly clustered with fungal riboflavin transporters. Functional analysis of this gene was performed by overexpression of MFS1 under the control of a strong, constitutive promoter. We successfully transformed the fungus with this overexpression plasmid using PEG-protoplast transformation, which generated nine transformants per µg of plasmid. RT-PCR indicated that the MFS1 expression level in the overexpressing strains increased 3- to 10-fold compared to the wild type. HPLC analysis of crude extracts of mutants and wild type demonstrated that four naphthoquinone derivatives, erythrostominone, epierythrostominol, deoxyerythrostominone, and deoxyerythrostominol, were the major naphthoquinones produced and excreted in staggering quantities (20- to 2300-fold) in 7-day old liquid cultures by the mutant C7, compared to the wild type. High resolution electrospray ionization mass spectrometry verified mass spectra of these purified metabolites. Three other naphthoquinone derivatives, whose structures have not been identified, were also detected in high amount in the mutant liquid cultures.

Analysis of a suppressive subtractive hybridization library of Alternaria alternata resistant to 2-propenyl isothiocyanate

Background: Isothiocyanates (ITCs) are natural products obtained from plants of the Brassicas family. They represent an environmentally friendly alternative for the control of phytopathogenic fungi. However, as it has been observed with synthetic fungicides, the possibility of inducing ITC-resistant strains is a major concern. It is, therefore, essential to understanding the molecular mechanisms of fungal resistance to ITCs. We analyzed a subtractive library containing 180 clones of an Alternaria alternata strain resistant to 2-propenyl ITC (2-pITC). After their sequencing, 141 expressed sequence tags (ESTs) were identified using the BlastX algorithm. The sequence assembly was carried out using CAP3 software; the functional annotation and metabolic pathways identification were performed using the Blast2GO program. Results: The bioinformatics analysis revealed 124 reads with similarities to proteins, involved in transcriptional control, defense and stress pathways, cell wall integrity maintenance, detoxification, organization and cytoskeleton destabilization; exocytosis, transport, DNA damage control, ribosome maintenance, and RNA processing. In addition, transcripts corresponding to enzymes as oxidoreductases, transferases, hydrolases, lyases, and ligases, were detected. Degradation pathways for styrene, aminobenzoate, and toluene were induced, as well as the biosynthesis of phenylpropanoid and several types of N-glycan. Conclusions: The fungal response showed that natural compounds could induce tolerance/resistance mechanisms in organisms in the same manner as synthetic chemical products. The response of A. alternata to the toxicity of 2-pITC is a sophisticated phenomenon including the induction of signaling cascades targeting a broad set of cellular processes. Whole-transcriptome approaches are needed to elucidate completely the fungal response to 2-pITC. Normal 0 21 false false false ES X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Calibri,sans-serif;}

Isolation of differentially expressed sex genes in garden asparagus using suppression subtractive hybridization.

Garden asparagus (Asparagus officinalis L.) is a dioecious species whose male and female flowers are found in separate unisexual individuals. A region called the M-locus, located on a pair of homomorphic sex chromosomes, controls sexual dimorphism in asparagus. To date, no sex determining gene has been isolated from asparagus. To identify more genes involved in flower development in asparagus, subtractive hybridization library of male flowers in asparagus was constructed by suppression subtraction hybridization. A total of 107 expressed sequence tags (ESTs) were identified. BLASTX analysis showed that the library contained several genes that could be related to flower development. The expression patterns of seven selected genes believed to be involved in the development of asparagus male flower were further analyzed by semi-quantitative or real-time reverse-transcription polymerase chain reaction (RT-PCR). Results showed that AOEST4-5, AOEST12-40, and AOEST13-38 were strongly expressed in the male flower stage, whereas no transcript level of AOEST13-38 was detected in the female flower stage. The expression levels of AOEST13-87, AOEST13-92, AOEST13-40, and AOEST18-87 in the male flower stage were also higher than those in the female flower stage, although these transcripts were also expressed in other tissues. The identified genes can provide a strong starting point for further studies on the underlying molecular differences between the male and female flowers of asparagus.

Construction and analysis of an Oryza sativa (cv. MR219) salinity-related cDNA library

To identify salt-responsive genes, we constructed a cDNA subtractive library from a salt-tolerant rice cultivar, MR219. Treatments of the whole plant were carried out with 150 mM sodium chloride for 0, 2, 4, 8, 12 and 24 h. The extraction of total RNA from 14-day-old seedlings was conducted on the shoots and roots of both treated and untreated plants. The treated plants were utilized as ‘drivers’ while the untreated plants were utilized as ‘testers’. Equal amounts of the extracted total RNA from the 12 samples were pooled together and subjected to mRNA isolation. A reverse transcription of tester and driver cDNAs from the mRNA of two pools of the samples was conducted. In total, 300 clones were sequenced and analysed using bioinformatics tools. The 260 high-quality sequences were assembled and 83 contigs and 42 singletons were generated, producing a total of 125 UTs. The majority of the UTs showed significant homology with sequences in the NCBI database. A total of 330 gene ontology terms were grouped into three categories, of which 144 UTs fell under biological process, 108 fell under cellular component and 78 fell under molecular function. Also, RT-PCR was utilized to substantiate the elevation in expression of six candidate genes ((MR219SAP8 (JZ532324) and salT (JZ532363), low molecular weight heat shock (JZ532334), phospholipid hydroperoxide glutathione (JZ532371), Acetamidase (JZ532408), Ferredoxin-1 and chloroplastic (JZ532374)) subjected to salinity stress at varying timepoints.

Gas1 is a pleiotropic regulator of cellular functions: from embryonic development to molecular actions in cancer gene therapy.

Cellular homeostasis is governed by a precise regulation of the molecular mechanisms of action of several proteins in a given time. There is a group of proteins that have a particular role depending on the cellular context in which they are present and are known as pleiotropic proteins. The Gas1 (Growth Arrest Specific 1) gene was isolated from a subtraction library from serum arrested versus growing NIH3T3 mouse fibroblast. Gas1 is a member of the alpha receptors (GFRα) for the family of GDNF ligands (GFL), we have previously shown that Gas1 acts as a negative modulator of the GDNF-induced intracellular signaling and induces cell arrest and apoptosis. This modulating activity is the cause of the capacity of Gas1 to act as a tumor suppressor. On the other hand, several studies have shown the interaction between Gas1 and Hh (Hedgehog) proteins to potentiate the positive regulation of this pathway, which is involved in the development of the nervous system, and in both the origin and progression of different tumors. This review summarizes our current understanding of the structure of Gas1 and the molecular mechanism of action in different cellular functions, both during embryonic development, in the adult and its effects inhibiting cell growth and inducing apoptosis of cancer cells.

Identification of genes differentially expressed in husk tomato (Physalis philadelphica) in response to whitefly (Trialeurodes vaporariorum) infestation

Plants respond to phloem-feeding whiteflies by extensive changes in gene expression. To identify differentially expressed genes in husk tomato plants (Physalis philadelphica) infested with Trialeurodes vaporariorum, young plants were challenged with adult whiteflies, and forward and reverse subtractive libraries were constructed from infested leaves at 5 and 15 days after infestation. Several genes were identified as up-regulated; these included a diversity of genes involved in plant defense responses, protein synthesis or degradation, and cell wall fortification or modification. Genes required for amino acid biosynthesis, lipid metabolism and synthesis, including cell surface components such as suberin, responses to stress, photosynthesis and other functions, were similarly induced. Down-regulated genes were also identified, most prominently kinases and aquaporin genes. Similarities in defense responses between tomato and P. philadelphica were noted regarding the expression of certain genes in response to nematode, aphid, or whitefly. A role for abscisic acid, brassinosteroids, and cytokinins in the regulated response to whitefly infestation in P. philadelphica was also implied by the expression pattern of phytohormone-associated genes, including genes coding for proteins containing F-box motifs. Differential expression of selected genes was validated by quantitative real-time PCR. The possible role played by some of these genes during whitefly infestation is discussed.

Construction and analysis of a suppression subtractive hybridization library of regeneration-related genes in soybean.

The development of a genetic transformation system is needed to address the problem of the low efficiency associated with soybean regeneration. To contribute to the enhancement of the soybean regenerative capacity, we explored the developmental mechanisms of soybean regeneration at the molecular level using a suppression subtractive hybridization cDNA library constructed from cotyledonary nodes of soybean cultivar DN50. A total of 918 positive clones were identified and screened, with most inserted fragments ranging from 100 to 750 bp. Of these, 411 differentially expressed functional expressed sequence tags were identified and annotated based on their similarity to orthologs and paralogs detected in GenBank using the nucleotide and translated nucleotide Basic Local Alignment Search Tools. Functional analysis revealed that the associated genes were involved in signal transduction, synthesis, and metabolism of macromolecules, glucose and protein synthesis and metabolism, light and leaf morphogenesis, regulation of apoptosis, cell defense, cell wall differentiation, and a variety of hormone and cytokinin-mediated signaling pathways. The information uncovered in our study should serve as a foundation for the establishment of an efficient and stable genetic transformation system for soybean regeneration.

Comparative transcript profiling of gene expression between self-incompatible and self-compatible mandarins by suppression subtractive hybridization and cDNA microarray

Self-incompatibility (SI) is an important trait of Citrus plants that is exploited by farmers to produce seedless fruit. However, the molecular mechanism of SI in Citrus is not well understood. Wuzishatangju (Citrus reticulata Blanco) (SI) is an excellent seedless cultivar selected from a seedy Shatangju cultivar (self-compatible, SC) through spontaneous bud mutation. The two cultivars are therefore excellent materials for studying the mechanisms of SI and/or SC in Citrus. In this study, an integrative strategy combining eight suppression subtractive hybridization libraries with cDNA microarray was used to study the molecular mechanisms that differ between Wuzishatangju and Shatangju (control) mandarins. A custom microarray screen resulted in a total of 1,830 up- or down-regulated clones (false discovery rate <0.05 and a fold change $${ \geqq }$$ 2) obtained from 9,810 positive clones. The expression of genes involved in embryonic development, ubiquitination pathway, Ca2+-signaling pathway, gibberellins, and auxin was significantly up-regulated in SI Wuzishatangju compared with SC Shatangju mandarin. The microarray analysis suggested that the ubiquitin-mediated proteolysis pathway might be involved in the SI reaction of Wuzishatangju. Additionally, our research highlighted some main genes (mitogen-activated protein kinase, SI S1 family protein, ubiquitin-conjugating factor E4-like, auxin transporter protein 1, and gibberellin receptor) that participate in the SI reaction of Wuzishatangju and could be beneficial for understanding the evolution of SI systems and for breeding seedless citrus fruits in the future.

Eleven &lt;i&gt;StOSMs&lt;/i&gt; Genes in the Potato Genome Response to Water Deficiency

Osmotin plays an important role in plant response to low temperature and pathogen infection . To date l ittle is known on existence and full function of OSM genes in genome-wide. I n this study, based on an OSM expression sequence tag identified from a drought-treated subtractive library, StOSMs in the potato genome were identified and characterized in response to a water deficit gradient. BLAST and bioinformatic analysis revealed that there are at least eleven StOSMs in the potato genome . Among eleven StOSMs ,eight StOSM mRNAs accumulated in leaf at the drought-lethal critical point (DLP ) were at least 4-fold higher than in the control. The peak StOSM- 8E mRNA accumulation was 49-fold higher than the control. Three StOSM mRNAs in leaf at DLP were 9.2-f old lower than in the control. This result of qRT-PCR quantification was identical with the FPKM values of all eleven StOSMs examined in the the Potato Genome Sequence database (PGSD) . In conclusion, water deficiency does induce the regulation of the expression of the eleven StOSMs. Either up or downregulated by water deficiency, StOSM s play a positive role in enhancing potato tolerance to drought stress. Therefore, osmotins can be considered to be drought responsive molecules involved in withstanding water deficits. The result provides an evidence to reveal how potato adapts to drought stress.

A novel mitochondrial membrane protein, Ohmm, limits fungal oxidative stress resistance and virulence in the insect fungal pathogen Beauveria bassiana.

The Hog1 mitogen-activated protein (MAP) kinase regulates environmental stress responses and virulence in the entomopathogenic fungus Beauveria bassiana. To further characterize this pathway, we constructed a subtraction library enriched for genes regulated by Hog1. One targeted gene, encoding a novel membrane protein, Ohmm (oxidative homeostasis membrane-protein-mitochondria), was uniquely identified as being downregulated in the ΔHog1 background during growth under non-stress and osmotic stress conditions, but upregulated under oxidative stress. Ohmm was an experimentally validated flavin-binding protein and targeted to the mitochondria. Deletion of Ohmm resulted in increased oxidative stress resistance, whereas overexpression caused an opposite phenotype. The ΔOhmm showed accumulation of reactive oxygen species with alterations in cell wall composition and compatible solute accumulation evident as compared with the wild type parent. Conidiation was reduced > 80%; however, conidia produced by the ΔOhmm strain germinated significantly faster than wild type cells. Insect bioassays using the greater wax moth revealed increased virulence for the ΔOhmm strain in both topical and intrahemocoel injection assays, indicating a negative effect of the presence of Ohmm with respect to pathogenesis. As predicted from our characterization, deletion of Ohmm in a ΔHog1 background rescued its oxidative sensitivity phenotype, confirming that Ohmm acts downstream of the Hog1 MAP-kinase.

Characterization of expressed sequence tags from Lilium longiflorum in vernalized and non-vernalized bulbs

In Lilium longiflorum, vernalization is both an obligatory requirement and the major factor affecting flowering time, however, little is known about the molecular regulation of this mechanism in Lilium and other flowering bulbs. Exposure of L. longiflorum bulbs to 9 weeks at 4°C greatly promoted stem elongation within the bulb, floral transition and flowering. Subtraction libraries of vernalized (V) and non-vernalized (NV) bulb meristems were constructed. 671 and 479 genes were sequenced, from which 72 and 82 proteins were inferred for the NV–V and the V–NV libraries, respectively. Much lower transcription levels and putative gene functions were recorded in the NV–V libraries compared the V–NV libraries. However, a large number of genes annotated to transposable elements (TEs), represented more than 20% of the sequenced cDNA were expressed in the NV–V libraries, as opposed to less than 2% in the V–NV libraries. The expression profile of several genes potentially involved in the vernalization pathway was assessed. Expression of LlSOC1, the lily homologue of SUPPRESSOR OF OVER-EXPRESSION OF CO1 (SOC1), an important flowering gene in several plant species, found in the V–NV library, was highly up-regulated during bulb meristem cold exposure. The subtraction libraries provided a fast tool for relevant gene isolation.

Comparative transcript profiling of gene expression of fresh and frozen–thawed bull sperm

Although frozen semen is widely used commercially in the cattle breeding industry, the resultant pregnancy rate is lower than that produced using fresh semen. Cryodamage is a major problem in semen cryopreservation; it causes changes to sperm transcripts that may influence sperm function and motility. We used suppression subtractive hybridization technology to establish a complementary DNA subtractive library, and combined microarray technology and sequence homology analysis to screen and analyze differentially expressed genes in the library, comparing fresh sperm with the frozen–thawed sperm of nine bulls. Overall, 19 positive differentially expressed unigenes were identified using microarray data and Significance Analysis of Microarrays software (|score (d)| ≥ 2, fold change > 1, and false discovery rate < 0.05). Of 15 differentially expressed unigenes exhibited high sequence homology (E-value ≤ 1 × 10−3), 12 were upregulated in frozen–thawed sperm, the remaining 3 were upregulated in fresh sperm, and 4 other clones were identified as unknown because of incomplete sequences or because there was no significant sequence homology (E-value > 1E−03) and were considered novel genes. The expression of five of these genes—RPL31, PRKCE, PAPSS2, PLP1, and R1G7—was verified by quantitative real-time reverse transcription–polymerase chain reaction. There was a significant differential expression of the RPL31 gene (P < 0.05). Our preliminary results provide an overview of differentially expressed transcripts between fresh and frozen–thawed sperm of Holstein bulls.

Comparative renal gene expression in response to abrupt hypoosmotic shock in spotted scat (Scatophagus argus)

Scatophagus argus, a euryhaline fish, is notable for its ability to tolerate a wide range of environmental salinities and especially for its tolerance to a rapid, marked reduction in salinity. Therefore, S. argus is a good model for studying the molecular mechanisms mediating abrupt hyperosmoregulation. The serum osmotic pressure decreased steeply within one hour after transferring S. argus from seawater (SW) to freshwater (FW) and remained at new balance throughout the duration of one week. To explain this phenomenon and understand the molecular responses to an abrupt hypoosmotic shock, hypoosmotic stress responsive genes were identified by constructing two suppression subtractive hybridization (SSH) cDNA libraries from the kidneys of S. argus that had been transferred from SW to FW. After trimming and blasting, 52 ESTs were picked out from the subtractive library. Among them, 11 genes were significantly up-regulated (p<0.05). The kinetics studies of gene expression levels were conducted for 1week after the transfer using quantitative real-time PCR. A significant variation in the expression of these genes occurred within 12h after the hypoosmotic shock, except for growth hormone (GH) and polyadenylate binding protein 1 (PBP1), which were significantly up-regulated 2days post-transfer. Our results suggest different functional roles for these genes in response to hypoosmotic stress during the stress response phase (1hpt–12hpt) and stable phase (12hpt–7dpt). Furthermore, the plasma growth hormone level was detected to be significantly elevated at 1hpt and 24hpt following abrupt hypoosmotic shock. Meanwhile, several hematological parameters, hemoglobin (HGB), red blood cell (RBC) and mean cellular hemoglobin concentration (MCHC), were observed to be significantly increased at 12hpt and 2dpt compared with that of control group. Our results provide a solid basis from which to conduct future studies on the osmoregulatory mechanisms in the euryhaline fish.

Identification of differentially expressed genes related to metabolic syndrome induced with high-fat diet in E3 rats.

Understanding the genes differentially expressing in aberrant organs of metabolic syndrome (MetS) facilitates the uncovering of molecular mechanisms and the identification of novel therapeutic targets for the disease. This study aimed to identify differentially expressed genes related to MetS in livers of E3 rats with high-fat-diet-induced metabolic syndrome (HFD-MetS). E3 rats were fed with high-fat diet for 24 weeks to induce MetS. Then, suppression subtractive hybridization (SSH) technology was used to identify the genes differentially expressed between HFD-MetS and control E3 rat livers. Twenty positive recombinant clones were chosen randomly from forward subtractive library and sent to sequence. BLAST analysis in GenBank database was used to determine the property of each cDNA fragment. In total, 11 annotated genes, 3 ESTs, and 2 novel gene fragments were identified by SSH technology. The expression of four genes (Alb, Pip4k2a, Scd1, and Tf) known to be associated with MetS and other five genes (Eif1, Rnase4, Rps12, Rup2, and Tmsb4) unknown to be relevant to MetS was significantly up-regulated in the livers of HFD-MetS E3 rats compared with control rats using real-time quantitative PCR (RT-qPCR). By analyzing the correlations between the expression of these nine genes and serum concentrations of TG, Tch, HDL-C, and LDL-C, we found that there were significant positive correlations between TG and the expression of five genes (Alb, Eif1, Pip4k2a, Rps12, and Tmsb4x), Tch and three genes (Rnase4, Scd1, and Tmsb4x), and LDL-C and two genes (Rnase4 and Scd1), as well there were significant negative correlations between HDL-C and the expression of three genes (Rup2, Scd1, and Tf). This study provides important clues for unraveling the molecular mechanisms of MetS.

Identification of a cold-inducible gene encoding calmodulin-binding protein from Eucalyptus dunnii through suppression subtractive hybridization

Low temperature is the main limiting factor for cultivation expansion, fast growth, and high yield of Eucalyptus species. To investigate the mechanism of their cold tolerance, a cDNA subtraction library representing the cold-induced genes of Eucalyptus dunnii was constructed using suppression subtractive hybridization (SSH) technique. A gene encoding a calmodulin-binding protein (CaMBP) was identified from the SSH library, and the expression pattern of CaMBP under cold stress was further evaluated through reverse transcription — quantitative polymerase chain reaction (RT-qPCR). The expressions of EguCBF1a and EdCaMBP increased already after 4 h of the cold stress, supporting the idea that the CaMBP gene may have a function in the survival of Eucalyptus during winter. A full-length mRNA sequence of 1 808 bp was obtained via a rapid amplification of cDNA ends method, and the sequence was subsequently deposited to GenBank (accession No. JX401571). The CaMBP cDNA of E. dunnii contained a single open reading frame of 1 362 bp, a 5t’ untranslated region of 175 bp, and a 3t’ untranslated region of 268 bp. Multiple sequence alignment and phylogenetic analysis indicated that CaMBP of E. dunnii shared 56 to 84 % identities with the CaMBPs of other plants and was similar to that of Ricinus communis, Medicago truncatula, and Gossypium hirsutum.

Cloning and expression of a tau class glutathione S-transferase (ZjGSTU1) from Chinese jujube in response to phytoplasma infection

Phytoplasmas are associated with diseases of several hundred plant species. Jujube witches’ broom disease (JWB) is a destructive phytoplasma disease in Chinese jujube (Ziziphus jujuba Mill.). Ziziphus jujuba Mill. ‘J5’, an excellent strain with extremely high resistance to JWB, was selected by us. In our previous study, a GST (EC 2.5.1.18) fragment was sequenced from suppression subtractive hybridization library of ‘J5’ under JWB phytoplasma stress. Based on this result, a GST gene (ZjGSTU1, HM345954) was first isolated from jujube by homology cloning and RACE. ZjGSTU1 contains a complete open reading frame of 702 bp encoding a protein of 233 amino acid residues. Sequence alignment showed that ZjGSTU1 shared a typical conserved structure and high identity with tau GSTs from other plant species. Relative RT-PCR demonstrated that the expression of ZjGSTU1 mRNA in jujube leaves and branches could be triggered by JWB phytoplasma. Moreover, the expression of ZjGSTU1 mRNA in resistant strain ‘J5’ increased sooner and higher than that in sensitive strain ‘J9’ under JWB phytoplasma stress (‘J5’ and ‘J9’ are two strains from the same cultivar). Western blotting analyses showed that the expressions of ZjGSTU1 in ‘J5’ and ‘J9’ were dramatically up-regulated under JWB phytoplasma stress and its expression in ‘J5’ was also higher than that in ‘J9’ at protein level. Collectively, this paper highlights that ZjGSTU1 gene is responsive to phytoplasma infection. The possible roles of this gene were discussed in terms of regulatory process in resistance to phytoplasma infection.

Identification of Genes Involved in the Biosynthesis of Tripterygium wilfordii Hook.f. Secondary Metabolites by Suppression Subtractive Hybridization

Tripterygium wilfordii Hook.f. (Celastraceae) produces a vast array of natural products, mainly terpenes and sesquiterpene pyridine alkaloids, which have strong biological activities. The limited gene information on the biosynthesis pathways restricts the metabolic engineering of this valuable natural resource. In this study, a suppression subtractive hybridization library of methyl jasmonate (MeJA)-elicited T. wilfordii Hook.f. adventitious root was successfully constructed. To clone the genes involved in the downstream of biosynthetic pathways, a long elicitation time of 12 days was applied. After evaluating the subtraction efficiency through polymerase chain reaction and reverse northern hybridization, 818 clones were randomly sequenced. Basic Local Alignment Search Tool (BLAST) analysis showed that 568 of the 735 successfully expressed sequence tags were annotated. Overrepresentation of gene ontology analysis indicated that the suppression subtractive hybridization library enriched genes that are related to biotic and abiotic stimuli. After BLAST and phylogenetic analyses, 11 genes were found to be putatively involved in the biosynthesis of terpene and sesquiterpene pyridine alkaloid, and nine transcription factors were putatively involved in modulation. The expression modulations of these promising genes were further examined after short MeJA treatment. This study provides valuable information for further research on the production improvement of terpene and sesquiterpene pyridine alkaloids through biotechnology methods.