The formation and accumulation of protein-networks by physical interactions in the rapid occlusion of laticifer cells in rubber tree undergoing successive mechanical wounding.

BACKGROUND: Although the wound response of plants has been extensively studied, little is known of the rapid occlusion of wounded cell itself. The laticifer in rubber tree is a specific type of tissue for natural rubber biosynthesis and storage. In natural rubber production, tapping is used to harvest the latex which flows out from the severed laticifer in the bark. Therefore, study of the rapid wound-occlusion of severed laticifer cells is important for understanding the rubber tree being protected from the continuously mechanical wounding. RESULTS: Using cytological and biochemical techniques, we revealed a biochemical mechanism for the rapid occlusion of severed laticifer cells. A protein-network appeared rapidly after tapping and accumulated gradually along with the latex loss at the severed site of laticifer cells. Triple immunofluorescence histochemical localization showed that the primary components of the protein-network were chitinase, ß-1,3-glucanase and hevein together with pro-hevein (ProH) and its carboxyl-terminal part. Molecular sieve chromatography showed that the physical interactions among these proteins occurred under the condition of neutral pH. The interaction of ß-1,3-glucanase respectively with hevein, chitinase and ProH was testified by surface plasmon resonance (SPR). The interaction between actin and ß-1,3-glucanase out of the protein inclusions of lutoids was revealed by pull-down. This interaction was pharmacologically verified by cytochalasin B-caused significant prolongation of the duration of latex flow in the field. CONCLUSIONS: The formation of protein-network by interactions of the proteins with anti-pathogen activity released from lutoids and accumulation of protein-network by binding to the cytoskeleton are crucial for the rapid occlusion of laticifer cells in rubber tree. The protein-network at the wounded site of laticifer cells provides not only a physical barrier but also a biochemical barrier to protect the wounded laticifer cells from pathogen invasion.

[Clinical features and treatment of macrolide-resistant Mycoplasma pneumoniae pneumonia in children].

OBJECTIVE: To study the clinical features of macrolide-resistant Mycoplasma pneumoniae pneumonia and its treatment regimens in children. METHODS: The samples of throat swab or bronchoalveolar lavage fluid were collected from 136 children with Mycoplasma pneumoniae pneumonia. Quantitative real-time PCR was used to detect 2063/2064 A:G mutation in 23S rRNA, and according to such results, the children were divided into drug-resistance group with 81 children and sensitive group with 55 children. The two groups were compared in terms of age composition, respiratory symptoms, extrapulmonary complications, laboratory markers, imaging changes, treatment regimens, and length of hospital stay. RESULTS: Compared with the sensitive group, the drug-resistance group had significantly longer duration of pyrexia and severe fever, a significantly higher percentage of children with reduced blood oxygen saturation, and significantly higher levels of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) (P<0.05). The conventional azithromycin treatment had a good clinical effect in the sensitive group, while corticosteroid therapy was usually needed in the drug-resistance group. CONCLUSIONS: Macrolide-resistant Mycoplasma pneumoniae infection cannot be identified based on a single clinical feature, but prolonged duration of pyrexia and severe fever, reduced blood oxygen saturation, and increased ALT and LDH can suggest the presence of this disease. Azithromycin combined with glucocorticoids may be a good treatment regimen for children with macrolide-resistant Mycoplasma pneumoniae pneumonia.

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.

Comparative transcriptome analysis of latex from rubber tree clone CATAS8-79 and PR107 reveals new cues for the regulation of latex regeneration and duration of latex flow.

BACKGROUND: Rubber tree (Hevea brasiliensis Muell. Arg.) is the primarily commercial source of natural rubber in the world. Latex regeneration and duration of latex flow after tapping are the two factors that determine rubber yield of rubber tree, and exhibit a huge variation between rubber tree clones CATAS8-79 and PR107. RESULTS: To dissect the molecular mechanism for the regulation of latex regeneration and duration of latex flow, we sequenced and comparatively analyzed latex of rubber tree clone CATAS8-79 and PR107 at transriptome level. More than 26 million clean reads were generated in each pool and 51,829 all-unigenes were totally assembled. A total of 6,726 unigenes with differential expression patterns were detected between CATAS8-79 and PR107. Functional analysis showed that genes related to mass of categories were differentially enriched between the two clones. Expression pattern of genes which were involved in latex regeneration and duration of latex flow upon successive tapping was analyzed by quantitative PCR. Several genes related to rubber biosynthesis, cellulose and lignin biosynthesis and rubber particle aggregation were differentially expressed between CATAS8-79 and PR107. CONCLUSIONS: This is the first report about probing latex regeneration and duration of latex flow by comparative transcriptome analysis. Among all the suggested factors, it is more important that the level of endogenous jasmonates, carbohydrate metabolism, hydroxymethylglutaryl-CoA reductase (HMGR) and Hevea rubber transferase (HRT) in mevalonate (MVA) parthway for latex regeneration while the level of endogenous ethylene (ETH), lignin content of laticifer cell wall, antioxidants and glucanases for the duration of latex flow. These data will provide new cues for understanding the molecular mechanism for the regulation of latex regeneration and duration of latex flow in rubber tree.

Comparative proteomics of primary and secondary lutoids reveals that chitinase and glucanase play a crucial combined role in rubber particle aggregation in Hevea brasiliensis.

Lutoids are specific vacuole-based organelles within the latex-producing laticifers in rubber tree Hevea brasiliensis. Primary and secondary lutoids are found in the primary and secondary laticifers, respectively. Although both lutoid types perform similar roles in rubber particle aggregation (RPA) and latex coagulation, they vary greatly at the morphological and proteomic levels. To compare the differential proteins and determine the shared proteins of the two lutoid types, a proteomic analysis of lutoid membranes and inclusions was performed, revealing 169 proteins that were functionally classified into 14 families. Biological function analysis revealed that most of the proteins are involved in pathogen defense, chitin catabolism, and proton transport. Comparison of the gene and protein changed patterns and determination of the specific roles of several main lutoid proteins, such as glucanase, hevamine, and hevein, demonstrated that Chitinase and glucanase appeared to play crucial synergistic roles in RPA. Integrative analysis revealed a protein-based metabolic network mediating pH and ion homeostasis, defense response, and RPA in lutoids. From these findings, we developed a modified regulation model for lutoid-mediated RPA that will deepen our understanding of potential mechanisms involved in lutoid-mediated RPA and consequent latex coagulation.

Genomic insight into domestication of rubber tree.

Understanding the genetic basis of rubber tree (Hevea brasiliensis) domestication is crucial for further improving natural rubber production to meet its increasing demand worldwide. Here we provide a high-quality H. brasiliensis genome assembly (1.58 Gb, contig N50 of 11.21 megabases), present a map of genome variations by resequencing 335 accessions and reveal domestication-related molecular signals and a major domestication trait, the higher number of laticifer rings. We further show that HbPSK5, encoding the small-peptide hormone phytosulfokine (PSK), is a key domestication gene and closely correlated with the major domestication trait. The transcriptional activation of HbPSK5 by myelocytomatosis (MYC) members links PSK signaling to jasmonates in regulating the laticifer differentiation in rubber tree. Heterologous overexpression of HbPSK5 in Russian dandelion (Taraxacum kok-saghyz) can increase rubber content by promoting laticifer formation. Our results provide an insight into target genes for improving rubber tree and accelerating the domestication of other rubber-producing plants.

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.

Molecular characterization of a novel 14-3-3 protein gene (Hb14-3-3c) from Hevea brasiliensis.

The cDNA encoding a 14-3-3 protein, designated as Hb14-3-3c, was isolated from Hevea brasiliensis. Hb14-3-3c was 1,269 bp long containing a 795 bp open reading frame encoding a putative protein of 264 amino acids, flanked by a 146 bp 5'UTR and a 328 bp 3' UTR. The predicted molecular mass of Hb14-3-3c is 29.67 kDa, with an isoelectric point of 4.52 and the deduced protein showed high similarity to the 14-3-3 protein from other plant species. Expression analysis revealed more significant accumulation of Hb14-3-3c transcripts in latex than in leaves, buds and flowers. The transcription of Hb14-3-3c in latex was induced by jasmonate and ethephon. Overproduction of recombinant Hb14-3-3c protein gave the Escherichia coli cells more tolerance on Co(2+), Cu(2+) and Zn(2+). Through yeast two-hybrid screening, 11 interaction partners of the Hb14-3-3c, which are involved in rubber biosynthesis, stress-related responses, defence etc., were identified in rubber tree latex. Taking these data together, it is proposed that the Hb14-3-3c may participate in regulation of rubber biosynthesis. Thus, the results of this study provide novel insights into the 14-3-3 signaling related to rubber biosynthesis, stress-related responses in rubber tree.

[Expression of RANTES in the lung tissue of asthmatic rats, and the intervention effect of vitamin D on RANTES expression].

OBJECTIVE: To investigate the effect of vitamin D on the expression of chemokine regulated on activation, normal T cells expressed and secreted (RANTES) in the lung tissue of asthmatic rats, and the role of vitamin D in the control of asthmatic airway inflammation and the synergistic action of hormones. METHODS: Forty female Wistar rats were randomly and equally divided into normal control, asthma, vitamin D intervention, budesonide intervention, and budesonide+vitamin D intervention groups. Hematoxylin and eosin staining was used to observe pathological changes in the lung tissue. Immunohistochemistry was used to measure the protein expression of RANTES in lung tissue. Enzyme-linked immunosorbent assay was used to measure the level of RANTES in bronchoalveolar lavage fluid (BALF). Real-time quantitative PCR was used to measure the mRNA expression of RANTES. RESULTS: The asthma group showed the most significant pathological changes in the lung tissue, including inflammatory cell infiltration, bronchial stenosis and distortion and smooth muscle rupture, while the intervention groups showed fewer pathological changes. Of the intervention groups, the budesonide intervention group showed fewer pathological changes than the vitamin D intervention group, and the budesonide+vitamin D intervention group showed the mildest pathological changes, which were similar to those observed in the normal control group. Protein expression of RANTES in the lung tissue and BALF was significantly higher in the asthma group than in the normal control group (P<0.05), while it was lower in the intervention groups than in the asthma group, exhibiting significant differences between each intervention group and the asthma group (P<0.05) (except the difference in protein expression of RANTES in BALF between the vitamin D intervention and asthma groups). The budesonide+vitamin D intervention group showed less protein expression of RANTES in the lung tissue and BALF than both the budesonide intervention and vitamin D intervention groups (P<0.05). The mRNA expression of RANTES was significantly higher in the asthma group than in the normal control group (P<0.05), while it was significantly lower in three intervention groups than in the asthma group (P<0.05), however no significant difference was found between the intervention groups in this regard. The budesonide+vitamin D intervention group showed the lowest level of RANTES mRNA, with no significant difference from the normal control group. CONCLUSIONS: The mRNA and protein expression of RANTES in BALF and lung tissue increases significantly in asthmatic rats. Vitamin D intervention can decrease the expression of RANTES, suggesting that vitamin D can reduce airway inflammation by regulating the expression of RANTES. Vitamin D can be used together with budesonide to further decrease the mRNA and protein expression of RANTES.

Three MADS-box genes of Hevea brasiliensis expressed during somatic embryogenesis and in the laticifer cells.

Three MADS-box genes, designated HbMADS1, HbMADS2 and HbMADS3, were isolated from Hevea brasiliensis. HbMADS1, HbMADS2 and HbMADS3 encode polypetides consisting of 245, 217 and 239 amino acids, respectively, containing conserved MADS-box motifs at N-terminus. Transcription pattern analysis revealed that three MADS-box genes had highly transcription in the laticifer cells. The transcriptions of HbMADS1and HbMADS3 were induced in the laticifer cells by jamonic acid, while HbMADS2 was not induction by jamonic acid. Ethephone is not effective in inducing their expression. The three genes were differentially expressed during somatic embryogenesis of rubber tree. Characterization of HbMADSs will attribute to understand their possible function in rubber tree.

Molecular characterization of a thioredoxin h gene (HbTRX1) from Hevea brasiliensis showing differential expression in latex between self-rooting juvenile clones and donor clones.

The cDNA code of thioredoxin h, designated as HbTRX1, was isolated from Hevea brasiliensis by rapid amplification of cDNA ends. HbTRX1 contained a 542-bp open reading frame encoding 123 amino acids. The deduced HbTRX1 protein showing high identity to thioredoxin h of other plant species was predicted to possess the conserved catalytic site WCXPC. Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that HbTRX1 was constitutively expressed in all tested tissues. HbTRX1 transcripts accumulated at relatively low levels in the flower, somatic embryo, and leaves, while HbTRX1 transcripts accumulated at relatively high levels in the callus and latex. The HbTRX1 transcript was expressed at different levels, with higher levels in self-rooting juvenile clones than in their donor clones. HbTRX1 was expressed in Escherichia coli, and its activity was demonstrated using the dithiothreitol-dependent insulin assay. This work provides a basis for studying the biological function of thioredoxin h in rubber tree.

The role and mechanism of 1,25-dihydroxyvitamin D3 in regulating the Rho-kinase signaling pathway in asthmatic rats.

BACKGROUND: Bronchial asthma (referred to as asthma in the present study) is the most common chronic airway inflammatory disease in childhood. The present study aimed to investigate the effect of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on VDR expression, which is closely associated with asthmatic airway smooth muscle cells (ASMCs), and explored its role and mechanism in the Rho-kinase signaling pathway. METHODS: The acute asthma model was induced by ovalbumin (OVA) and pertussis bacillus, and ASMCs obtained from asthmatic rats were cultured in vitro. These cells were randomly divided into five groups: control (N) group, TNF-α (TNF) group, 1,25-(OH)2D3 (VD) group, dexamethasone (DXM) group, and 1,25-(OH)2D3 + DXM (L) group. The protein expression levels of VDR, ROCK, MLC20 and P-MLC20 were detected by western blot, and the mRNA expression levels of VDR, ROCK, MLC20 and P-MLC20 were detected by real-time quantitative PCR. RESULTS: The expression of ROCK, MLC20 and P-MLC20 in each treatment group were significantly lower, when compared to the TNF group (P<0.05), but this remained stronger than (P<0.05) or similar to (P>0.05) that in the N group. CONCLUSIONS: The regulation mechanism of 1,25-(OH)2D3 in alleviating asthma should be correlated to its regulation of the expression of related signaling molecules in the Rho-kinase signaling pathway, and this effect may be achieved by regulating the mRNA and protein expression of the VDR gene.

Molecular characterization of HbCDPK1, an ethephon-induced calcium-dependent protein kinase gene of Hevea brasiliensis.

Calcium-dependent protein kinases (CDPKs), as major primary Ca(2+) sensors, have been implicated in the regulation of stress and developmental signals in plants. In this study, a novel CDPK gene, designated HbCDPK1, was isolated from Hevea brasiliensis. The HbCDPK1 cDNA had 2,400 bp with an open reading frame of 1,671 bp encoding 556 amino acids, and the deduced HbCDPK1 protein contained four characteristic domains identified in CDPKs, showing a high level of sequence similarity to CDPKs from other plants. Expression analysis revealed more significant accumulation of the transcripts of HbCDPK1 in latex than in the leaves, bark, and roots in H. brasiliensis. In addition, transcription of HbCDPK1 was strongly induced by mechanical wounding, jasmonic acid (JA), and ethephon. Recombinant HbCDPK1 was expressed in E. coli, and its activity was assayed. The assay indicated that HbCDPK1 had the kinase and Ca(2+)-binding activity in vitro as a calcium-dependent protein. The potential roles of the HbCDPK1 are discussed as to latex production and rubber biosynthesis.

Cloning and molecular characterization of HbCOI1 from Hevea brasiliensis.

The full-length cDNA encoding a coronatine insensitive-1 (COI1) protein, designated HbCOI1, was isolated for the first time from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbCOI1 contained a 2,187 bp open reading frame encoding 597 amino acids. The deduced HbCOI1 protein, which showed high identity to COI1 protein of other plant species, was predicted to possess F-box and LRRs domains. The promoter region of HbCOI1 was isolated by the PCR-based DNA walking method. TATA box and other core configurations were found in the promoter. Several sequences similar to the eukaryotic cis regulatory element were found in the 5'-untranslated region (UTR) proximal 5' flanking sequence of HbCOI1. Southern blot analysis indicated that the HbCOI1 is present as a single copy in Hevea brasiliensis. Transcription pattern analysis revealed that HbCOI1 had high transcription in laticifer, low in barks and leaf. Transcription of HbCOI1 in latex was induced by jasmonate and tapping.

Vegetative storage protein with trypsin inhibitor activity occurs in Sapindus mukorassi, a sapindaceae deciduous tree.

A vegetative storage protein (VSP) with trypsin inhibitor activity in a deciduous tree, Sapindus mukorassi, was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot, immuno-histochemical localization, light- and electro-microscopy, together with analysis of proteinase inhibitor activity of the purified VSP in vitro. There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S. mukorassi in leafless periods. The proteins decreased markedly during young shoot development, indicating their role in seasonal nitrogen storage. Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells. The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope. So, the 23 kDa protein was a typical VSP in S. mukorassi. The 23 and 27 kDa proteins shared no immuno-relatedness, whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity. The 23 kDa protein may confer dual functions: nitrogen storage and defense.

Genome-Wide Identification and Characterization of the JAZ Gene Family in Rubber Tree (Hevea brasiliensis).

Jasmonate signaling plays a vital role in the regulation of secondary laticifer differentiation and natural rubber biosynthesis in Hevea brasiliensis. Jasmonate ZIM-domain (JAZ) proteins are the master regulators of jasmonate signaling. Although several JAZs have been reported in the laticifer cells of H. brasiliensis, the genome-wide screening of HbJAZ members has not yet been explored. In the present study, 18 HbJAZs were identified based on the recent H. brasiliensis genome. Phylogenetic construction revealed that the HbJAZs were clustered into five subgroups and that members within the same subgroup shared highly conserved gene structures and protein motifs. Cis-element analysis of HbJAZ promoters suggested the presence of hormone, stress and development-related cis-elements. HbJAZ1.0, HbJAZ2.0, and HbJAZ5.0 interacted with CORONATINE INSENSITIVE1 (COI1) in the presence of coronatine (COR, a JA mimic). HbJAZ1.0, HbJAZ2.0, HbJAZ5.0, and HbJAZ12.0 could also interact with each other. Of the 18 HbJAZs, transcripts of 15 HbJAZs were present in the vascular cambium region except for that of HbJAZ7.0, HbJAZ8.0d, and HbJAZ13.0. Fourteen of the 15 HbJAZs were significantly up-regulated upon COR treatment. The transcripts of three genes that were absent from vascular cambium region were also absent from the latex. Among the 15 HbJAZs in the latex, the expression patterns of 13 HbJAZs were different between the tapping and ethrel treatments. Eight of the 14 COR-up-regulated HbJAZs in the vascular cambium region were also activated by tapping in latex. Of the eight tapping-activated HbJAZs, 5 HbJAZs were repressed by ethrel application. Based on the computational analyses and gene expression patterns described in this study, the HbJAZ5.0 and HbJAZ10.0b may be associated with laticifer differentiation while the HbJAZ8.0b is a negative regulator for natural rubber biosynthesis in H. brasiliensis.

Cloning and characterization of a novel cysteine protease gene (HbCP1) from Hevea brasiliensis.

The full-length cDNA encoding a cysteine protease,designated HbCP1, was isolated for the first time from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbCP1 contained a 1371 bp open reading frame encoding 457 amino acids.The deduced HbCP1 protein,which showed high identity to cysteine proteases of other plant species,was predicted to possess a putative repeat in toxin (RTX) domain at the N-terminal and a granulin (GRAN) domain at the C-terminal.Southern blot analysis indicated that the HbCP1 gene is present as a single copy in the rubber tree.Transcription pattern analysis revealed that HbCP1 had high transcription in laticifer,and low transcription in bark and leaf.The transcription of HbCP1 in latex was induced by ethylene and tapping.Cloning of the HbCP1 gene will enable us to further understand the molecular characterization of cysteine protease and its possible function in the rubber tree.

Vegetative storage protein in Litchi chinensis, a subtropical evergreen fruit tree, possesses trypsin inhibitor activity.

BACKGROUND AND AIMS: Vegetative storage proteins (VSPs) are commonly bioactive in herbaceous plants but few VSPs with bioactivity have been identified in trees. In addition, information on the characterization of VSPs in evergreen trees is limited. The objective of this study was to characterize the VSPs with bioactivity in evergreen trees. Methods The VSP in lychee (Litchi chinensis), an evergreen fruit tree, was characterized by a combination of cytological, biochemical and molecular biological techniques. KEY RESULTS: The VSP in lychee was a 22-kDa protein. It accumulated in the large central vacuoles of protein-storing cells (PSCs) in two distinguishable forms, granular and floccular. The PSCs were of a novel type. The 22-kDa protein is distributed in mature leaves, bark tissues of branches, trunk and large roots, paralleling the distribution of PSCs. Its homologues were present in mature seed. During young shoot development and fruiting, the 22-kDa protein decreased apparently, suggesting a nitrogen-storage function. The 22-kDa protein had several isoforms encoded by a small multigene family. One gene member, LcVSP1, was cloned. The LcVSP1 had no intron and contained a 675 bp open reading frame encoding a putative protein of 225 amino acids. LcVSP1 was homologous to Kunitz trypsin inhibitors. The 22-kDa protein inhibited trypsin and chymotrypsin, but had no inhibitory effect on subtilisin. CONCLUSIONS: Lychee is rich in a 22-kDa VSP with trypsin inhibitor activity. The VSP plays an important role in nitrogen storage while its possible defensive function remains to be elucidated.

Transcript Profiling of Hevea brasiliensis during Latex Flow.

Latex exploitation enhances latex regeneration in rubber trees. The latex exploitation-caused latex flow lasts from 10 min to a few hours, which is convenient for exploring the transcript profiling of latex metabolism-related genes at the different stages of latex flow. In the present study, the expression pattern of 62 latex metabolism-related genes involved in water transportation, carbohydrate metabolism, natural rubber biosynthesis, hormone signaling, ROS generation and scavenging, and latex coagulum across three stages of latex flow between rubber tree clones CATAS7-33-97 and CATAS8-79 were comparatively analyzed by quantitative real-time PCR. The two clones show differences in latex regeneration and have a different duration of latex flow. The results showed that the expression levels of 38 genes were significantly higher in CATAS8-79 latex than in CATAS7-33-97 during latex regeneration, while 45 genes had a notably higher expression level in CATAS8-79 latex during latex flow. Together with the activation of the MEP pathway and jasmonate pathway in CATAS8-79 latex, HbPIP1;3, HbPIP1;4, HbSUT3, HbSus3, HbHMGS1-2, HbMK should contribute to the high latex regeneration ability. The up-regulation of ethylene signaling and Hb44KD and the down-regulation of latex coagulation-related genes in CATAS8-79 latex might contribute to its longer latex flow duration. This study provides some cues for revealing the regulation of latex metabolism in rubber trees.

Cloning and molecular characterization of a RING zinc-finger gene of Hevea brasiliensis.

Using the information about the sequence from a differentially expressed clone (designated as HbSSH10) encodes a protein specifying a cysteine-rich sequence containing a putative "RING finger" or "C3HC4" consensus motif that was cloned recently by the subtractive hybridization between latex and leaves from rubber tree (Hevea brasiliensis). A full-length cDNA encoding C3HC4 type zinc-finger protein was isolated and characterized from rubber tree. Sequence analysis revealed that the ORFs of HbRZF encode 156 amino acid residues with a total predicted molecular mass of 17.2 kD, HbRZF protein having a putative "RING finger" segment (amino acid residues 100-144). The deduced amino acid sequences of HbRZF showed high identities of 48%, 52% and 50% to those of the ring zinc protein from Poncirus trifoliata, Arabidopsis thaliana, Thellungiella halophila. The result of Northern blot analysis indicated that the transcripts of the HbRZF were expressed more in the latex than in the leaves, whereas little expression was detected in roots and flowers. The transcription of HbRZF was induced by jasmonic acid, whereas ethylene had little effect.