Regulatory mechanism of GA3 application on grape (Vitis vinifera L.) berry size.

Gibberellin A3 (GA3) is often used as a principal growth regulator to increase plant size. Here, we applied Tween-20 (2%)-formulated GA3 (T1:40 mg/L; T2:70 mg/L) by dipping the clusters at the initial expansion phase of 'Red Globe' grape (Vitis vinifera L.) in 2018 and 2019. Tween-20 (2%) was used as a control. The results showed that GA3 significantly increased fruit cell length, cell size, diameter, and volume. The hormone levels of auxin (IAA) and zeatin (ZT) were significantly increased at 2 h (0 d) -1 d after application (DAA0-1) and remained significantly higher at DAA1 until maturity. Conversely, ABA exhibited an opposite trend. The mRNA and non-coding sequencing results yielded 436 differentially expressed mRNA (DE_mRNAs), 79 DE_lncRNAs and 17 DE_miRNAs. These genes are linked to hormone pathways like cysteine and methionine metabolism (ko00270), glutathione metabolism (ko00480) and plant hormone signal transduction (ko04075). GA3 application reduced expression of insensitive dwarf 2 (GID2, VIT_07s0129g01000), small auxin-upregulated RNA (SAUR, VIT_08s0007g03120) and 1-aminocyclopropane-1-carboxylate synthase (ACS, VIT_18s0001g08520), but increased SAUR (VIT_04s0023g00560) expression. These four genes were predicted to be negatively regulated by vvi-miR156, vvi-miR172, vvi-miR396, and vvi-miR159, corresponding to specific lncRNAs. Therefore, miRNAs could affect grape size by regulating key genes GID2, ACS and SAUR. The R2R3 MYB family member VvRAX2 (VIT_08s0007g05030) was upregulated in response to GA3 application. Overexpression of VvRAX2 in tomato transgenic lines increased fruit size in contrast to the wild type. This study provides a basis and genetic resources for elucidating the novel role of ncRNAs in fruit development.

Insights on the stem elongation of spur-type bud sport mutant of 'Red Delicious' apple.

MAIN CONCLUSION: The decreased capacity of auxin-, CTK-, and BR-mediated cell division and cell enlargement pathways, combined with the enhanced capacity of GA and ETH-, JA-, ABA-, SA-mediated stress-resistant pathways were presumed to be the crucial reasons for the formation of spur-type 'Red Delicious' mutants. Vallee Spur', which exhibit short internodes and compact tree shape, is the fourth generation of the spur-type bud sport mutant of 'Red Delicious'. However, the underlying molecular mechanism of these properties remains unclear. Here, comparative phenotypic, full-length transcriptome and phytohormone analyses were performed between 'Red Delicious' (NSP) and 'Vallee Spur' (SP). The new shoot internode length of NSP was ˃ 1.53-fold higher than that of the SP mutant. Cytological analysis showed that the stem cells of the SP mutant were smaller and more tightly arranged relative to the NSP. By Iso-Seq, a total of 1426 differentially expressed genes (DEGs) were detected, including 808 upregulated and 618 downregulated genes in new shoot apex with 2 leaves of the SP mutant. Gene expressions involved in auxin, cytokinin (CTK), and brassinosteroid (BR) signal transduction were mostly downregulated in the SP mutant, whereas those involved in gibberellin (GA), ethylene (ETH), jasmonate (JA), ABA, and salicylic acid (SA) signal transduction were mostly upregulated. The overall thermogram analysis of hormone levels in the shoot apex carrying two leaves detected by LC-MS/MS absolute quantification showed that the levels of IAA-Asp, IAA, iP7G, OPDA, and 6-deoxyCS were significantly upregulated in the SP mutant, while the remaining 28 hormones were significantly downregulated. It is speculated that the decreased capacity of auxin, CTK, and BR-mediated cell division and cell enlargement pathways is crucial for the formation of the SP mutant. GA and stress-resistant pathways of ETH, JA, ABA, and SA also play vital roles in stem elongation. These results highlight the involvement of phytohormones in the formation of stem elongation occurring in 'Red Delicious' spur-type bud sport mutants and provide information for exploring its biological mechanism.

Comprehensive genomic identification and expression analysis 4CL gene family in apple.

To clarify the structural characteristics, phylogeny, biological function and regulation of 4-coumarate-CoAligase (4CL) in anthocyanin synthesis, the 4CL gene family members in apples were identified and bioinformatic analysis was performed. qRT-PCR was used to analyze the expression levels of this gene family members in different apple varieties, and the role of the 4CL gene in apple anthocyanin synthesis was preliminaries clarified, which provided a certain theoretical basis for the regulatory network of apple anthocyanin synthesis. The results showed that a total of 69 members of the 4CL gene family were identified in the apple (Malus domestica Brokh.), encoding amino acids ranging from 97 to 2310 with theoretical isoelectric points ranging from 5.28 to 9.84. The 69 4CL family members were distributed on 17 chromosomes in the apple, among which chromosome 17 had the largest distribution (9 members), followed by chromosome 9 (7 members), chromosomes 16 and 14 (6 members each), and chromosomes 15 and 13 (5 members each). The subcellular localization prediction showed that apple 4CL gene family members were mainly expressed in cytoplasm, chloroplast, nucleus and cell membrane, with a small amount of expression in mitochondria, vacuoles, peroxisomes, cytoskeleton, golgi and cell matrix, but not in endoplasmic reticulum. The secondary structures are mainly α-helices and irregular coils. Microarray expression profile analysis showed that the expression levels of each member in apple were related to fruit variety and tissue structure, and the expression levels were mainly higher in fruit, flower and leaf. Real-time PCR analysis showed that the expression level of each member was directly proportional to the degree of fruit coloring and anthocyanin accumulation. The expression levels of Md4CL10 and Md4CL23 in 'Astar' (G4) apple fruit skin with the highest anthocyanin content were 516, 20 and 2 times higher than those in 'Chengji NO.1' (G1), 'Golden Delicious' (G2) and 'Ruixue' (G3), respectively.

Changes and response mechanism of sugar and organic acids in fruits under water deficit stress.

The content and the ratio of soluble sugars and organic acids in fruits are significant indicators for fruit quality. They are affected by multiple environmental factors, in which water-deficient is the most concern. Previous studies found that the content of soluble sugars and organic acids in fruit displayed great differences under varied water stress. It is important to clarify the mechanism of such difference and to provide researchers with systematic knowledge about the response to drought stress and the mechanism of sugar and acid changes in fruits, so that they can better carry out the study of fruit quality under drought stress. Therefore, the researchers studied dozens of research articles about the content of soluble sugar and organic acid, the activity of related metabolic enzymes, and the expression of related metabolic genes in fruits under water stress, and the stress response of plants to water stress. We found that after plants perceived and transmitted the signal of water deficit, the expression of genes related to the metabolism of soluble sugars and organic acids changed. It was then affected the synthesis of metabolic enzymes and changed their metabolic rate, ultimately leading to changes in soluble sugar and organic acid content. Based on the literature review, we described the pathway diagrams of sugar metabolism, organic acid metabolism, mainly malic acid, tartaric acid, and citric acid metabolism, and of the response to drought stress. From many aspects including plants' perception of water stress signal, signal conversion and transmission, induced gene expression, the changes in soluble sugar and the enzyme activities of organic acids, as well as the final sugar and acid content in fruits, this thesis summarized previous studies on the influence of water stress on soluble sugars and the metabolism of organic acids in fruits.

Thin layer drying kinetics and quality dynamics of persimmon (Diospyros kaki) treated with preservatives and solar dried under different temperatures.

Poor postharvest handling, microbial infestation, and high respiration rate are some the factors are responsible for poor storage life of perishable commodities. Therefore, effective preservation of these commodities is needed to lower the damages and extend shelf life. Preservation is regarded as the action taken to maintain desired properties of a perishable commodity as long as possible. Persimmon (Diospyros kaki) is perishable fruit with high nutritive value; however, has very short shelf-life. Therefore, effective preservation and drying is needed to extend its storage life. Drying temperature and preservatives significantly influence the quality of perishable vegetables and fruits during drying. The current study investigated the effect of different temperatures and preservatives on drying kinetics and organoleptic quality attributes of persimmon. Persimmon fruits were treated with preservatives (25% honey, 25% aloe vera, 2% sodium benzoate, 1% potassium metabisulfite, and 2% citric acid solutions) under different drying temperatures (40, 45, and 50°C). All observed parameters were significantly affected by individual effects of temperatures and preservatives, except ash contents. Similarly, interactive effects were significant for all parameters except total soluble sugars, ash contents, and vitamin C. Generally, fruits treated with citric acid and dried under 50°C had 8.2% moisture loss hour-1, 14.9 drying hours, 0.030 g H2O g-1 hr-1, 1.23° Brix of total soluble solids, 6.71 pH, 1.35% acidity, and 6.3 mg vitamin C. These values were better than the rest of the preservatives and drying temperatures used in the study. Therefore, treating fruits with citric acid and drying at 50°C was found a promising technique to extend storage life of persimmon fruits. It is recommended that persimmon fruits dried at 50°C and preserved in citric acid can be used for longer storage period.

Genome-wide identification and expression analysis of the EXO70 gene family in grape (Vitis vinifera L).

EXO70 is the pivotal protein subunit of exocyst, which has a very crucial role in enhancing the shielding effect of the cell wall, resisting abiotic and hormonal stresses. This experiment aims to identify family members of the EXO70 gene family in grape and predict the characteristics of this gene family, so as to lay the foundation of further exploring the mechanism of resisting abiotic and hormone stresses of VvEXO70s. Therefore, the Vitis vinifera 'Red Globe' tube plantlet were used as materials. Bioinformatics was used to inquire VvEXO70 genes family members, gene structure, system evolution, cis-acting elements, subcellular and chromosomal localization, collinearity, selective pressure, codon bias and tissue expression. All of VvEXO70s had the conserved pfam03081 domain which maybe necessary for interacting with other proteins. Microarray analysis suggested that most genes expressed to varying degrees in tendrils, leaves, seeds, buds, roots and stems. Quantitative Real-Time PCR (qRT-PCR) showed that the expression levels of all genes with 5 mM salicylic acid (SA), 0.1 mM methy jasmonate (MeJA), 20% PEG6000 and 4 °C for 24 h were higher than for 12 h. With 20% PEG6000 treatment about 24 h, the relative expression of VvEXO70-02 was significantly up-regulated and 361 times higher than CK. All genes' relative expression was higher at 12 h than that at 24 h after treatment with 7 mM hydrogen peroxide (H2O2) and 0.1 mM ethylene (ETH). In conclusion, the expression levels of 14 VvEXO70 genes are distinguishing under these treatments, which play an important role in the regulation of anti-stress signals in grape. All of these test results provide a reference for the future research on the potential function analysis and plant breeding of VvEXO70 genes.

MYB_SH[AL]QKY[RF] transcription factors MdLUX and MdPCL-like promote anthocyanin accumulation through DNA hypomethylation and MdF3H activation in apple.

Heritable DNA methylation is a highly conserved epigenetic mark that is important for many biological processes. In a previous transcriptomic study on the fruit skin pigmentation of apple (Malus domestica Borkh.) cv. 'Red Delicious' (G0) and its four continuous-generation bud sport mutants including 'Starking Red' (G1), 'Starkrimson' (G2), 'Campbell Redchief' (G3) and 'Vallee spur' (G4), we identified MYB transcription factors (TFs) MdLUX and MdPCL-like involved in regulating anthocyanin synthesis. However, how these TFs ultimately determine the fruit skin color traits remains elusive. Here, bioinformatics analysis revealed that MdLUX and MdPCL-like contained a well-conserved motif SH[AL]QKY[RF] in their C-terminal region and were located in the nucleus of onion epidermal cells. Overexpression of MdLUX and MdPCL-like in 'Golden Delicious' fruits, 'Gala' calli and Arabidopsis thaliana promoted the accumulation of anthocyanin, whereas MdLUX and MdPCL-like suppression inhibited anthocyanin accumulation in 'Red Fuji' apple fruit skin. Yeast one-hybrid assays revealed that MdLUX and MdPCL-like may bind to the promoter region of the anthocyanin biosynthesis gene MdF3H. Dual-luciferase assays indicated that MdLUX and MdPCL-like activated MdF3H. The whole-genome DNA methylation study revealed that the methylation levels of the mCG context at the upstream (i.e., promoter region) of MdLUX and MdPCL-like were inversely correlated with their mRNA levels and anthocyanin accumulation. Hence, the data suggest that MYB_SH[AL]QKY[RF] TFs MdLUX and MdPCL-like promote anthocyanin biosynthesis in apple fruit skins through the DNA hypomethylation of their promoter regions and the activation of the structural flavonoid gene MdF3H.

Whole-genome DNA methylation patterns and complex associations with gene expression associated with anthocyanin biosynthesis in apple fruit skin.

MAIN CONCLUSION: DNA methylation of anthocyanin biosynthesis-related genes and MYB/bHLH transcription factors was associated with apple fruit skin color revealed by whole-genome bisulfite sequencing. DNA methylation is a common feature of epigenetic regulation and is associated with various biological processes. Anthocyanins are among the secondary metabolites that contribute to fruit colour, which is a key appearance and nutrition quality attribute of apple fruit. Although few studies reported that DNA methylation in the promoter of MYB transcription factor was associated with fruit skin color, there is a general lack of understanding of the dynamics of global and genic DNA methylation in apple fruit. Here, whole-genome bisulfite sequencing was carried out in fruit skin of apple (Malus domestica Borkh.) cv. 'Red Delicious' (G0) and its four-generation bud sport mutants, including 'Starking Red' (G1), 'Starkrimson' (G2), 'Campbell Redchief' (G3) and 'Vallee spur' (G4) at color break stage. Correlation and linear-regression analysis between DNA methylation level and anthocyanin content, as well as the transcription levels of genes related to anthocyanin biosynthesis were carried out. The results showed that the number of differentially methylated regions (DMRs) and differentially methylated genes (DMGs) was considerably increased from G1 to G4 versus the number observed in G0. The mCHH context was dominant in apple, but the levels of mCG and mCHG of DMGs were significantly higher than that of the mCHH. Genetic variation of bud sport mutants from 'Red Delicious' was associated with differential DNA methylation. Additionally, hypomethylation of mCG and mCHG contexts in flavonoid biosynthesis pathway genes (PAL, 4CL, CYP98A, PER, CCoAOMT, CHS, and F3'H), mCHG context in MYB10 at upstream, led to transcriptional activation and was conductive to anthocyanin accumulation. However, hypermethylation of mCG context in bHLH74 at upstream led to transcriptional inhibition, inhibiting anthocyanin accumulation.

Synthesis of light-inducible and light-independent anthocyanins regulated by specific genes in grape 'Marselan' (V. vinifera L.).

Anthocyanin is an important parameter for evaluating the quality of wine grapes. However, the effects of different light intensities on anthocyanin synthesis in grape berry skin and its regulation mechanisms are still unclear. In this experiment, clusters of wine grape cv. 'Marselan' were bagged using fruit bags with different light transmittance of 50%, 15%, 5%, and 0, designated as treatment A, B, C and D, respectively. Fruits that were not bagged were used as the control, designated as CK. The anthocyanin composition and concentration, as well as gene expression profiles in the berry skin were determined. The results showed that the degree of coloration of the berry skin reduced with the decrease of the light transmittance, and the veraison was postponed for 10 days in D when compared with the CK. Total anthocyanin concentration in the berry skin treated with D decreased by 51.50% compared with CK at the harvest stage. A total of 24 and 21 anthocyanins were detected in CK and D, respectively. Among them, Malvidin-3-O-coumaroylglucoside (trans), which showed a significant positive correlation with the total concentration of anthocyanins at the harvest stage (r = 0.775) and was not detected in D, was presumed to be light-induced anthocyanin. Other anthocyanins which were both synthesized in CK and D were considered to be light-independent anthocyanins. Among them, Malvidin-3-O-coumaroylglucoside (cis) and Malvidin-3-O-acetylglucoside were typical representatives. Remarkably, the synthesis of light-inducible anthocyanins and light-independent anthocyanins were regulated by different candidate structural genes involved in flavonoid biosynthesis pathway and members of MYB and bHLH transcription factors.

Elevated CO2 concentration promotes photosynthesis of grape (Vitis vinifera L. cv. 'Pinot noir') plantlet in vitro by regulating RbcS and Rca revealed by proteomic and transcriptomic profiles.

BACKGROUND: Plant photosynthesis can be improved by elevated CO2 concentration (eCO2). In vitro growth under CO2 enriched environment can lead to greater biomass accumulation than the conventional in micropropagation. However, little is know about how eCO2 promotes transformation of grape plantlets in vitro from heterotrophic to autotrophic. In addition, how photosynthesis-related genes and their proteins are expressed under eCO2 and the mechanisms of how eCO2 regulates RbcS, Rca and their proteins have not been reported. RESULTS: Grape (Vitis vinifera L. cv. 'Pinot Noir') plantlets in vitro were cultured with 2% sucrose designated as control (CK), with eCO2 (1000 µmol·mol- 1) as C0, with both 2% sucrose and eCO2 as Cs. Here, transcriptomic and proteomic profiles associated with photosynthesis and growth in leaves of V. vinifera at different CO2 concentration were analyzed. A total of 1814 genes (465 up-regulated and 1349 down-regulated) and 172 proteins (80 up-regulated and 97 down-regulated) were significantly differentially expressed in eCO2 compared to CK. Photosynthesis-antenna, photosynthesis and metabolism pathways were enriched based on GO and KEGG. Simultaneously, 9, 6 and 48 proteins were involved in the three pathways, respectively. The leaf area, plantlet height, qP, ΦPSII and ETR increased under eCO2, whereas Fv/Fm and NPQ decreased. Changes of these physiological indexes are related to the function of DEPs. After combined analysis of proteomic and transcriptomic, the results make clear that eCO2 have different effects on gene transcription and translation. RbcS was not correlated with its mRNA level, suggesting that the change in the amount of RbcS is regulated at their transcript levels by eCO2. However, Rca was negatively correlated with its mRNA level, it is suggested that the change in the amount of its corresponding protein is regulated at their translation levels by eCO2. CONCLUSIONS: Transcriptomic, proteomic and physiological analysis were used to evaluate eCO2 effects on photosynthesis. The eCO2 triggered the RbcS and Rca up-regulated, thus promoting photosynthesis and then advancing transformation of grape plantlets from heterotrophic to autotrophic. This research will helpful to understand the influence of eCO2 on plant growth and promote reveal the mechanism of plant transformation from heterotrophic to autotrophic.

Anthocyanin accumulation correlates with hormones in the fruit skin of 'Red Delicious' and its four generation bud sport mutants.

BACKGROUND: Bud sport mutants of apple (Malus domestica Borkh.) trees with a highly blushed colouring pattern are mainly caused by the accumulation of anthocyanins in the fruit skin. Hormones are important factors modulating anthocyanin accumulation. However, a good understanding of the interplay between hormones and anthocyanin synthesis in apples, especially in mutants at the molecular level, remains elusive. Here, physiological and comparative transcriptome approaches were used to reveal the molecular basis of color pigmentation in the skin of 'Red Delicious' (G0) and its mutants, including 'Starking Red' (G1), 'Starkrimson' (G2), 'Campbell Redchief' (G3) and 'Vallee spur' (G4). RESULTS: Pigmentation in the skin gradually proliferated from G0 to G4. The anthocyanin content was higher in the mutants than in 'Red Delicious'. The activation of early phenylpropanoid biosynthesis genes, including ASP3, PAL, 4CL, PER, CHS, CYP98A and F3'H, was more responsible for anthocyanin accumulation in mutants at the color break stage. In addition, IAA and ABA had a positive regulatory effect on the synthesis of anthocyanins, while GA had the reverse effect. The down-regulation of AACT1, HMGS, HMGR, MVK, MVD2, IDI1 and FPPS2 involved in terpenoid biosynthesis influences anthocyanin accumulation by positively regulating transcripts of AUX1 and SAUR that contribute to the synthesis of IAA, GID2 to GA, PP2C and SnRK2 to ABA. Furthermore, MYB and bHLH members, which are highly correlated (r=0.882-0.980) with anthocyanin content, modulated anthocyanin accumulation by regulating the transcription of structural genes, including CHS and F3'H, involved in the flavonoid biosynthesis pathway. CONCLUSIONS: The present comprehensive transcriptome analyses contribute to the understanding of the the relationship between hormones and anthocyanin synthesis as well as the molecular mechanism involved in apple skin pigmentation.

[Situation of distribution and utilization of crop straw resources in seven western provinces, China].

Based on agricultural statistical and investigating data on farmers, amount, distribution density and amount per capita of crop straw resources of seven western provinces of China were estimated, the trends of amount dynamics from 1997 to 2011 and distribution and utilization of crop straw resources were analyzed, and the constraints of comprehensive utilizing straw resources and possible ways to improve straw utilization were discussed. Results showed that theoretical amount of crop straw resources in seven western provinces was 8.82 x 10(7) t in 2009, in which straw of cereals accounted for 63.1% of the total, and straw nutrients returned back to fields was 1.20 x 10(6) t, accounting for 50.5% of the total. The distribution density of straw resources of the seven western provinces was less than the national mean, while the amount per capita of straw resources was higher. According to the survey, the straw was mainly used as fuel, feed, industrial materials, matrix and returned field directly, accounting for 33.8%, 29.3%, 5.2%, 1.8% and 13.5% of the total amount, respectively. In addition, the amounts of straw burned and abandoned were 11.1% and 5.3% of the total, respectively.

[Effects of different drip irrigation modes on root distribution of wine grape 'Cabernet Sauvignon' in desert area of Northwest China].

To study the effects of different drip irrigation modes on the wine grape root distribution is the basis of formulating fertilization, irrigation, and over-wintering management practices for wine grape. Taking the wine grape "Cabernet Sauvignon" as test material, this paper studied the effects of different water-saving irrigation modes (drip irrigation under straw mulching, drip irrigation under plastic mulching, double-tube drip irrigation, and single-tube drip irrigation) on the root distribution of wine grape in the desert area of Northwest China, with the conventional furrow irrigation as the control. The root system of the "Cabernet Sauvignon" was distributed from 0 to 70 cm vertically, and from 0 to 120 cm horizontally. With double-tube drip irrigation, the root amount was the largest (138.3 roots per unit profile), but the root vertical distribution scope was narrowed by 20 cm, as compared to the control. Drip irrigation with straw mulching increased the root amount significantly, and increased the root horizontal distribution scope by 9.1%, as compared to the control. No significant difference was observed in the root number and root horizontal distribution scope between the drip irrigation under plastic mulching and the control, but the root vertical distribution scope with the drip irrigation under plastic mulching decreased by 20 cm. Single-tube drip irrigation increased the root number significantly, but had lesser effects on the root vertical or horizontal distribution, as compared to the conventional irrigation. It was suggested that the drip irrigation under straw mulching could be the best water-saving practice for the wine grape "Cabernet Sauvignon" in the study area.

[Construction of a recombinant lentiviral vector of p38 MAPK and establishment of a human prostatic carcinoma cell line stably expressing p38 MAPK].

OBJECTIVE: To construct a recombinant lentiviral vector for p38 MAPK and establish a human prostatic carcinoma cell line that stably expresses p38 MAPK. METHODS: EGFP/p38 fusion gene was subcloned into the lentiviral vector pTYF- EF1α-IRES-EGFP. The recombinant lentiviral vector pTYF-EF1α-EGFP/p38 was indentified by restriction enzyme digestion, and packaged in HEK 293T cells using lipofectamintm2000 with the packaging plasmid psPAX2 and envelope plasmid pMD2.G. The viral titer was tested according to the expression level of GFP. The resulting recombinant lentiviral vector was transduced into human prostatic carcinoma DU145 cells, and stably transduced cells were selected by limiting dilution analysis. The intracellular expression level of total p38 was detected by Western blotting and the cell growth curve was drawn. RESULTS: DNA restriction enzyme digestion demonstrated that the recombinant lentiviral vector of the fusion gene EGFP/p38 (pTYF-EF1α-EGFP/p38) was constructed successfully. The recombinant lentiviral vector was packaged in 293T with a viral titer of 4.7×10(6) TU/ml. A stable cell line, EGFP/p38-DU145, was established, which stably expressed exogenous EGFP/p38 MAPK fusion protein as detected by Western blotting and showed a lowered growth rate compared to the control cells. CONCLUSION: We have successfully constructed a recombinant lentiviral vector of the fusion gene EGFP/p38 and established a stable cell line EGFP/p38-DU145. Overexpression of p38 has a significant inhibitory effect on the proliferation of DU145 cells in vitro.

[A comparative analysis of the methods for titering adenoviruses].

OBJECTIVE: To compare different methods commonly used for titering adenovirus and analyze the advantages and limitations of each method. METHODS: Four recombined adenoviruses (Ad-G-AT2R-EGFP, Ad-CMV-EGFP, Ad-mif-shRNA-EGFP and Ad-CBA-GFP) were amplified and purified, and each was titered by optical absorbance, real-time PCR, green fluorescent protein (GFP)-labeled method, immunoassay, and cytopathic effect (CPE). The results were then comparatively analyzed. RESULTS: No significant difference was found in the titer amounts derived from GFP-labeled method, immunoassay, and cytopathic effect method (P>0.1). A positive correlation was noted in the titer amounts determined by real-time PCR and immunoassay (r=0.965), even though the value (vg/ml) obtained by real-time PCR was 10 times higher than that by immunoassay (ifu/ml). CONCLUSION: GFP-labeled method and immunoassay allow rapid determination of the adenoviral titer. Real-time PCR can not directly determine the real infectious titer of the adenovirus, but the result is well correlated to that of immunoassay and reflects, though indirectly, the actual infectious titer of adenovirus. Considering the procedural convenience and shorter time consumption, real-time PCR is still a practical method for adenoviral titration.

[Development of the recombinant SAG1 antigen of Toxoplasma gondii by high-density fermentation and identification of its immunoreactivity].

OBJECTIVE: To develop a technology for production of recombinant SAG1 of Toxoplasma gondii (T.g) in batches. METHODS: The rSAG1 of T.g was expressed in E.coli by high-density fermentation and purified by Sephadex G-75 column chromatography after Ni-NTA agarose at native condition. The activity of rSAG1 and its efficacy in T.g diagnosis were identified by Western blotting and ELISA, respectively. RESULTS: The optical density (OD) of the bacteria reached 20.21 after induction, and 300 g bacteria were harvested from 11.5 L broth. The rSAG1 was highly expressed in E.coli as a fusion protein, accounting for about 25.82% of the total bacterial protein. The purity of rSAG1 reached 98.54% after purification by Ni-NTA combined with Sephadex G-75 column chromatography. Western blotting revealed a distinct band reacting with the sera of rabbits vaccinated by T.g. Twenty-four of the 25 sera of mice infected with T.g and 36 of the 38 sera of human subjects with IgG antibody against T.g were detected by rSAG1-ELISA. CONCLUSION: A large-scale production of immunoreactive SAG1 of T.g is developed by high-density fermentation and purification with Ni-NTA combined with Sephadex G-75 column chromatography.

Advanced oxidation protein products activate vascular endothelial cells via a RAGE-mediated signaling pathway.

The accumulation of advanced oxidation protein products (AOPPs) has been linked to vascular lesions in diabetes, chronic renal insufficiency, and atherosclerosis. However, the signaling pathway involved in AOPPs-induced endothelial cells (ECs) perturbation is unknown and was investigated. AOPPs modified human serum albumin (AOPPs-HSA) bound to the receptor for advanced glycation end products (RAGE) in a dose-dependent and saturable manner. AOPPs-HSA competitively inhibited the binding of soluble RAGE (sRAGE) with its preferential ligands advanced glycation end products (AGEs). Incubation of AOPPs, either prepared in vitro or isolated from uremic serum, with human umbilical vein ECs induced superoxide generation, activation of NAD(P)H oxidase, ERK 1/2 and p38, and nuclear translocation of NF-kappaB. Activation of signaling pathway by AOPPs-ECs interaction resulted in overexpression of VCAM-1 and ICAM-1 at both gene and protein levels. This AOPPs-triggered biochemical cascade in ECs was prevented by blocking RAGE with either anti-RAGE IgG or excess sRAGE, but was not affected by the neutralizing anti-AGEs IgG. These data suggested that AOPPs might be new ligands of endothelial RAGE. AOPPs-HSA activates vascular ECs via RAGE-mediated signals.

[Screening and preliminary identification of mimetic peptides of Plasmodium falciparum-infected erythrocyte membrane surface protein 1].

OBJECTIVE: To screen and identify mimetic peptides of Plasmodium falciparun-infected erythrocyte membrane surface protein 1 in order to explore anti-adhesive agent against cerebral malaria. METHODS: Phage-borne peptide KLYLIAEGSVAA was used as panning targets to select target binders in a disulfide-constrained heptapeptides library. Three rounds of biopanning were carried out and then ELISA and competitive ELISA were used to evaluate the binding character between phage-borne peptides and ICAM-1. The insert DNA sequences of positive clones were determined and their amino acid sequences were deduced. RESULTS: After three-round panning, 22 clones were randomly chosen from the third panning and analyzed. Three clones showed positive interaction with ICAM-1, and two of them possessed the amino acid sequence C-ITAVPVR-C, the other one was C-DIMGGYN-C. These peptides specifically inhibited the binding of 15.2 antibody to ICAM-1 detected by competitive ELISA. CONCLUSION: Two kinds of mimetic peptides of PfEMP-1 have been obtained, which can bind with ICAM-1 specifically.

[Immunogenicity of a multiple epitope DNA vaccine against hepatitis B virus].

AIM: To study the immunogenicity of a multiple epitope DNA vaccine against hepatitis B virus(HBV). METHODS: A multiple epitope HBV antigen gene BPT was synthesized and cloned into eukaryotic expression vector pcDNA3.1 and then BALB/c mice were immunized with the DNA vaccine. The specific humoral and cellular immune responses were detected by indirect ELISA, cytotoxicity of CTL, and lymphocyte proliferation. The immunized mice were also observed for the possible toxicity and side effects after administration of the DNA vaccine. RESULTS: Immunization with pcDNA3.1/BPT elicited high-level antigen-specific IgG and antigen-specific CTL response, and stimulated lymphocyte proliferation. RT-PCR analysis of spleen lymphocytes showed that levels of IL-12 mRNA in immunized mice were notably higher than that in control mice. CONCLUSION: The multiple epitope HBV DNA vaccine can induce specific humoral and cellular immune responses, which lays a certain foundation for development of prophylactic and therapeutic HBV vaccine.

[Preparation and identification of phage-displayed ICAM-1-mimic peptide].

AIM: To obtain bioactive ICAM-1 mimetic peptide. METHODS: Phages displaying P1 and P2 were prepared by phage amplication and PEG precipitation. The binding between phage-displayed peptides and anti-ICAM-1 mAb 15.2 was evaluated by sandwich ELISA and competitive ELISA. Bioactivities of P1 and P2 was detected by immunohistochemical staining. RESULTS: Phage-displayed peptides P1 and P2 could specifically bind to mAb 15.2, and the binding could be competitively inhibited by ICAM-1. Immunohistochemical staining showed that P1 and P2 could mimic the binding of ICAM-1 to its receptor LFA-1. CONCLUSION: Phage-displayed peptides P1 and P2 are bioactive just as native ICAM-1.