In vitro selection of DNA aptamer against abrin toxin and aptamer-based abrin direct detection

Abrin toxin as the target protein, belongs to class II ribosome-inactivating proteins family, has high toxicity to eukaryotic cells. Here, we firstly report the DNA aptamers, isolated by in vitro selection, recognize abrin toxin with high affinity and specificity, and have the advantage of no cross-reaction with structure-similar protein ricin toxin over antibodies. Then, a highly selective and sensitive aptamer-based abrin assay was established using a molecular light switching reagent [Ru(phen)(2)(dppz)](2+) with a limit of detection of 1 nM and a wide linear range from 1 to 400 nM with the correlation coefficient of 0.993. This assay can be successfully directly performed not only in physiological buffer but also in more complicated biological matrix, such as diluted serum. (c) 2006 Elsevier B.V. All rights reserved

Dual-Color Fluorescent Hydrogel Microspheres Combined with Smartphones for Visual Detection of Lactate

Since it is difficult for human eyes to distinguish between two identical colors with only <15% variation in brightness, mono-color fluorescent hydrogel microspheres have some limitations in the detection of lactate. Herein, we prepared novel dual-color fluorescent hydrogel microspheres, which can achieve hue transformation. Microspheres were prepared by introducing a fluorescent nanoparticle as the reference signal while CdTe QDs were used as the response signal. We used smartphones with image processing software to collect and analyze data. In this way, the signal of lactate was converted to RGB (red, green, and blue) values, which can be quantitatively read. Within 10 to 1500 μM, the R/G values of the microspheres had a linear relationship with the logarithm of the lactate concentration. Moreover, color cards for lactate detection were prepared, from which the color change and concentration of lactate could be easily read by the naked eye. It is worth mentioning that this method was successfully applied to screen patients with hyperlactatemia

<i>HuNAC20</i> and <i>HuNAC25</i>, Two Novel NAC Genes from Pitaya, Confer Cold Tolerance in Transgenic <i>Arabidopsis</i>

NAC transcription factors are one of the largest families of transcriptional regulators in plants, and members of the gene family play vital roles in regulating plant growth and development processes including biotic/abiotic stress responses. However, little information is available about the NAC family in pitaya. In this study, we conducted a genome-wide analysis and a total of 64 NACs (named HuNAC1-HuNAC64) were identified in pitaya (Hylocereus). These genes were grouped into fifteen subgroups with diversities in gene proportions, exon–intron structures, and conserved motifs. Genome mapping analysis revealed that HuNAC genes were unevenly scattered on all eleven chromosomes. Synteny analysis indicated that the segmental duplication events played key roles in the expansion of the pitaya NAC gene family. Expression levels of these HuNAC genes were analyzed under cold treatments using qRT-PCR. Four HuNAC genes, i.e., HuNAC7, HuNAC20, HuNAC25, and HuNAC30, were highly induced by cold stress. HuNAC7, HuNAC20, HuNAC25, and HuNAC30 were localized exclusively in the nucleus. HuNAC20, HuNAC25, and HuNAC30 were transcriptional activators while HuNAC7 was a transcriptional repressor. Overexpression of HuNAC20 and HuNAC25 in Arabidopsis thaliana significantly enhanced tolerance to cold stress through decreasing ion leakage, malondialdehyde (MDA), and H2O2 and O2− accumulation, accompanied by upregulating the expression of cold-responsive genes (AtRD29A, AtCOR15A, AtCOR47, and AtKIN1). This study presents comprehensive information on the understanding of the NAC gene family and provides candidate genes to breed new pitaya cultivars with tolerance to cold conditions through genetic transformation

A Genome-Wide Identification Study Reveals That <i>HmoCYP76AD1</i>, <i>HmoDODA</i><i>α1</i> and <i>HmocDOPA5GT</i> Involved in Betalain Biosynthesis in <i>Hylocereus</i>

Betalains are water-soluble nitrogen-containing pigments with multiple bioactivities. Pitayas are the only at large-scale commercially grown fruit containing abundant betalains for consumers. Currently, the key genes involved in betalain biosynthesis remain to be fully elucidated. Moreover, genome-wide analyses of these genes in betalain biosynthesis are not available in betalain-producing plant species. In this study, totally 53 genes related to betalain biosynthesis were identified from the genome data of Hylocereus undatus. Four candidate genes i.e., one cytochrome P-450 R gene (HmoCYP76AD1), two L-DOPA 4,5-dioxygenase genes (HmoDODAα1 and HmoDODAα2), and one cyclo-DOPA 5-O glucosyltransferase gene (HmocDOPA5GT) were initially screened according to bioinformatics and qRT-PCR analyses. Silencing HmoCYP76AD1, HmoDODAα1, HmoDODAα2 or HmocDOPA5GT resulted in loss of red pigment. HmoDODAα1 displayed a high level of L-DOPA 4,5-dioxygenase activity to produce betalamic acid and formed yellow betaxanthin. Co-expression of HmoCYP76AD1, HmoDODAα1 and HmocDOPA5GT in Nicotiana benthamiana and yeast resulted in high abundance of betalain pigments with a red color. These results suggested that HmoCYP76AD1, HmoDODAα1, and HmocDOPA5GT play key roles in betalain biosynthesis in Hylocereus. The results of the present study provide novel genes for molecular breeding programs of pitaya

Genome-Wide Identification of WRKY Gene Family in Pitaya Reveals the Involvement of HmoWRKY42 in Betalain Biosynthesis

The WRKY gene family is a plant-specific transcription factor (TF) that regulates many physiological processes and (a) biotic stress responses. Despite this, little is known about the molecular properties and roles of WRKY TFs in pitaya betalain biosynthesis. Here we report the identification of 70 WRKY in Hylocereus undatus, their gene structure, locations on each chromosome, systematic phylogenetic analysis, conserved motif analysis, and synteny of HuWRKY genes. HmoWRKY42 is a Group IIb WRKY protein and contains a coiled-coil motif, a WRKY domain and a C2H2 zinc-finger motif (CX5CX23HXH). Results from yeast one-hybrid and transient dual-luciferase assays showed that HmoWRKY42 was a transcriptional repressor and could repress HmocDOPA5GT1 expression by binding to its promoter. Yeast two-hybrid assays showed that HmoWRKY42 could interact with itself to form homodimers. Knocking out the coiled-coil motif of HmoWRKY42 prevented its self-interaction and prevented it from binding to the HmocDOPA5GT1 promoter. Knocking out the WRKY domain and C2H2 zinc-finger motif sequence of HmoWRKY42 also prevented it from binding to the HmocDOPA5GT1 promoter. The coiled-coil motif, the WRKY domain and the C2H2 zinc finger motif are key motifs for the binding of HmoWRKY42 to the HmocDOPA5GT1 promoter. HmoWRKY42 is localized in the nucleus and possesses trans-activation ability responsible for pitaya betalain biosynthesis by repressing the transcription of HmocDOPA5GT1. As far as we know, no reports are available on the role of HmoWRKY42 in pitaya betalain biosynthesis. The results provide an important foundation for future analyses of the regulation and functions of the HuWRKY gene family

Metabolic Profiling of Organic Acids Reveals the Involvement of HuIPMS2 in Citramalic Acid Synthesis in Pitaya

Pitayas are rich in organic acids, especially citramalic acid, which is significantly higher than the plants. However, the mechanism of citramalic acid biosynthesis remains to be fully elucidated. In this study, organic acid compositions and contents, as well as expression patterns of key genes related to organic acid metabolism were analyzed during fruit maturation of four different pitaya cultivars i.e., &lsquo;Guanhuabai&rsquo; (GHB), &lsquo;Guanhuahong&rsquo; (GHH), &lsquo;Wucihuanglong&rsquo; (WCHL), and &lsquo;Youcihuanglong&rsquo; (YCHL). The total organic acid contents increased first and then declined during fruit maturation. The main organic acids were citramalic acid during the early stages of GHB, GHH, and WCHL pitayas, and dominated by malic acid as fruit maturation. In comparison, citric acid and malic acid were main organic acid for &lsquo;YCHL&rsquo; pitaya. Citramalate synthase (IPMS) was involved in the synthesis of citramalic acid, and three types of HuIPMS i.e., HuIPMS1, HuIPMS2, and HuIPMS3, were obtained in our study. Highest expression levels of HuIPMS1 were detected in sepals, while HuIPMS2 and HuIPMS3 exhibited preferential expression in tender stems and ovaries. The expression levels of HuIPMS2 and HuIPMS3 were positively correlated with the content of citramalic acid in the four pitaya cultivars. HuIPMS2 was a chloroplast-localized protein, while HuIPMS3 presented a cytoplasmic-like and nuclear subcellular localization. These findings provide an important basis for further understanding of the molecular mechanism that leads to citramalic acid metabolism during pitaya fruit maturation

Quality Analysis and Evaluation of Different Batches of Pitaya Fruit (Hylocereus) in South China

ABSTRACTPitaya is a tropical and subtropical fruit; it can produce several batches fruit in one year. To find out the fruit quality differences between various batches in the same year in Guangzhou, South China, 11 pitaya varieties were used as the materials. Comparative analysis was performed between these varieties of each batch by 14 indexes, comprehensive evaluation and ranking were evaluated by the principal component analysis (PCA). Results showed that the red-peel and red-pulp pitaya has the longer fruit period and could obtain more batches fruits. By comparing the fruit quality of these 11 varieties in different batches: Except “Guanhuahong,” fruit weight is significant different between other 10 varieties. The edible rate of fruits from 2nd and 3rd batches is significantly higher than others. The hardness, total sugar, total acid, betalain, total phenol, and flavonoids were significant difference between batches. The PCA results indicated that in most varieties, the 1st, 8th, 9th batches are generally with heavier fruit, better color, harder and sweeter; more stable antioxidant compounds were shown in 6th, 7th, 8th, 9th batches; the 3rd, 4th, 5th, and 6th batches are smaller, softer, lower soluble sugar and higher titratable acid. Pitaya fruit quality and tastes from various batches are different in the same year, the climate may be the main factor. The fruits of 7th, 8th and 9th batches picking from Sep to Nov has better quality and higher economic value. This research has practical application value and could provide theoretical basis for the production of pitaya

Metabolic Profiling of Organic Acids Reveals the Involvement of <i>HuIPMS2</i> in Citramalic Acid Synthesis in Pitaya

Pitayas are rich in organic acids, especially citramalic acid, which is significantly higher than the plants. However, the mechanism of citramalic acid biosynthesis remains to be fully elucidated. In this study, organic acid compositions and contents, as well as expression patterns of key genes related to organic acid metabolism were analyzed during fruit maturation of four different pitaya cultivars i.e., ‘Guanhuabai’ (GHB), ‘Guanhuahong’ (GHH), ‘Wucihuanglong’ (WCHL), and ‘Youcihuanglong’ (YCHL). The total organic acid contents increased first and then declined during fruit maturation. The main organic acids were citramalic acid during the early stages of GHB, GHH, and WCHL pitayas, and dominated by malic acid as fruit maturation. In comparison, citric acid and malic acid were main organic acid for ‘YCHL’ pitaya. Citramalate synthase (IPMS) was involved in the synthesis of citramalic acid, and three types of HuIPMS i.e., HuIPMS1, HuIPMS2, and HuIPMS3, were obtained in our study. Highest expression levels of HuIPMS1 were detected in sepals, while HuIPMS2 and HuIPMS3 exhibited preferential expression in tender stems and ovaries. The expression levels of HuIPMS2 and HuIPMS3 were positively correlated with the content of citramalic acid in the four pitaya cultivars. HuIPMS2 was a chloroplast-localized protein, while HuIPMS3 presented a cytoplasmic-like and nuclear subcellular localization. These findings provide an important basis for further understanding of the molecular mechanism that leads to citramalic acid metabolism during pitaya fruit maturation

Metabolic Profiling of Sugars and Organic Acids, and Expression Analyses of Metabolism-Associated Genes in Two Yellow-Peel Pitaya Species

Sugar and organic acids are important factors determining pitaya fruit quality. However, changes in sugars and acids, and expressions of metabolism-associated genes during fruit maturation of yellow-peel pitayas are not well-documented. In this study, metabolic and expression analyses in pulps of different fruit developmental stages of &lsquo;Wucihuanglong&rsquo; (&lsquo;WCHL&rsquo;, Hylocereus undatus) and &lsquo;Youcihuanglong&rsquo; pitaya (&lsquo;YCHL&rsquo;, Hylocereus megalanthus) were used to explore the sugar and organic acid metabolic process. Total phenols and flavonoids were mainly accumulated at S1 in pitaya pulps. Ascorbic acid contents of &lsquo;WCHL&rsquo; pitaya were higher than that of &lsquo;YCHL&rsquo; pitaya during fruit maturation. Starch was mainly accumulated at early fruit development stages while soluble sugars were rich in late stages. Sucrose, fructose, and glucose were the main sugar components of &lsquo;YCHL&rsquo; pitaya while glucose was dominant in &lsquo;WCHL&rsquo; pitaya. Malic and citric acids were the main organic acids in &lsquo;WCHL&rsquo; and &lsquo;YCHL&rsquo; pitayas, respectively. Based on the transcriptome analyses, 118 genes involved in pitaya sugar and organic acid metabolism were obtained. Results from the correlation analyses between the expression profiling of candidate genes and the contents of sugar and organic acid showed that 51 genes had a significant correlation relationship and probably perform key role in pitaya sugar and organic acid metabolism processes. The finding of the present study provides new information for quality regulation of pitayas