Molecular Cloning and Characterization of an Anthocyanidin Synthase Gene in Prunus persica (L.) Batsch

To elucidate the effect of anthocyanidin synthase (ANS) gene on anthocyanin accumulation in fruit skin of Prunus persica (L.) Batsch cv. 'Chunmei', this study cloned and characterized an ANS gene (PpANS) from peach. PpANS (GenBank accession No. KX760117) was encoded by a 1074 bp-long open reading frame (ORF) corresponding to a polypeptide consisting of 358 amino acids with a molecular mass of 40.45 kD and an isoelectric point of 5.46. PpANS contains a conserved 2-oxoglutarate- and iron-dependent dioxygenases and non-haem dioxygenase binding regions. PpANS shared high similarities to angiosperm ANS and displayed the closest genetic relationship to Prunus domestica. Real-time PCR analysis indicated that PpANS was highly expressed in fruit skin, flesh and flowers, and peach fruit skin showed the highest transcript level of PpANS. Anthocyanin accumulation analysis indicated that it was highly accumulated in fruit skin and flesh of peach. Changes in the transcript level were highly correlated with anthocyanin content in the different tissues of peach. Prokaryotic expression analysis showed PpANS that protein can be expressed correctly in E. coli, and the size of PpANS recombinant protein was consistent with its predicted size. In vitro enzyme activity assay revealed that recombinant PpANS protein could catalyze the formation the cyanidin from leucocyanidin. These results indicated that PpANS was responsible for anthocyanin accumulation in P. persica.

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MdMYB3 helps regulate anthocyanin accumulation in apple calli under moderately acidic conditions

10.1101/429456 ◽

2018 ◽

Author(s):

Yi-Cheng Wang ◽

Jing-Jing Sun ◽

Yan-Fen Qiu ◽

Xiao-Jun Gong ◽

Li Ma ◽

...

Keyword(s):

Anthocyanin Biosynthesis ◽

Myb Transcription Factor ◽

Anthocyanin Content ◽

Anthocyanin Accumulation ◽

Mobility Shift ◽

In The Wild ◽

Acidic Conditions ◽

Electrophoretic Mobility Shift Assays

AbstractAnthocyanins are the key factors controlling the coloration of plant tissues. However, the molecular mechanism underlying the effects of environmental pH on the synthesis of apple anthocyanins is unclear. In this study, we analyzed the anthocyanin contents of apple calli cultured in media at different pHs (5.5, 6.0, and 6.5). The highest anthocyanin content was observed at pH 6.0. Additionally, the moderately acidic conditions up-regulated the expression of MdMYB3 as well as specific anthocyanin biosynthesis structural genes (MdDFR and MdUFGT). Moreover, the anthocyanin content was higher in calli overexpressing MdMYB3 than in the wild-type controls at different pHs. Yeast one-hybrid assay results indicated that MdMYB3 binds to the MdDFR and MdUFGT promoters in vivo. An analysis of the MdDFR and MdUFGT promoters revealed multiple MYB-binding sites. Meanwhile, electrophoretic mobility shift assays confirmed that MdMYB3 binds to the MdDFR and MdUFGT promoters in vitro. Furthermore, GUS promoter activity assays suggested that the MdDFR and MdUFGT promoter activities are enhanced by acidic conditions, and the binding of MdMYB3 may further enhance activity. These results implied that an acid-induced apple MYB transcription factor (MdMYB3) promotes anthocyanin accumulation by up-regulating the expression of MdDFR and MdUFGT under moderately acidic conditions.

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A cRNA Probe Detects Prunus Necrotic Ringspot Virus in Three Peach Cultivars after Micrografting and in Peach Shoots following Long-term Culture at 4 °C

HortScience ◽

10.21273/hortsci.34.2.346 ◽

1999 ◽

Vol 34 (2) ◽

pp. 346-347 ◽

Cited By ~ 6

Author(s):

K. Heuss ◽

Q. Liu ◽

F.A. Hammerschlag ◽

R.W. Hammond

Keyword(s):

Prunus Persica ◽

Blot Hybridization ◽

Ringspot Virus ◽

Shoot Cultures ◽

Crna Probe ◽

Reading Frame ◽

Prunus Necrotic Ringspot Virus ◽

Repeated Use ◽

Graft Success

As part of a program to develop transgenic peach (Prunus persica L. Batsch) cultivars with resistance to Prunus necrotic ringspot virus (PNRSV), we are testing a system for measuring virus in peach shoot cultures. Micrografting in vitro is used for inoculation and slot-blot hybridization, with a digoxigenin (DIG)-labeled cRNA probe complementary to the 5′ open reading frame (ORF) of PNRSV RNA 3, for detection. In this study, we investigated whether infected shoots maintain virus infection over long periods of culture at 4 °C and if PNRSV-infected `Suncrest' shoot cultures can serve as graft bases to transmit virus equally well into cultivars Nemaguard, Springcrest, and Suncrest. The results of RNA hybridization analysis showed that virus was present in extracts of leaf samples from 2-year-old PNRSV-infected `Suncrest' shoots that had been subjected to varying lengths of incubation at 4 °C in the dark, suggesting that infected shoots can be maintained for repeated use. Rates of graft success were higher in heterografts between `Suncrest' bases and tips of `Springcrest' or `Nemaguard' than in autografts between `Suncrest' and `Suncrest', and there was equal efficacy of graft inoculation from `Suncrest' into these three cultivars.

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Characterisation and preliminary functional analysis of N-acetyltransferase 13 from Schistosoma japonicum

BMC Veterinary Research ◽

10.1186/s12917-021-03045-y ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Yalan Tang ◽

Kerou Zhou ◽

Qingqing Guo ◽

Cheng Chen ◽

Jing Jia ◽

...

Keyword(s):

Schistosoma Japonicum ◽

Egg Production ◽

Developmental Stages ◽

Pcr Analysis ◽

Female Adult ◽

Male Adult ◽

Transcript Levels ◽

Reading Frame ◽

Interfering Rna

Abstract Background N-acetyltransferase 13 (NAT13) is a probable catalytic component of the ARD1A-NARG1 complex possessing alpha (N-terminal) acetyltransferase activity. Results In this study, a full-length complementary DNA (cDNA) encoding Schistosoma japonicum NAT13 (SjNAT13) was isolated from schistosome cDNAs. The 621 bp open reading frame of SjNAT13 encodes a polypeptide of 206 amino acids. Real-time PCR analysis revealed SjNAT13 expression in all tested developmental stages. Transcript levels were highest in cercariae and 21-day-old worms, and higher in male adult worms than female adult worms. The rSjNAT13 protein induced high levels of anti-rSjNAT13 IgG antibodies. In two independent immunoprotection trials, rSjNAT13 induced 24.23% and 24.47% reductions in the numbers of eggs in liver. RNA interference (RNAi) results showed that small interfering RNA (siRNA) Sj-514 significantly reduced SjNAT13 transcript levels in worms and decreased egg production in vitro. Conclusions Thus, rSjNAT13 might play an important role in the development and reproduction of schistosomes.

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4-Coumarate:coenzyme A ligase isoform 3 from Piper nigrum (Pn4CL3) catalyzes the CoA thioester formation of 3,4-methylenedioxycinnamic and piperic acids

Biochemical Journal ◽

10.1042/bcj20190527 ◽

2020 ◽

Vol 477 (1) ◽

pp. 61-74

Author(s):

Zhehao Jin ◽

Juraithip Wungsintaweekul ◽

Sang-Hoon Kim ◽

Jeong-Han Kim ◽

Yongho Shin ◽

...

Keyword(s):

Catalytic Efficiency ◽

Transcript Level ◽

Binding Pocket ◽

Phenylpropanoid Pathway ◽

Black Pepper ◽

Piper Nigrum ◽

Pcr Analysis ◽

Coumarate:Coa Ligase ◽

Ferulic Acids

Black pepper, dried green fruit of Piper nigrum L., is a household spice most popular in the world. Piperine, the pungency compound of black pepper, is proposed to partially arise from phenylpropanoid pathway. In the biosynthesis of piperine, 4-coumarate:CoA ligase (4CLs) must play a pivotal role in activating intermediate acids to corresponding CoA thioesters to serve as substrates. Based on transcriptome data, we isolated three P. nigrum 4CL isoforms (Pn4CL1, -2, and -3) from unripe peppercorn. These Pn4CLs were expressed in E. coli for in vitro enzyme assay with putative substrates, namely cinnamic, coumaric, ferulic, piperonylic, 3,4-methylenedioxycinnamic (3,4-MDCA), and piperic acids. Phylogenetic analysis and substrate usage study indicated that Pn4CL1, active towards coumaric and ferulic acids, belongs to class I 4CL for lignin synthesis. Pn4CL2 was a typical cinnamate-specific coumarate:CoA ligase-like (CLL) protein. The Pn4CL3, as class II enzyme, exhibited general 4CL activity towards coumaric and ferulic acids. However, Pn4CL3 was also active towards piperonylic acid, 3,4-MDCA, and piperic acid. Pn4CL3 possessed ∼2.6 times higher catalytic efficiency (kcat/KM) towards 3,4-MDCA and piperic acid than towards coumaric and ferulic acids, suggesting its specific role in piperine biosynthesis. Different substrate preference among the Pn4CL isoforms can be explained by 3-dimensional protein structure modeling, which demonstrated natural variants in amino acid residues of binding pocket to accommodate different substrates. Quantitative PCR analysis of these isoforms indicated that Pn4CL1 transcript level was highest in the roots whereas Pn4CL2 in the fruits and Pn4CL3 in the leaves.

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A novel NAC transcription factor, MdNAC42, regulates anthocyanin accumulation in red-fleshed apple by interacting with MdMYB10

Tree Physiology ◽

10.1093/treephys/tpaa004 ◽

2020 ◽

Vol 40 (3) ◽

pp. 413-423

Author(s):

Shuangyi Zhang ◽

Yixi Chen ◽

Lingling Zhao ◽

Chenqi Li ◽

Jingyun Yu ◽

...

Keyword(s):

Anthocyanin Biosynthesis ◽

Expression Patterns ◽

Apple Fruit ◽

Bimolecular Fluorescence Complementation ◽

Anthocyanin Content ◽

Anthocyanin Accumulation ◽

Flavonoid Pathway ◽

Anthocyanin Pigmentation

Abstract Anthocyanin pigmentation is an important consumption trait of apple (Malus domestica Borkh.). In this study, we focused on the identification of NAC (NAM, ATAF1/2 and CUC2) proteins involved in the regulation of anthocyanin accumulation in apple flesh. A group of MdNACs was selected for comparison of expression patterns between the white-fleshed cultivar ‘Granny Smith’ and red-fleshed ‘Redlove’. Among them, MdNAC42 was screened, which exhibited a higher expression level in red-fleshed than in white-fleshed fruit, and has a positive correlation with anthocyanin content as fruits ripened. Moreover, overexpression of MdNAC42 in apple calli resulted in the up-regulation of flavonoid pathway genes, including MdCHS, MdCHI, MdF3H, MdDFR, MdANS and MdUFGT, thereby increasing the accumulation of anthocyanins, which confirmed the roles of MdNAC42 in anthocyanin biosynthesis. Notably, MdNAC42 was demonstrated to have an obvious interaction with MdMYB10 either in vitro or in vivo by yeast two-hybrid combined with bimolecular fluorescence complementation, further suggesting that MdNAC42 is an important part of the regulatory network controlling the anthocyanin pigmentation of red-fleshed apples. To the best of our knowledge, this is the first report identifying the MdNAC gene as related to anthocyanin accumulation in red-fleshed apples. This study provides valuable information for improving the regulatory model of anthocyanin biosynthesis in apple fruit.

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Dose-dependent Inhibition of Gynecophoral Canal Protein Gene Expression in Vitro in the Schistosome (Schistosoma japonicum) by RNA Interference

Acta Biochimica et Biophysica Sinica ◽

10.1111/j.1745-7270.2005.00058.x ◽

2005 ◽

Vol 37 (6) ◽

pp. 386-390 ◽

Cited By ~ 26

Author(s):

Guo-Feng Cheng ◽

Jiao-Jiao Lin ◽

Yi Shi ◽

You-Xin Jin ◽

Zhi-Qiang Fu ◽

...

Keyword(s):

Schistosoma Japonicum ◽

Protein Gene ◽

Female Worm ◽

Transcript Level ◽

Pcr Analysis ◽

Rt Pcr ◽

Male Worm ◽

Dependent Inhibition ◽

Dose Dependent

Abstract The gynecophoral canal protein gene SjGCP of Schistosoma japonicum that is necessary for the pairing between the male and female worms is specifically expressed in the adult male worm. This protein is widely distributed in the adult female worm after pairing. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence were employed to analyze the relationship between the RNAi effect and dsRNA dosage in the parasites. The results revealed that the inhibition of SjGCP expression by siRNA is dose-dependent. RT-PCR analysis showed that the SjGCP transcript level was reduced by 75% when 100 nM dsRNA was applied.

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Effects of Fruit Bagging on Anthocyanin Accumulation and Related Gene Expression in Peach

Journal of the American Society for Horticultural Science ◽

10.21273/jashs05019-20 ◽

2021 ◽

pp. 1-8

Author(s):

Yingtao Ma ◽

Mengmeng Zhao ◽

Hongxia Wu ◽

Congying Yuan ◽

Huiyun Li ◽

...

Keyword(s):

Gene Expression ◽

Prunus Persica ◽

Critical Role ◽

Light Transmittance ◽

Anthocyanin Synthesis ◽

Anthocyanin Content ◽

Agricultural Practice ◽

Anthocyanin Accumulation ◽

Related Gene ◽

Fruit Bagging

Fruit bagging is a popular agricultural practice that has been widely used to physically protect fruit. However, the application of fruit bags usually has various effects on fruit quality. In this study, three kinds of paper bags with different colors and transmittance were applied to investigate their effects on the skin coloration and related gene expression of peach (Prunus persica). Our findings showed that bagging treatment inhibited anthocyanin accumulation and the expression of related structural and regulatory genes in the peach pericarp. To a certain extent, the inhibitory effects were negatively correlated with the light transmittance of these paper bags. The expression of MYB10.1 was also suppressed by fruit bagging and was highly consistent with anthocyanin content in peach pericarps, which indicated that MYB10.1 might have a critical role in the light-mediated regulation of anthocyanin production in peach pericarps. These findings further enrich our theoretical knowledge of the regulation of anthocyanin synthesis in peach fruit and provide a theoretical basis for common horticultural practices.

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Characterization of a Human Foamy Virus 170-Kilodalton Env-Bet Fusion Protein Generated by Alternative Splicing

Journal of Virology ◽

10.1128/jvi.72.5.4088-4094.1998 ◽

1998 ◽

Vol 72 (5) ◽

pp. 4088-4094 ◽

Cited By ~ 36

Author(s):

Dirk Lindemann ◽

Axel Rethwilm

Keyword(s):

Fusion Protein ◽

Foamy Virus ◽

Protein A ◽

Wild Type Virus ◽

Pcr Analysis ◽

Exact Nature ◽

Acceptor Pair ◽

Reading Frame ◽

Humoral Immune

ABSTRACT Primate foamy viruses (FVs) express, in addition to the 130-kDa envelope protein, a 170-kDa glycoprotein, which reacts with antisera specific for the envelope and Bel proteins. We determined the exact nature of this 170-kDa glycoprotein by using the molecularly cloned human FV (HFV). Radioimmunoprecipitation analysis of 293T cells transfected with appropriate expression constructs by using antisera specific for the HFV Env, Bel1, and Bel2 proteins, as well as reverse transcription-PCR analysis of HFV-infected cells, demonstrated that this protein is an Env-Bet fusion protein that is secreted into the supernatant. However, it is only loosely associated, or not associated, with viral particles. gp170 is generated by an alternatively spliced Env mRNA using a splice donor and splice acceptor pair localized within the env open reading frame (ORF), which is normally used to generate Bel1 and Bet transcripts derived from the internal promoter within the env ORF. gp170 is expressed at a level 30 to 50% of the Env precursor gp130. However, it alone does not confer infectivity to HFV particles, because capsids derived from proviruses expressing only the gp170 were not released into the supernatant. In contrast, viruses derived from proviral clones deficient in gp170 expression showed similar in vitro infectivity and replication kinetics to wild-type virus. Furthermore, both types of viruses were inactivated to a similar extent by neutralizing sera, indicating that shedding of gp170 probably does not affect the humoral immune response in the infected host.

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Inhibitory effects of bioaccessible anthocyanins and procyanidins from apple, red grape, cinnamon on α-amylase, α-glucosidase and lipase

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000652 ◽

2020 ◽

pp. 1-9

Author(s):

Pınar Ercan ◽

Sedef Nehir El

Keyword(s):

Inhibitory Activity ◽

Digestive Enzymes ◽

Positive Control ◽

Anthocyanin Content ◽

Inhibitory Effects ◽

Total Anthocyanin ◽

Total Anthocyanin Content ◽

Before And After ◽

Red Grape

Abstract. The goals of this study were to determine and evaluate the bioaccessibility of total anthocyanin and procyanidin in apple (Amasya, Malus communis), red grape (Papazkarası, Vitis vinifera) and cinnamon (Cassia, Cinnamomum) using an in vitro static digestion system based on human gastrointestinal physiologically relevant conditions. Also, in vitro inhibitory effects of these foods on lipid (lipase) and carbohydrate digestive enzymes (α-amylase and α-glucosidase) were performed with before and after digested samples using acarbose and methylumbelliferyl oleate (4MUO) as the positive control. While the highest total anthocyanin content was found in red grape (164 ± 2.51 mg/100 g), the highest procyanidin content was found in cinnamon (6432 ± 177.31 mg/100 g) (p < 0.05). The anthocyanin bioaccessibilities were found as 10.2 ± 1%, 8.23 ± 0.64%, and 8.73 ± 0.70% in apple, red grape, and cinnamon, respectively. The procyanidin bioaccessibilities of apple, red grape, and cinnamon were found as 17.57 ± 0.71%, 14.08 ± 0.74% and 18.75 ± 1.49%, respectively. The analyzed apple, red grape and cinnamon showed the inhibitory activity against α-glucosidase (IC50 544 ± 21.94, 445 ± 15.67, 1592 ± 17.58 μg/mL, respectively), α-amylase (IC50 38.4 ± 7.26, 56.1 ± 3.60, 3.54 ± 0.86 μg/mL, respectively), and lipase (IC50 52.7 ± 2.05, 581 ± 54.14, 49.6 ± 2.72 μg/mL), respectively. According to our results apple, red grape and cinnamon have potential to inhibit of lipase, α-amylase and α-glucosidase digestive enzymes.

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Healing effects of curcumin nanoparticles in deep tissue injury mouse model.

Current Drug Delivery ◽

10.2174/1567201818666201214125237 ◽

2020 ◽

Vol 18 ◽

Author(s):

Zirui Zhang ◽

Shangcong Han ◽

Panpan Liu ◽

Xu Yang ◽

Jing Han ◽

...

Keyword(s):

Wound Healing ◽

Mrna Expression ◽

Tissue Injury ◽

Inflammatory Cells ◽

Pcr Analysis ◽

Protective Effects ◽

Deep Tissue ◽

Deep Tissue Injury

Background: Chronic inflammation and lack of angiogenesis are the important pathological mechanisms in deep tissue injury (DTI). Curcumin is a well-known anti-inflammatory and antioxidant agent. However, curcumin is unstable under acidic and alkaline conditions, and can be rapidly metabolized and excreted in the bile, which shortens its bioactivity and efficacy. Objective: This study aimed to prepare curcumin-loaded poly (lactic-co-glycolic acid) nanoparticles (CPNPs) and to elucidate the protective effects and underlying mechanisms of wound healing in DTI models. Methods: CPNPs were evaluated for particle size, biocompatibility, in vitro drug release and their effect on in vivo wound healing. Results : The results of in vivo wound closure analysis revealed that CPNP treatments significantly improved wound contraction rates (p<0.01) at a faster rate than other three treatment groups. H&E staining revealed that CPNP treatments resulted in complete epithelialization and thick granulation tissue formation, whereas control groups resulted in a lack of compact epithelialization and persistence of inflammatory cells within the wound sites. Quantitative real-time PCR analysis showed that treatment with CPNPs suppressed IL-6 and TNF-α mRNA expression, and up-regulated TGF-β, VEGF-A and IL-10 mRNA expression. Western blot analysis showed up-regulated protein expression of TGF-β, VEGF-A and phosphorylatedSTAT3. Conclusion: Our results showed that CPNPs enhanced wound healing in DTI models, through modulation of the JAK2/STAT3 signalling pathway and subsequent upregulation of pro-healing factors.

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