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CiT Galaxy White Mid-Tower PC Gaming Case with 1 x LED Strip 1 x 120mm Rainbow RGB Fan Included Tempered Glass Side Panel Howarth, C. J., Ougham, H. J. (1993). Gene expression under temperature stress. New Phytol. 125, 1–26. doi:10.1111/j.1469-8137.1993.tb03862.x First-strand cDNA was synthesized from each RNA sample (0.2 μg). Specific primers ( Supplementary Table S1), designed by NCBI Primer-BLAST using genome sequences, were used for quantitative real-time PCR (qPCR) cycling on a CFX96 Real-Time PCR Detection System. Real-Time PCR System (Hercules, CA, USA). The qPCR cycling conditions were 95°C for 2 minutes followed by 39 cycles of 95°C for 5 seconds and 57°C for 40 seconds. Actin was utilized as an internal control ( Supplementary Table S1), and biological replicates were triplicated. Statistical analysis

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CiT Vento White Micro-ATX PC Gaming Case with 4 x 120mm ARGB Fans Included 1 x 6-Port Fan Hub Tempered Glass Side Panel Zhai, R., Wang, Z. M., Zhang, S. W., Meng, G., Song, L. Y., Wang, Z. G., et al. (2016). Two MYB transcription factors regulate flavonoid biosynthesis in pear fruit (Pyrus bretschneideri rehd.). J. Exp. Bot. 67, 1275–1284. doi:10.1093/jxb/erv524

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Free WiFi Dongle provided to enable WiFi onto the computer. Please note if streaming or gaming online we recommend upgrading the dongle or connecting by Ethernet cable. Zhou, H., Lin-Wang, K., Wang, H., Gu, C., Dare, A. P., Espley, R. V., et al. (2015). Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors. Plant J. 82, 105–121. doi:10.1111/tpj.12792 Misra, P., Pandey, A., Tiwari, M., Chandrashekar, K., Sidhu, O. P., Asif, M. H., et al. (2010). Modulation of transcriptome and metabolome of tobacco by arabidopsis transcription factor, AtMYB12, leads to insect resistance. Plant Physiol. 152, 2258–2268. doi:10.1104/pp.109.150979 Correlation analysis and canonical correlation analysis between flavonoids content and expression of synthesis-related genes CiT S8-13 SFF Micro ATX Desktop Case with Mesh Front Panel 8.3 Litre 1x USB3.0 1x USB2.0 1 x 80mm Fan

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Long, A., Zhang, J., Yang, L. T., Ye, X., Lai, N. W., Tan, L. L., et al. (2017). Effects of low pH on photosynthesis, related physiological parameters, and nutrient profiles of citrus. Front. Plant Sci. 8. doi:10.3389/fpls.2017.00185 Peng, Y., Hu, M. J., Lu, Q., Tian, Y., He, W. Y., Chen, L., et al. (2019). Flavonoids derived from exocarpium citri grandis inhibit LPS-induced inflammatory response via suppressing MAPK and NF-kappa b signalling pathways. Food Agric. Immunol. 30, 564–580. doi:10.1080/09540105.2018.1550056 Ramakrishna, A., Ravishankar, G. A. (2011). Influence of abiotic stress signals on secondary metabolites in plants. Plant Signal Behav. 6, 1720–1731. doi:10.4161/psb.6.11.17613 Collins, M. E., Obreza, T. A., Morgan, K. T., Graham, J., Alferez, F. (2020). “Nutrition of Florida citrus trees,” in Chapter 2. production areas, soils, and land preparation, 3rd Edition (University of Florida George A Smathers Libraries). In GO annotation analysis, a total of 38,397 DEGs were annotated in three categories: biological process, molecular function, and cellular component categories ( Supplementary Table7). These DEGs were further divided into 47 categories based on gene function, with 722 genes related to biological processes such as signaling. TopGO analysis revealed that the most enriched molecular function terms were monooxygenase activity (GO0004497), oxidoreductase activity (GO0016705), heme binding (GO0020037), iron ion binding (GO0005506), and tetrapyrrole binding (GO0046906) ( Supplementary Figure7). The most enriched biological process terms were flavonoid metabolic process (GO0009812) and flavonoid biosynthetic process (GO0009813). The most enriched cellular component terms were chloroplast thylakoid (GO0009534) and plastid thylakoid (GO0031976). KEGG enrichment analysis identified the top 20 enriched metabolic pathways, including metabolic pathways (ko01100), biosynthesis of secondary metabolites (ko01110), MAPK signaling pathway-plant (ko04016), plant hormone signal transduction (ko04075), phenylpropanoid biosynthesis (ko00940), and flavonoid biosynthesis (ko00941) under magnesium stress ( Figure5D). These results were presented in a bubble diagram. Metabolic and gene co-expression networks in SOPs at different developmental stagesAdobe Acrobat Reader DC - The leading PDF Viewer for reliably viewing, printing, signing and commenting on PDF documents. Liu, X. M., Hu, C. X., Liu, X. D., Riaz, M., Liu, Y., Dong, Z. H., et al. (2022). Effect of magnesium application on the fruit coloration and sugar accumulation of navel orange (Citrus sinensis osb.). Scientia Hortic. 304. doi:10.1016/j.scienta.2022.111282 Guo, Y., Gao, C. Y., Wang, M. K., Fu, F. F., El-Kassaby, Y. A., Wang, T. L., et al. (2020). Metabolome and transcriptome analyses reveal flavonoids biosynthesis differences in ginkgo biloba associated with environmental conditions. Ind. Crops Products 158. doi:10.1016/j.indcrop.2020.112963

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Ferrer, J. L., Austin, M. B., Stewart, C., Jr., Noel, J. P. (2008). Structure and function of enzymes involved in the biosynthesis of phenylpropanoids. Plant Physiol. Biochem. 46, 356–370. doi:10.1016/j.plaphy.2007.12.009 Kwon, M. C., Kim, Y. X., Lee, S., Jung, E. S., Singh, D., Sung, J., et al. (2019). Comparative metabolomics unravel the effect of magnesium oversupply on tomato fruit quality and associated plant metabolism. Metabolites 9. doi:10.3390/metabo9100231 Garcia, C., Arroyo, J. M., Godoy, J. A., Jordano, P. (2005). Mating patterns, pollen dispersal, and the ecological maternal neighbourhood in a prunus mahaleb l. population. Mol. Ecol. 14, 1821–1830. doi:10.1111/j.1365-294X.2005.02542.x Deng, Y. X., Lu, S. F. (2017). Biosynthesis and regulation of phenylpropanoids in plants. Crit. Rev. Plant Sci. 36, 257–290. doi:10.1080/07352689.2017.1402852Flavonoids are a significant group of secondary polyphenolic metabolites with a chemical structure of 3-C (C6-C3-C6) ( Li etal., 2020b; Nabavi etal., 2020). These compounds are widely distributed throughout the plant kingdom, and more than 6,000 distinct flavonoids have been identified to date ( Lepiniec etal., 2006; Ferrer etal., 2008). The pathways involved in flavonoid metabolism have been extensively studied in model plants ( Deng and Lu, 2017; Tohge etal., 2017). The flavonoid biosynthesis process begins with the primary glucose produced through photosynthesis, which is then converted into phenylalanine through glycolysis, pentose phosphate, and shikimic acid pathways. Phenylalanine enters the phenylpropane metabolic pathway through the action of phenylalanine ammonylase (PAL). In this pathway, phenylalanine is converted into p-coumaryl CoA through a series of reactions catalyzed by PAL, cinnamate 4-hydroxylase (C4H), and 4-coumaroyl CoA ligase (4CL) ( Forkmann and Martens, 2001; Du etal., 2010; Saito etal., 2013). The flavonoid biosynthesis pathway is initiated by condensation of one molecule of 4-coumaroyl-CoA with three molecules of malonyl CoA by chalcone synthase (CHS) enzyme. Subsequently, chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) lead to the synthesis of anthocyanidin pigments. Flavone synthase (FNS) and isoflavone synthase (IFS) produce flavones and isoflavones, respectively. Flavonol synthase (FLS) catalyzes dihydroflavonols to flavonols, while leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) synthesize cis- or trans-flavan-3-ols, which are precursors of proanthocyanidin (PA) polymers ( Owens etal., 2008; Li etal., 2020a). Regulation of genes involved in flavonoid biosynthesis is controlled through specific mechanisms that vary depending on the species or tissue involved ( Zhang etal., 2022). The accumulation of flavonoids is mainly controlled by structural biosynthetic genes, regulatory MYB transcription factors (TFs) and the MYB-bHLH-WD40 (MBW) complex ( Appelhagen etal., 2011; Hichri etal., 2011; Song etal., 2019; Yang etal., 2023). These factors are responsible for regulating the expression of genes involved in flavonoid biosynthesis, leading to their accumulation. Keywords: Citrus sinensis, flavonoid biosynthesis, magnesium stress, flavones, CHS, regulatory mechanism

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Nabavi, S. M., Samec, D., Tomczyk, M., Milella, L., Russo, D., Habtemariam, S., et al. (2020). Flavonoid biosynthetic pathways in plants: versatile targets for metabolic engineering. Biotechnol. Adv. 38. doi:10.1016/j.biotechadv.2018.11.005 Zhang, B., Yang, H. J., Qu, D., Zhu, Z. Z., Yang, Y. Z., Zhao, Z. Y. (2022). The MdBBX22-miR858-MdMYB9/11/12 module regulates proanthocyanidin biosynthesis in apple peel. Plant Biotechnol. J. 20, 1683–1700. doi:10.1111/pbi.13839 Ahmed, O. M., Hassan, M. A., Abdel-Twab, S. M., Azeem, M. N. A. (2017). Navel orange peel hydroethanolic extract, naringin and naringenin have anti-diabetic potentials in type 2 diabetic rats. Biomed. Pharmacother. 94, 197–205. doi:10.1016/j.biopha.2017.07.094 Li, C. P., Qi, Y. P., Zhang, J., Yang, L. T., Wang, D. H., Ye, X., et al. (2017). Magnesium-deficiency-induced alterations of gas exchange, major metabolites and key enzymes differ among roots, and lower and upper leaves of citrus sinensis seedlings. Tree Physiol. 37, 1564–1581. doi:10.1093/treephys/tpx067 CiT LT100 1 Litre USB3.0 Ultra-Thin Mini-ITX Computer Case With 120W Laptop Adpater CPU Cooler WIFI Antenna 5cm IncludedWinkel-Shirley, B. (2001). Flavonoid biosynthesis. a colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 126, 485–493. doi:10.1104/pp.126.2.485 RNA isolation and sequencing were performed by Metware Biotechnology Co., Ltd. (Wuhan, China). The quality of the cDNA libraries was examined, and PCR amplification and sequencing were performed using an Illumina HiSeq™ 2500 platform. The obtained reads were processed to remove contaminants and mapped to the reference genome sequence of C. sinensis v3.0 ( http://www.hzau.edu.cn) using HISAT 2.2.4. StringTie was used for transcript assembly. Fragments per kilobase per million (FPKM) values were calculated to determine gene abundance and normalize data. Differential expression analysis of mRNAs was performed using DESeq2 software. DEGs were identified based on fold change > 2 and false discovery rate (FDR) < 0.05 as cutoff values (P < 0.05). Weighted gene co-expression network analysis Yu, F. Q., Xu, X. Y., Lin, S., Peng, T., Zeng, S. H. (2022). Integrated metabolomics and transcriptomics reveal flavonoids glycosylation difference in two citrus peels. Scientia Hortic. 292. doi:10.1016/j.scienta.2021.110623 Hu, H., Fei, X., He, B., Luo, Y., Qi, Y., Wei, A. (2021). Integrated analysis of metabolome and transcriptome data for uncovering flavonoid components of zanthoxylum bungeanum maxim. leaves under drought stress. Front. Nutr. 8. doi:10.3389/fnut.2021.801244