AbstractBackgroundThe HongXin radish (Raphanus sativus L.), which contains the natural red pigment (red radish pigment), is grown in the Fuling district of Chongqing City. However, the molecular mechanisms underlying anthocyanin synthesis for the formation of natural red pigment in the fleshy roots of HongXin radish are not well studied.ResultsDe novo transcriptome of HX-1 radish, as well as that of the advanced inbred lines HX-2 and HX-3 were characterized using next generation sequencing (NGS) technology. In total, approximately 66.22 million paired-end reads comprising 34, 927 unigenes (N50 = 1, 621 bp) were obtained. Based on sequence similarity search with known proteins, total of 30, 127 (about 86.26%) unigenes were identified. Additionally, functional annotation and classification of these unigenes indicated that most of the unigenes were predominantly enriched in the metabolic process-related terms, especially for the biosynthetic pathways of secondary metabolites. Moreover, majority of the anthocyanin biosynthesis-related genes (ABRGs) involved in the regulation of anthocyanin biosynthesis were identified by targeted search for their annotation. Subsequently, the expression of 15 putative ABRGs involved in the anthocyanin synthesis-related pathways were validated using quantitative real-time polymerase chain reaction (qRT-PCR). Of those, RsPAL2, RsCHS-B2, RsDFR1, RsDFR2, RsFLS, RsMT3 and RsUFGT73B2-like were identified significantly associated with anthocyanin biosynthesis. Especially for RsDFR1, RsDFR2 and RsFLS, of those, RsDFR1 and RsDFR2 were highest enriched in the HX-3 and WG-3, but RsFLS were down-regulated in HX-3 and WG-3. We proposed that the transcripts of RsDFR1, RsDFR2 and RsFLS might be act as key regulators in anthocyanin biosynthesis pathway.ConclusionsThe assembled radish transcript sequences were analysed to identify the key ABRGs involved in the regulation of anthocyanin biosynthesis. Additionally, the expression patterns of candidate ABRGs involved in the anthocyanin biosynthetic pathway were validated by qRT-PCR. We proposed that the transcripts of RsDFR1, RsDFR2 and RsFLS might be acted as key regulators in anthocyanin biosynthesis pathway. This study will enhance our understanding of the biosynthesis and metabolism of anthocyanin in radish.

1993-02-01·Acta virologica4区 · 医学

Detection of viral antigens on the surface of cells infected with Japanese encephalitis virus by modified immunofluorescent technique.

4区 · 医学

Article

作者: Lad, V J ; Gupta, A K ; Ghosh, S N

A modified immunofluorescent method employing anti-Japanese encephalitis (JE) virus monoclonal (MoAbs) and polyclonal (immune PF) antibodies was evaluated for the detection of viral antigens expressed on the surface of porcine stable kidney cells infected with JE virus (733913, India). The infected cells showed granular immunofluorescence on the surface with both the MoAbs, Hs-1 and Hx-3 and immune PF 24 hr and 48 hr post virus infection. Interestingly, two strains of JE virus viz. Yoken (Japan) and 755468 (India) which did not react with the MoAb Hs-1 in the standard indirect fluorescent antibody (FA) technique, were positive for surface immunofluorescence (IF) with the same MoAb. Thus, the modified technique will be useful for the detection of more labile and conformational-dependent epitopes which might get lost or denatured by prior fixation of infected cells with acetone.

1991-05-01·Acta virologica4区 · 医学

Nuclear immunofluorescence in porcine kidney cells infected with Japanese encephalitis virus.

4区 · 医学

Article

作者: Ghosh, S N ; Lad, V J ; Gupta, A K ; Gore, M M

Acetone-fixed porcine stable kidney (PS) cells infected with Japanese encephalitis (JE) virus were stained in indirect fluorescent antibody (FA) assay with anti-JE virus monoclonal (MoAb) and polyclonal (immune PF) antibodies. First positive immunofluorescence (IF) occurred in the cytoplasm with MoAb Hs-1 (anti-envelope, JE-specific) and immune PF after 7 hr post-infection (p. i.); it became prominent by 15 hr to 48 hr (maximum) when cells reacted strongly also with MoAb Hx-3 (flavivirus crossreactive epitope). In addition, 15 to 20% of the infected cells, which revealed positive cytoplasmic IF, showed intranuclear IF with Hs-1, Hx-3, and immune PF by 20 to 24 hr p.i. By 48 hr, the intranuclear IF was not observed or became diminished. These observations indicate that the JE virus specific epitope Hs-1 appeared first followed by the flavivirus cross-reactive epitope Hx-3. Nuclei of the infected cells seem to play some role in the replication of JE virus.

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