Chalcone-Synthase-Encoding RdCHS1 Is Involved in Flavonoid Biosynthesis in Rhododendron delavayi.

Flower color is an important ornamental feature that is often modulated by the contents of flavonoids. Chalcone synthase is the first key enzyme in the biosynthesis of flavonoids, but little is known about the role of R. delavayi CHS in flavonoid biosynthesis. In this paper, three CHS genes (RdCHS1-3) were successfully cloned from R. delavayi flowers. According to multiple sequence alignment and a phylogenetic analysis, only RdCHS1 contained all the highly conserved and important residues, which was classified into the cluster of bona fide CHSs. RdCHS1 was then subjected to further functional analysis. Real-time PCR analysis revealed that the transcripts of RdCHS1 were the highest in the leaves and lowest in the roots; this did not match the anthocyanin accumulation patterns during flower development. Biochemical characterization displayed that RdCHS1 could catalyze p-coumaroyl-CoA and malonyl-CoA molecules to produce naringenin chalcone. The physiological function of RdCHS1 was checked in Arabidopsis mutants and tobacco, and the results showed that RdCHS1 transgenes could recover the color phenotypes of the tt4 mutant and caused the tobacco flower color to change from pink to dark pink through modulating the expressions of endogenous structural and regulatory genes in the tobacco. All these results demonstrate that RdCHS1 fulfills the function of a bona fide CHS and contributes to flavonoid biosynthesis in R. delavayi.

Characterization of Two Key Flavonoid 3-O-Glycosyltransferases Involved in the Formation of Flower Color in Rhododendron Delavayi.

Flower color, largely determined by anthocyanin, is one of the most important ornamental values of Rhododendron delavayi. However, scant information of anthocyanin biosynthesis has been reported in R. delavayi. We found that anthocyanidin 3-O-glycosides were the predominant anthocyanins detected in R. delavayi flowers accounting for 93.68-96.31% of the total anthocyanins during its development, which indicated the key role of flavonoid 3-O-glycosyltransferase (3GT) on R. delavayi flower color formation. Subsequently, based on correlation analysis between anthocyanins accumulation and Rd3GTs expressions during flower development, Rd3GT1 and Rd3GT6 were preliminarily identified as the pivotal 3GT genes involved in the formation of color of R. delavayi flower. Tissue-specific expressions of Rd3GT1 and Rd3GT6 were examined, and their function as 3GT in vivo was confirmed through introducing into Arabidopsis UGT78D2 mutant and Nicotiana tabacum plants. Furthermore, biochemical characterizations showed that both Rd3GT1 and Rd3GT6 could catalyze the addition of UDP-sugar to the 3-OH of anthocyanidin, and preferred UDP-Gal as their sugar donor and cyanidin as the most efficient substrate. This study not only provides insights into the biosynthesis of anthocyanin in R. delavayi, but also makes contribution to understand the mechanisms of its flower color formation.

Functional analysis of a dihydroflavonol 4-reductase gene in Ophiorrhiza japonica (OjDFR1) reveals its role in the regulation of anthocyanin.

Dihydroflavonol 4-reductase (DFR), a key regulatory enzyme, participated in the biosynthesis of anthocyanins, proanthocyanidins and other flavonoids that essential for plant survival and human health. However, the role of this enzyme in Ophiorrhiza japonica is still unknown. Here, three putative DFR-like genes were firstly isolated from O. japonica. Phylogenetic analysis indicated that OjDFR1 was classified into DFR subgroup, while the rest two were clustered into other NADPH-dependent reductases. Then, functions of the three genes were further characterized. Expression analysis showed that OjDFR1 transcripts had strong correlations with the accumulation pattern of anthocyanin during the flower developmental, whereas other two were not, this suggested the potential roles of OjDFR1 in anthocyanin biosynthesis. Subsequently, all three clones were functionally expressed in Escherichia coli, but confirming that only OjDFR1 encode active DFR proteins that catalyzed the reduction of dihydroflavonols to leucoanthocyanidin. Consistant with the biochemical assay results, overexpressing OjDFR1 in Arabidopsis tt3-1 mutant successfully restored the deficiency of anthocyanin and proanthocyanidin, hinting its function as DFR in planta. Additionally, heterologous expression of OjDFR1 in transgenic tobacco contributed to darker flower color via up-regulating the expressions of endogenous NtANS and NtUFGT, which suggested that OjDFR1 was involved in flower color development. In summary, this study validates the functions of OjDFR1 and expands our understanding of anthocyanin biosynthesis in O. japonica.

Characterization and functional analysis of RdDFR1 regulation on flower color formation in Rhododendron delavayi.

Rhododendron delavayi is a popular ornamental plant with globular flowers noted for their bright red color, but very limited studies have been reported on its flower color formation. In this study, we successfully isolated a novel DFR gene (RdDFR1) from red flowers of Rhododendron delavayi. Multiple sequence alignments revealed that RdDFR1 had the conserved NADP and substrate binding domain, and was classified into Asn-type DFR. Meanwhile, quantitative real-time PCR analysis showed that transcript levels of RdDFR1 matched the accumulation patterns of anthocyanins during flower development, hinting its potential role involved in anthocyanin biosynthesis. Then in vitro enzymatic analysis indicated that recombinant RdDFR1 protein could catalyze the production of leucoanthocyanidins from dihydroquercetin and dihydromyricetin. Furthermore, the in planta assay, using Arabidopsis thaliana dfr mutant (tt3-1) and tobacco, displayed that RdDFR1 transgenes recovered the defective proanthocyanidin and anthocyanin biosynthesis at seed coats, hypocotyl as well as cotyledon, and altered the flowers color of tobacco from pale pink to dark pink which demonstrated its function as dihydroflavonol 4-reductase in vivo. In summary, our findings suggest that RdDFR1 plays a crucial role in the biosynthesis of anthocyanin and will also make a contribution to understand the mechanisms of flower color formation in Rhododendron delavayi.

Ultrasensitive rapid detection of human serum antibody biomarkers by biomarker-capturing viral nanofibers.

Candida albicans (C. albicans) infection causes high mortality rates within cancer patients. Due to the low sensitivity of the current diagnosis systems, a new sensitive detection method is needed for its diagnosis. Toward this end, here we exploited the capability of genetically displaying two functional peptides, one responsible for recognizing the biomarker for the infection (antisecreted aspartyl proteinase 2 IgG antibody) in the sera of cancer patients and another for binding magnetic nanoparticles (MNPs), on a single filamentous fd phage, a human-safe bacteria-specific virus. The resultant phage is first decorated with MNPs and then captures the biomarker from the sera. The phage-bound biomarker is then magnetically enriched and biochemically detected. This method greatly increases the sensitivity and specificity of the biomarker detection. The average detection time for each serum sample is only about 6 h, much shorter than the clinically used gold standard method, which takes about 1 week. The detection limit of our nanobiotechnological method is approximately 1.1 pg/mL, about 2 orders of magnitude lower than that of the traditional antigen-based method, opening up a new avenue to virus-based disease diagnosis.

Nontoxic virus nanofibers improve the detection sensitivity for the anti-p53 antibody, a biomarker in cancer patients.

The presence of anti-p53 antibody in serum is a biomarker for cancer. However, its high sensitivity detection is still an issue in cancer diagnosis. To tackle this challenge, we used fd phage, a human-safe bacteria-specific virus nanofiber that can be mass-produced by infecting host bacteria in an error-free manner, and genetically engineered it to display a peptide capable of recognizing and capturing anti-p53 antibody on its side wall. We employed the resultant phage nanofibers as a capture probe to develop a modified version of the enzyme-linked immunosorbent assay (ELISA) method, termed phage-ELISA. We compared it to the traditional ELISA method for the detection of anti-p53 antibody, p53-ELISA, which uses recombinant wild-type p53 protein to capture anti-p53 antibody. We applied phage-ELISA to detect anti-p53 antibody in an experimental group of 316 patients with various types of malignant tumors. We found that a detection rate of 17.7% (56 positive cases) was achieved by phage-ELISA, which was comparable to the detection rate of 20.6% for p53-ELISA (65 positive cases). However, when both phage and p53 were combined to form antibody-capturing probes for phage/p53-ELISA, a detection rate of 30.4% (96 positive cases) was achieved. Our work showed that owing to the combined capture of the anti-p53 antibody by both phage nanofibers and p53, the phage/p53-ELISA achieved the highest diagnostic accuracy and detection efficiency for the anti-p53 antibody in patients with various types of cancers. Our work suggests that a combination of nanofibers and antigens, both of which capture antibody, could lead to increased detection sensitivity, which is useful for applications in the life sciences, clinical medicine, and environmental sciences.

Detection of serum anti-P53 antibodies from patients with colorectal cancer in China using a combination of P53- and phage-ELISA: correlation to clinical parameters.

BACKGROUND: Colorectal cancer is one of the most common malignant tumors in China. The aims of this research were to increase the sensitivity of anti-p53 antibody detection in the sera of patients with colorectal cancer and to assist in their diagnosis. METHODS: Sixty-seven non-selected Chinese with colorectal cancer were involved in this study. Anti-p53 antibodies in serum were detected by ELISA using recombinant human wild- type p53 protein and hybrid phage as the coating antigen. Correlations between the anti-p53 antibodies and clinicopathological parameters were also analyzed. RESULTS: The detection efficiency of anti-p53 antibodies in the patients with colorectal cancer was increased (46.3%, 31/67) through the combination of the two ELISA methods compared with each method alone. The titer of serum anti-p53 antibodies was not associated with clinicopathological parameters, but there was a significant correlation between their presence, the CEA level, and the stage of the patient's colorectal cancer. CONCLUSIONS: These results demonstrate that combination of the two ELISA methods increased the detection rate of anti-p53 antibodies in patients with colorectal cancer. This research may provide a useful method to complement conventional clinical diagnosis.