Cloning and expression analysis of ANR genes from different species of Lonicera japonica Thunb / 药学学报

Anthocyanidin reductase (ANR) is one of the key enzyme in the flavonoid biosynthetic pathway, and its catalytic activity is important for the synthesis of plant anthocyanin. In this study, specific primers were designed according to the transcriptome data of Lonicera japonica Thunb., and the CDS, gDNA and promoter sequences of ANR genes from Lonicera japonica Thunb. and Lonicera japonica Thunb. var. chinensis (Wats.) Bak. were cloned. The results showed that the CDS sequences of LjANR and rLjANR were 1 002 bp, the gDNA sequences were 2 017 and 2 026 bp respectively, and the promoter sequences were 1 170 and 1 164 bp respectively. LjANR and rLjANR both contain 6 exons and 5 introns, which have the same length of exons and large differences in introns. The promoter sequences both contain a large number of light response, hormone response and abiotic stress response elements. Bioinformatics analysis showed that both LjANR and rLjANR encoded 333 amino acids and were predicted to be stable hydrophobic proteins without transmembrane segments and signal peptides. The secondary structures of LjANR and rLjANR were predicted to be mainly consisted of α-helix and random coil. Sequence alignment and phylogenetic analysis showed that LjANR and rLjANR had high homology with Actinidia chinensis var. chinensis, Camellia sinensis and Camellia oleifera, and were closely related to them. The expression levels of LjANR and rLjANR were the highest in flower buds and the lowest in roots. The expression patterns at different flowering stages were similar, with higher expression levels in S1 and S2 stages and then gradually decreased until reaching the lowest level in S4 stage, after a slow increase in S5 stage, the expression levels decreased again. The expression levels of ANR genes in the two varieties showed significant differences in roots, S2 and S5 stages, while the differences in stems, flower buds, S1, S3 and S6 stages were extremely significant. The prokaryotic expression vector pET-32a-LjANR was constructed for protein expression. The target protein was successfully expressed of about 59 kD. This study lays a foundation for further study on the function of ANR gene and provides theoretical guidance for breeding new varieties of Lonicera japonica Thunb.

Function analysis of anthocyanidin synthase from Morus alba L. by expression in bacteria and tobacco

Background: Flavonoids are a kind of important secondary metabolite and are commonly considered to provide protection to plants against stress and UV-B for a long time. Anthocyanidin synthase (ANS), which encodes a dioxygenase in the flavonoid pathway, catalyzes the conversion of leucoanthocyanidins to anthocyanidins, but there is no direct evidence indicating that it provides tolerance to stress in plants. Results: To investigate whether ANS can increase tolerance to abiotic stress, MaANS was isolated from mulberry fruits and transformed into tobacco. Our results suggested that the bacterially expressed MaANS protein can convert dihydroquercetin to quercetin. Overexpression of MaANS remarkably increased the accumulation of total flavonoids in transgenic lines and anthocyanins in corollas of flowers. Transgenic lines showed higher tolerance to NaCl and mannitol stress. Conclusions: These results indicated that MaANS participates in various dioxygenase activities, and it can protect plants against abiotic stress by improving the ROS-scavenging ability. Thus, this alternative approach in crop breeding can be considered in the improvement of stress tolerance by enriching flavonoid production in plants

Protective effect of anthocyanidin extracts on light-induced retinal functional and structural damage in rat / 中华实验眼科杂志

Background Light leads to the damage of retinal function and structure by promoting the reproduction of radicals and lipid peroxides when retina is exposed to an intense light environment for a long time.It is necessary to study how to protect the retina against light-induced injury in ophthalmology.Objective The aim of this study was to evaluate the effect of anthocyanidin extracts in preventing retinal photic damage.Methods Eighteen clean male Sprague-Dawley (SD) rats were randomly assigned to normal saline group,anthocyanidin group and mixed (hydrogen-rich saline (HRS) + anthocyanidin,1:1 in volume) group and 6 rats for each.The electroretinogram (ERG) with international standardized 5 items was recorded from all the rats before experiment.Normal saline,anthocyanidin extracts or mixed solution of 5 ml/kg were intraperitoneally injected in the three groups,respectively,for consecutive 5 days.Then the diffused light with the luminance intensity of (5000±300)lx was used to irradiate the right eyes of the rats for consecutive 3 hours during 19:00 through 7:00 in a device made by our laboratory,and the left eyes were covered at the same time.The ERG was repeatedly recorded 5 days after light irradiation.The rats were sacrificed at the end of the experiment and retinal sections were prepared for the histopathological examination.The functional and structural changes of retinas were compared among the different groups.The use of the rats followed the Statement of ARVO.Results The differences of rat body weight were not statistically significant among the three groups (before experiment:F =0.472,P =0.841 ; after experiment:F =0.658,P=0.762).No any significant difference was found in scotopic 0.01 ERG b wave,scotopic 3.0 ERG a and b waves,scotopic 3.0 oscillatory potentials,photopic 3.0 ERG b wave and 3.0 flicker P1wave between the left eyes and the right eyes in the three groups before experiment (P＞0.05).The amplitudes of various waves of ERG were significantly declined in the right eyes compared with the left eyes (P＜0.05).The mean differential values of scotopic 0.01 ERG b wave,scotopic 3.0 ERG a and b waves,scotopic 3.0 oscillatory potentials were significantly different(F =4.594,P=0.029; F=3.834,P=0.037; F=12.823,P=0.000; F=3.976,P=0.032),but those in photopic 3.0 ERG b wave were not statistically significant (F =1.488,P =0.259).Compared with the normal saline group,the differential values of scotopic 0.01 ERG b wave,scotopic 3.0 ERG a and b waves,scotopic 3.0 oscillatory potentials were all reduced in the anthocyanidin group and mixed group (P ＜ 0.05).The cells of outer nuclear layer were decreased in the right eyes in the three groups compared with the left eyes,especially around the optic nerve head and the upside of the retina,with significant differences between them (P＜0.05),and those in the anthocyanidin group and mixed group were significantly less than normal saline group (P＜0.05).Conclusions Anthocyanidin has a protective effect on light-induced retinal damage of SD rats.The protective effect of anthocyanidin extracts is similar to the integrated effect of the mixed group.

Identification of Anthocyanin from The Extract of Soybean Seedcoat

Anthocyanins are naturally occuring phytochemicals and the main components of the coloring of plants, flowers and fruits. They are known to elicit antioxidative, anti-inflammatory and cancer preventive activity. In this study, we investigated anthocyanins in black / yellow soybean seedcoats using different methods of detection - thin layer chromatography (TLC), capillary zone electrophoresis (CZE) and HPLC analysis. The anthocyanins in soybean seedcoats were extracted by five independent methods of extraction and the aglycons (anthocyanidins) of the corresponding anthocyanins were prepared by acid mediated hydrolysis. The anthocyanin / anthocyanidin in black soybean seedcoat showed characteristic TLC mobility, CZE electrophoretic retention and HPLC migration time while little of anthocyanins were detected from yellow soybean seedcoat. The extracted anthocyanins showed pH dependent retention time in CZE and spectral change in UV-Vis spectrum. HPLC analysis of the hydrolyzed extract of black soybean seedcoat identified the presence of four anthocyanidins. The major anthocyanin in black soybean seedcoat was cyanin (cyanidin-3-O-glucoside), with the relative order of anthocyanidin in cyanidin > delphinidin > petunidin > pelargonidin.

Studies on the properties of the anthocyanidin in Suaeda hete-roptera of halophyte / 中国海洋药物

Objective The properties of anthocyanins in Suaeda heteroptera Kitag.were investigated.Methods Properties of the extract were determined via spectral absorption,physco-chemical analyses.Results Spectrogram results showed that the red pigment was classified to anthocyanins.The color properties of the pigment were similar to plant anthocyanins and pH changes could affect the tone of the color.The pigment was soluble in water,alcohol;slightly soluble in acetone;and insoluble in petroleum ether.Under natural light,degradation of the pigment could be accelerated.High temperature was disadvantage to the pigment stability.Redox stability was very low.Fe~(2+),Mg~(2+),Cu~(2+) and Mn~(2+) did not significant affect the tone of the color.Conclusion The pigment from Suaeda heteroptera Kitag.is water-soluble anthocyanins,and may be important nature edible pigment resource.