Identification and expression analysis of WRKY gene family in eukaryotic algae / 生物工程学报

WRKY is a superfamily of plant-specific transcription factors, playing a critical regulatory role in multiple biological processes such as plant growth and development, metabolism, and responses to biotic and abiotic stresses. Although WRKY genes have been characterized in a variety of higher plants, little is known about them in eukaryotic algae, which are close to higher plants in evolution. To fully characterize algal WRKY family members, we carried out multiple sequence alignment, phylogenetic analysis, and conserved domain prediction to identify the WRKY genes in the genomes of 30 algal species. A total of 24 WRKY members were identified in Chlorophyta, whereas no WRKY member was detected in Rhodophyta, Glaucophyta, or Bacillariophyta. The 24 WRKY members were classified into Ⅰ, Ⅱa, Ⅱb and R groups, with a conserved heptapeptide domain WRKYGQ(E/A/H/N)K and a zinc finger motif C-X4-5-C-X22-23-H-X-H. Haematococcus pluvialis, a high producer of natural astaxanthin, contained two WRKY members (HaeWRKY-1 and HaeWRKY-2). Furthermore, the coding sequences of HaeWRKY-1 and HaeWRKY-2 genes were cloned and then inserted into prokaryotic expression vector. The recombinant vectors were induced to express in Escherichia coli BL21(DE3) cells and the fusion proteins were purified by Ni-NTA affinity chromatography. HaeWRKY-1 had significantly higher expression level than HaeWRKY-2 in H. pluvialis cultured under normal conditions. High light stress significantly up-regulated the expression of HaeWRKY-1 while down-regulated that of HaeWRKY-2. The promoters of HaeWRKY genes contained multiple cis-elements responsive to light, ethylene, ABA, and stresses. Particularly, the promoter of HaeWRKY-2 contained no W-box specific for WRKY binding. However, the W-box was detected in the promoters of HaeWRKY-1 and the key enzyme genes HaeBKT (β-carotene ketolase) and HaePSY (phytoene synthase) responsible for astaxanthin biosynthesis. Considering these findings and the research progress in the related fields, we hypothesized that the low expression of HaeWRKY-2 under high light stress may lead to the up-regulation of HaeWRKY-1 expression. HaeWRKY-1 may then up-regulate the expression of the key genes (HaeBKT, HaePSY, etc.) for astaxanthin biosynthesis, consequently promoting astaxanthin enrichment in algal cells. The findings provide new insights into further analysis of the regulatory mechanism of astaxanthin biosynthesis and high light stress response of H. pluvialis.

Transcriptome analysis of signal transduction pathway involved in light inducing astaxanthin accumulation in Haematococcus pluvialis / 生物工程学报

The unicellular green alga Haematococcus pluvialis is the best source of natural astaxanthin (AST) in the world due to its high content under stress conditions. Although high light (HL) can effectively induce AST biosynthesis, the specific mechanisms of light signal perception and transduction are unclear. In the current study, we used transcriptomic data of normal (N), high white light (W), and high blue light (B) to study the mechanisms of light inducing AST accumulation from the point of photoreceptors. The original data of 4.0 G, 3.8 G, and 3.6 G for N, W, and B were obtained, respectively, by the Illumina Hi-seq 2000 sequencing technology. Totally, 51 954 unigenes (at least 200 bp in length) were generated, of which, 20 537 unigenes were annotated into at least one database (NR, NT, KO, SwissProt, Pfam, GO, or KOG). There were 1 255 DEGs in the W vs N, 1 494 DEGs in the B vs N, and 1 008 DEGs in the both W vs N and B vs N. KEGG enrichment analysis revealed that photosynthesis, oxidative phosphorylation, carotenoid biosynthesis, fatty acids biosynthesis, DNA replication, nitrogen metabolism, and carbon metabolism were the significantly enriched pathways. Moreover, a large number of genes encoding photoreceptors and predicted interacting proteins were predicted in Haematococcus transcriptome data. These genes showed significant differences at transcriptional expression levels. In addition, 15 related DEGs were selected and tested by qRT-PCR and the results were significantly correlated with the transcriptome data. The above results indicate that the signal transduction pathway of "light signal - photoreceptors - interaction proteins - (interaction proteins - transcription factor/transcriptional regulator) - gene expression - AST accumulation" might play important roles in the regulation process, and provide reference for further understanding the transcriptional regulation mechanisms of AST accumulation under HL stress.

Preparation and Stability of Levocetirizine Hydrochloride Chewable Tablets / 中国药房

OBJECTIVE:To prepare Levocetirizine hydrochloride chewable tablets,and to investigate the stability. METH-ODS:Levocetirizine hydrochloride chewable tablets were prepared with wet granulation. Using accumulative dissolution rate within 45 min as index,the formulation of Levocetirizine hydrochloride chewable tablets was optimized by orthogonal design with the in-ternal and external ratio of MCC and carboxymethyl starch sodium,the amount of magnesium stearate as factors. The dissolution rate and content uniformity of optimized tablet were verified. The appearance,dissolution rate,related substance and content change of the tablet were investigated within 10 d under the condition of high temperature(60 ℃),high light(4 500 lx)and high moisture (92.5%). RESULTS:The optimized formulation of the tablet was as levocetirizine hydrochloride 5 mg,lactose 30 mg, microcrystalline cellulose 90 mg(internal-external ratio 4∶5),mannitol 60 mg,aspartame 10 mg,carboxymethyl starch sodium 12 mg(internal-external ratio 1∶1)and magnesium stearate 1.0%. The accumulative dissolution rates of 3 batches of optimized tablet were(97.23±1.21)%,(98.49±1.28)% and(98.15±1.94)%. The content uniformity were 2.30,2.34 and 2.60. Those indicators had no significant change except related substance increased slightly under high temperature on 10th day and high moisture on 5th day. CONCLUSIONS:Levocetirizine hydrochloride chewable tablets is prepared successfully with good stability.

The comparison of reticulocyte parameters on the XN-1000 and LH780 hematology analyzer / 国际检验医学杂志

Objective To analyze the comparability and correlation of reticulocyte parameters on the Beckman‐Coulter LH780 (LH780)and Sysmex XN‐1000(XN‐1000)hematology analyzers .Methods 80 blood samples were measured by the two instru‐ments ,the RBC count ,percentage of Ret(Ret% ) ,Ret value(Ret#)and immature reticulocyte fraction(IRF) were analyzed ,and the correlation of Ret% ,Ret# and IRF between two instruments were also analysed .The correlation between percentage of high laser reticulocyte(HLR% ) of LH780 and percentage of middle fluorescent reticulogyte(MFR% )+percentage of high fluorescent reticu‐locyte(HFR% ) of XN‐1000 were compared using two calculation methods of each instrument ,and the application value of HLR%were analysed .Results No significant differences were founded in RBC count ,Ret% ,and Ret# between the two instruments(P>0 .05) ,while there was statistical difference in IRF between the the two instruments (P<0 .05) .The relative deviation coincidence rate of RBC count was 97 .5% ,the correlation coefficent (r) of Ret% ,Ret# and IRF were 0 .912 ,0 .895 and 0 .666 respectively . There were statistical differences and correlations between HLR% and MFR% + HFR% when using calculation method of XN‐1000 and LH780 respectively(r were 0 .666 and 0 .767 respectively ,P<0 .05) .Conclusion The RBC count could meet the matc‐hing requirement ,and the Ret% and Ret # may be highly correlated on the two instruments .While the reference range of IRF should be established in each instrument .

Alteration in the acceptor side of photosystem II of chloroplast by high light.

The effect of high light on the acceptor side of photosystem II of chloroplasts and core particles of spinach was studied. Both Vmax and apparent Km for DCIP were altered in photoinhibited photosystem II core particles. The double reciprocal plot analysis as a function of actinic light showed increased slope in chloroplasts photoinhibited in the presence of DCMU. Exposure of chloroplasts to high light in the presence of DCMU did not protect the chloroplast against high light induced decrease in Fm, level. Further the high light stress induced decrease in Fm level was not restored by the addition of DCMU. These results suggest that the high light stress induced damage to chloroplast involves alteration in the binding site for QB on the DI protein on the acceptor side of photosystem II.