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In recent years, methods of microalgal direct PCR have been used frequently, which simplified the DNA isolation procedure and also facilitated identification of unknown natural microalgal isolates. However, our recent attempts to amplify the 18S rRNA gene of Phaeodactylum tricornutum using the protocols reported led to poor repeatability. To explore the reasons for the instability of these methods, one-way analysis of variance was used to analyze the effects of lysis buffer type, cell number, growth stage, and lysis boiling time on the crude DNA content in the lysate. The results showed that the highest content of crude DNA (344 ng μL−1) was obtained with lysis buffer 1% NP-40 (v/v) by using stationary phase cells, which was nearly twice of the content of crude DNA with buffer 0.5% Triton X-100 (207.5 ng μL−1) and three times of those with buffers TE (107.5 ng μL−1) and 1% SDS (108 ng μL−1). It was also found that increase of the number of microalgal cells led to non-linear elevation of the crude DNA content, and the amount of crude DNA from the cells within logarithmic phase was significantly higher than those within stationary phase or decline phase. The effect of lysis boiling time was insignificant when boiling time ranged from 5 to 15 min. The results of PCR amplification with the crude DNAs isolated with different buffers indicated that efficient and repeatable amplification could be realized only when the amount of crude DNA added in 20 μL PCR reaction ranged from 50 to 200 ng, while impacts of lysis buffers themselves on the PCR reactions were negligible. Hence, it was found that the amount of crude DNA in the lysate should be measured and this was a key factor to ensure the positive PCR amplification regardless of which lysis buffer, cell number, growth stage, and lysis boiling time were used. To check the reliability and efficiency of the improved method, a pair of 18S rRNA universal primers for eukaryotic microalgae were designed, which worked very well in the PrimeBlast test and 18S rRNA amplification of microalgal species kept in the laboratory so that the primers could be widely used for microalgal screening combined with improved microalgal direct PCR. Furthermore, transformants of P. tricornutum harboring exogenous gene were screened out successfully via the improved method. Fluorescence microscope observation confirmed the screening results. This improved method can provide reliable technique for the molecular identification of microalgae species and the screen of microalgae transformants.

In Arabidopsis (Arabidopsis thaliana) plants growing under normal conditions, DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2A (DREB2A) is present at low levels because it is ubiquitinated and destabilized by DREB2A INTERACTING PROTEIN1 (DRIP1) and DRIP2 through 26S proteasome-mediated proteolysis. Drought stress counteracts the ubiquitination and proteolysis of DREB2A, thus allowing the accumulation of sufficient amounts of DREB2A protein to activate downstream gene expression. The mechanisms leading to drought stress-mediated DREB2A accumulation are still unclear. Here, we report that the wheat (Triticum aestivum) TaSAP5 protein, which contains an A20/AN1 domain, acts as an E3 ubiquitin ligase to mediate DRIP degradation and thus increase DREB2A protein levels. Drought induces TaSAP5 expression in wheat, and TaSAP5 overexpression in Arabidopsis and wheat seedlings increased their drought tolerance, as measured by survival rate and grain yield under severe drought stress. TaSAP5 can interact with and ubiquitinate TaDRIP, as well as AtDRIP1 and AtDRIP2, leading to their subsequent degradation through the 26S proteasome pathway. Consistent with this, TaSAP5 overexpression enhances DRIP degradation and increases the levels of DREB2A protein and its downstream targets. These results suggest that TaSAP5 acts to link drought with DREB2A accumulation and illustrate the molecular mechanisms involved in this process.

Astaxanthin is widely applied as a nutraceutical, pharmaceutical, and aquaculture feed additive because of its high antioxidant activity. Haematococcus pluvialis is a microalgal species that can largely accumulate astaxanthin under adverse environmental conditions. Here we review the research progress of astaxanthin biosynthesis in H. pluvialis, including the induction and regulation of massive astaxanthin, the relationship between astaxanthin synthesis, photosynthesis and lipid metabolism.虾青素是一种重要的次级类胡萝卜素，具有高活性的抗氧化功能，广泛应用于食品保健、医药、水产养殖等领域。雨生红球藻是一种在胁迫条件下能够大量积累虾青素的微藻。文中回顾了雨生红球藻虾青素的生物合成研究的进展，包括虾青素生物合成的诱导与调控、虾青素合成与光合作用及脂类代谢的关系等研究现状。.

Phytoene desaturase (PDS) is an essential constituent of several carotenoid biosynthetic enzymes, which catalyzes phytoene into ζ-carotenoids. Here, a cDNA encoding PDS gene named UpPDS in Ulva pertusa was cloned by the approaches of expressed sequence tag sequencing of suppression subtractive hybridization cDNA library and rapid amplification of cDNA ends technique. The results showed that the full-length cDNA of UpPDS was 2029 bp, containing 1599 bp open reading frame and encoding 532 amino acids. Homology analysis showed that the deduced UpPDS protein was highly homologous to the PDS proteins from some algal species. Phylogenetic analysis indicated that the UpPDS was clustered together with PDS of Ectocarpus siliculosus firstly and was more close to PDS proteins from Chlamydomonas reinhardtii and Volvox carteri. The RT-qPCR analysis indicated that the transcript level of UpPDS was upregulated during the period of emersion.

The low lipid content is one of the major bottlenecks to realize the industrialization of the algae biodiesel. Improvement of lipid content through global regulation to get high-yield generating algae is a good strategy. Leafy Cotyledon 2 (LEC2) is an important transcription factor for seed maturation and oil accumulation in Arabidopsis. However, there are few reports regarding adoption of LEC2 for lipid accumulation until now. In this study, LEC2 from Arabidopsis was cloned into the plant expression vector pCIMBIA1300 and transformed into C. sorokiniana through particle bombardment. One recombinant was screened by PCR, RT-PCR and Western blot analyses. Compared with the wild type one, the total lipid content in the recombinant increased one fold, which did not show effect on cell growth, indicating that LEC2 can efficiently enhance the lipid accumulation in C. sorokiniana.油脂含量是影响微藻产业化生产生物柴油的因素之一。基因工程的方法是培育高产藻株的一个重要手段。Leafy Cotyledon 2 (LEC2) 在拟南芥中是调节种子成熟及油脂积累的重要转录因子，在藻类植物中尚无相关的报道。本研究从拟南芥中获取LEC2 基因，构建表达载体pCIMBIA1300-35s-GFP-ATLEC2，通过基因枪介导法转入小球藻C. sorokiniana。经过PCR、RT-PCR、Western blotting 分析，筛选出一株转ATLEC2 基因藻株。对总脂肪酸含量的分析发现，转ATLEC2 基因藻株的脂肪酸含量比原始藻株提高了1 倍且没有明显影响生长。以上结果表明，ATLEC2 能促进小球藻油脂的积累。.

Hexaploid wheat (Triticum aestivum L.) is an important food crop but it is vulnerable to heat. The heat-responsive proteome of wheat remains to be fully elucidated because of previous technical and genomic limitations, and this has hindered our understanding of the mechanisms of wheat heat adaptation and advances in improving thermotolerance. Here, flag leaves of wheat during grain filling stage were subjected to high daytime temperature stress, and 258 heat-responsive proteins (HRPs) were identified with iTRAQ analysis. Enrichment analysis revealed that chlorophyll synthesis, carbon fixation, protein turnover, and redox regulation were the most remarkable heat-responsive processes. The HRPs involved in chlorophyll synthesis and carbon fixation were significantly decreased, together with severe membrane damage, demonstrating the specific effects of heat on photosynthesis of wheat leaves. In addition, the decrease in chlorophyll content may result from the decrease in HRPs involved in chlorophyll precursor synthesis. Further analysis showed that the accumulated effect of heat stress played a critical role in photosynthesis reduction, suggested that improvement in heat tolerance of photosynthesis, and extending heat tolerant period would be major research targets. The significantly accumulation of GSTs and Trxs in response to heat suggested their important roles in redox regulation, and they could be the promising candidates for improving wheat thermotolerance. In summary, our results provide new insight into wheat heat adaption and provide new perspectives on thermotolerance improvement.

Transcriptional regulation plays a key role in plant response to abiotic stresses. Study in barley indicated that a stress-responsive gene (Srg6), as potential transcriptional regulator, was induced by various abiotic stresses. However, functional roles of Srg proteins in stress tolerance are unknown. In this study, we isolated a wheat homolog of barley Srg6 gene, and designated TaSrg6. Expression analysis indicated that expression of TaSrg6 gene is induced by various stress treatments, including PEG, salt, low temperature and ABA. The fusion protein GFP-TaSrg6 was concentrated in the nucleus when transiently expressed in onion epidermal cells. Overexpression of TaSrg6 gene in Arabidopsis conferred water stress tolerance in terms of survival rate, CMS value and relative water loss. We concluded that TaSrg6 protein played an important role in the enhancement of water stress tolerance in plants.

To explore the cyclic changes of S100A10 expression in human endometrium.The gene and protein expression of S100A10 in human endometrium was examined by real-time polymerase chain reaction (PCR), immunohistochemical staining and Western blot.S100A10 protein was predominantly expressed in cytoplasm and cytomembrane of luminal and glandular epithelia. The S100A10 protein was continuously expressed from proliferative phase to secretory phase of menstrual cycle. However the expression in the secretory phase was much higher than in the proliferative phase (2.26 ± 0.17, 3.75 ± 0.22, 2.65 ± 0.20 vs 1.17 ± 0.11, 1.32 ± 0.14, both P0.05). Specifically, the highest level of S100A10 expression occurred during the mid-secretory phase. Then real-time PCR was used to analyze the mRNA expression of S100A10 in human endometrial samples at various points during the menstrual cycle. And the results confirmed the corresponding changes at the genetic level (0.50 ± 0.05, 1.02 ± 0.10, 0.55 ± 0.05 vs 0.25 ± 0.02, 0.30 ± 0.03, both P0.05).The endometrial expression of S100A10 increases during the window of embryo implantation. It suggests that it may increase the endometrial receptivity of uterine epithelial cells for embryos.

Enhanced fucosyltransferase IV (FUT4) expression correlates with increased tumor malignancy in many carcinomas. However, little is known about the regulation of FUT4 expression, and whether FUT4 expression is influenced by the methylation status of the FUT4 promoter is unclear. In this study, we demonstrated that FUT4 expression is negatively correlated with the methylation degree of a CpG island in the FUT4 promoter, suggesting that the methylation status of FUT4 promoter regulates the expression of FUT4. The results indicate that manipulating the methylation status of the FUT4 promoter to regulate FUT4 expression may be a novel approach in the treatment of malignant tumors. Electronic Supplementary Material Supplementary material is available for this article at 10.2478/s11658-012-0003-x and is accessible for authorized users.