The Role of Malic Enzyme on Promoting Total Lipid and Fatty Acid Production in Phaeodactylum tricornutum.

To verify the function of malic enzyme (ME1), the ME1 gene was endogenously overexpressed in Phaeodactylum tricornutum. Overexpression of ME1 increased neutral and total lipid content and significantly increased saturated fatty acids (SFAs) and polyunsaturated fatty acids (PUFAs) in transformants, which varied between 23.19 and 25.32% in SFAs and between 49.02 and 54.04% in PUFAs, respectively. Additionally, increased ME1 activity was accompanied by elevated NADPH content in all three transformants, indicating that increased ME1 activity produced additional NADPH comparing with that of WT. These results indicated that ME1 activity is NADP-dependent and plays an important role in the NADPH levels required for lipid synthesis and fatty acid desaturation in P. tricornutum. Furthermore, our findings suggested that overexpression of endogenous ME1 represents a valid method for boosting neutral-lipid yield in diatom.

Silencing UDP-glucose pyrophosphorylase gene in Phaeodactylum tricornutum affects carbon allocation.

The effects of the suppression of UDP-glucose pyrophosphorylase (UGPase) on chrysolaminaran biosynthesis and carbon allocation were investigated in Phaeodactylum tricornutum. The 69% decrease in UGPase activity was accompanied by a 4.89 fold reduction in Ugp transcript abundance. Inactivation of UGPase in P. tricornutum led to a significant decrease in chrysolaminaran content and an increase in lipid synthesis. These findings suggest that UGPase is a rate-limiting enzyme and may play an important role in chrysolaminarin biosynthesis and carbon allocation. Our results support a theoretical deduction that Ugp is a good candidate for improving lipid synthesis in diatoms.

Identification of chilling-responsive transcripts in peanut (Arachis hypogaea L. )

To isolate differentially expressed peanut genes responsive to chilling, a suppression subtractive hybridization (SSH) cDNA library was constructed for a chilling tolerant peanut cultivar A4 with mRNAs extracted from the seeds imbibed at 2ºC and 15ºC, respectively, for 24 hrs. A total of 466 cDNA clones were sequenced, from which 193 unique transcripts (73 contigs and 120 singlets) were assembled. Of these unique transcripts, 132 (68.4 percent) were significantly similar to the sequences in GenBank non-redundant (nr) protein database, which belonged to diverse functional categories including metabolism, signal transduction, stress response, cell defense and transcriptional regulation. The remaining 61 (31.6 percent) showed no similarity to either hypothetical or known proteins. Six differentially expressed transcripts were further confirmed with real-time quantitative PCR (RT-qPCR).

[Microbial degradation of soil polycyclic aromatic hydrocarbons (PAHs) and its relations to soil bacterial population diversity].

A laboratory simulation test was conducted to study the microbial remediation of soils contaminated by medium and low concentration polycyclic aromatic hydrocarbons (PAHs), and the relationships between the degradation of PAHs and the inoculated and indigenous microbes. The addition of high-effective PAHs-degrading bacteria promoted the biodegradation of soil PAHs, and the effect was remarkable in the first two weeks. The biodegradation of test PAHs was phenanthrene < anthracene < pyrene < benzo [a] pyrene < chrysene, and negatively correlated with the diversity/abundance of soil bacterial population. In the same treatments, soil bacterial population structure varied less with time, and hence, to increase the activity of indigenous microbes would be an effective way to remediate the farmland soils contaminated by medium and low concentration PAHs.

Microsatellite DNA variation of the gametophyte clones isolated from introduced Laminaria japonica (Phaeophyta) and L. longissima of China and varieties derived from them.

The variation of 90 Laminaria gametophyte clones representing the introduced Laminaria japonica (Group 1) and Laminaria longissima (Group 2), the varieties of L. japonica (Group 3) and the varieties derived from interspecific hybrids (Group 4) was determined with 18 microsatellite markers. The allelic diversity and Nei's gene diversity of Group 1 were significantly higher than those of Group 2 (2.9 vs. 1.8 and 0.414 vs. 0.161, respectively), demonstrating that the variation of the introduced L. japonica is richer than that of L. longissima. Both allelic diversity and Nei's gene diversity of Group 3 were lower than those of Group 1, indicating that only a portion of variation of L. japonica was incorporated into the varieties of L. japonica. Significant genetic differentiation was detected between four groups and between female (Population 1) and male (Population 2) gametophyte clones in each group. The variation among groups accounted for 39.95%, while that among populations accounted for 21.65% of the total. The genetic distance between Group 1 and Group 4 was obviously longer than that between Group 2 and Group 4 (0.686 vs. 0.291), indicating that maternal gametophyte clone contributed more variation to the hybrids than the paternal gametophyte clone did.

Cloning of beta-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expression in Escherichia coli BL21 (DE3).

Beta-1,3-1,4-glucanase has been applied in the brewing and animal feed additive industry. It can effectively improve digestibility of barley-based diets and reduce enteritis. It also reduces viscosity during mashing for high-quality brewers malt. The aim of this work is to clone beta-1,3-1,4-glucanase-encoding gene and express it heterogeneously. The gene was amplified by polymerase chain reaction using Bacillus licheniformis genomic DNA as the template and ligated into the expression vector pET28a. The recombinant vector was transformed into Escherichia coli. The estimated molecular weight of the recombinant enzyme with a six-His tag at the N terminus was about 28 kDa, and its activities in cell lysate supernatant were 1,286 and 986 U ml(-1) for 1% (w/v) barley beta-glucan and 1% (w/v) lichenan, respectively. Accordingly, the specific activities were 2,479 and 1,906 U mg(-1) for these two substrates. The expression level of recombinant beta-1,3-1,4-glucanase was about 60.9% of the total protein and about 12.5% of the total soluble protein in crude cell lysate supernatant. Acidity and temperature optimal for this recombinant enzyme was pH 5.6 and 40 degrees C, respectively.

Fusion expression of bovine lactoferricin in Escherichia coli.

The drug resistance problem has been growing with the utilization of current antibiotics in feed and medical industries. LfcinB, a 25-amino acid antibacterial peptide derived from bovine lactoferrin, is one of potential alternatives of antibiotics. According to the bias of codon utilization of Escherichia coli, a fragment encoding LfcinB has been chemically synthesized, inserted into vector pGEX-4T-2 and expressed in E. coli. The antibacterial peptide was fused with GST with a protease cleavage site located between them. Two constructs with different cleavage sites were made. One construct, pGEX-Th-LfcinB, contains a thrombin cleavage site carried by the vector, and the other, pGEX-Th-Xa-LfcinB, contains a Factor Xa cleavage site which was introduced after the thrombin cleavage site. Fusion protein GST-Th-LfcinB protein was efficiently cleaved by thrombin, yielding recombinant LfcinB showing antibacterial activity. However, fusion protein GEX-Th-Xa-Lfcin B containing Factor Xa recognition site could not be cleaved by Factor Xa at the conditions tried in this study. Successful expression of LfcinB in E. coli provides a possible method to produce LfcinB in large amounts.