miR5298b regulated taxol biosynthesis by acting on TcNPR3, resulting in an alleviation of the strong inhibition of the TcNPR3-TcTGA6 complex in Taxus chinensis.

Taxol, a valuable but rare secondary metabolite of the genus Taxus, is an effective anticancer drug. Understanding the regulation of taxol biosynthesis may provide a means to increase taxol content. The microRNA miR5298b was found to promote the accumulation of taxol and upregulate several taxol biosynthesis genes, including DBAT, TASY, and T5H, as demonstrated by experiments using the overexpression and mimicry of transient leaves. Moreover, miR5298b cleaves the mRNA sequence of TcNPR3, a homolog of the salicylic acid receptor AtNPR3/4. Overexpression and knockdown by RNA interference of TcNPR3 confirmed that it repressed taxol biosynthesis. These results indicate that miR5298b enhances taxol biosynthesis via the cleavage of TcNPR3. Yeast two-hybrid bimolecular fluorescence complementation and pull-down assays revealed that TcTGA6, a TGA transcription factor, physically interacted with TcNPR3. Functional experiments showed that TcTGA6 negatively regulates taxol biosynthesis by directly combining with the TGACG motif in the promoters of TASY, T5H, and T10H. TcNPR3 enhances TcTGA6 inhibition Luciferase assays showed that miR5298b alleviated the repression of the TcNPR3-TcTGA6 complex. In summary, miR5298b can cleave TcNPR3, thereby alleviating the inhibition of the TcNPR3-TcTGA6 complex to upregulate taxol biosynthesis genes.

Camptothecin distribution and content in Nothapodytes nimmoniana.

Nothapodytes nimmoniana is an important anti-cancer medicinal plant which possesses various bioactive substances, of which camptothecin (CPT) is the most important. CPT is mainly obtained by extraction from plants. However, extraction is problematic because of the lack of reference information, which leads to a large number of trees being felled. In this study, CPT was detected in different parts (shoots, two- and three-year-old stems, tender leaves, mature leaves, senescent leaves, roots and bark) of N. nimmoniana. A high performance liquid chromatographic (HPLC) method was used to determine the CPT content. All parts analyzed contained CPT, but concentrations depended on the different growth phases of the stems and leaves. The highest CPT content was 1.87 mg/g from roots, followed by the shoots, with a content of 1.07 mg/g. With a view to resource utilization, we concluded that the shoots were the best tissues for exploitation. Suggestions for shoot picking are also provided.

TLC and HPLC methods to follow the synthesis of vinorelbine.

The thin-layer chromatography (TLC) combined with high-performance liquid chromatography (HPLC) has been proved to be a quick and valid method to detect the intermediates and the end-product created during the chemosynthesis process of vinorelbine (VB). This paper gives a detailed investigation on the results of two determination methods when the condition of the detection changed. It shows that when TLC developer is consisted of petroleum ether, chloroform, acetone, and diethyl amine (23.5:12:2:2.5, v/v/v/v), vinblastine sulfate (VBS), anhydrovinblastine (AHVB), and VB can be separated specifically. When the mobile phase of HPLC is a mixture of methyl alcohol, acetonitrile, diethyl amine, and high purity water (420:252:3:225; v/v/v/v), adjusted with orthophosphoric acid to pH 6.5, the intermediates and the resultants of the chemosynthesis of VB can be determined effectively. It can also be used to fix quantify of the resultants. The calibration curve for VB shows good linearity in the two mass concentration ranges of 0.0100-0.0500 mg/mL (r = 0.9956) and 0.00600-0.0100 mg/mL (r = 0.9978), respectively. The limit of detection of HPLC for VB is 0.200 microg/mL.

Isolation of magnetotactic bacterium WM-1 from freshwater sediment and phylogenetic characterization.

The magnetotactic bacterium was isolated from freshwater sediment from North Lake of Wuhan. The isolate, designated WM-1, was Gram-negative, helical shaped, and studied by means of electron microscopy. The strain WM-1 was 0.2-0.4 microm in diameter and 3-4 microm in length. The DNA G + C content was found to be 65.7 mol%. Phylogenetic analysis of the 16S rDNA gene (Accession number DQ899734 in GeneBank) revealed that this isolate was a member ofalphasubdivision of the Proteobacteria. Strain WM-1 was closely related (97.7%) to Magnetospirillum sp. AMB-1. Randomly amplified polymorphic DNA analysis showed that these two strains were in fact different strains. Electron diffraction patterns of WM-1 magnetosomes indicated that the magnetosomes were composed of magnetite. The magnetosomes from WM-1 were cuboidal in shape as observed by electron microscopy. Statistical analysis of magnetite crystals from WM-1 showed narrow asymmetric size distribution. The average number of magnetosomes in each WM-1 bacterium was 8 +/- 3.4. The average length of magnetosomes in WM-1 was 54 +/- 12.3 nm and the average width is 43 +/- 10.9 nm. These data showed that the grains in WM-1 were single-domain crystals.

A simple DNA extraction method for PCR amplification from dry seeds of Brassica napus.

A simple and reliable DNA extraction method for dry seeds of Brassica napus has been developed in our laboratory. The NaCl and PVP were used to remove polysaccharides and polyphenols during DNA purification. The oil and proteins of dry seeds were removed only through centrifugation in this method. The RAPD amplification patterns have no obviously difference between the DNA extracted from dry seeds and fresh leaves extracted with control method. The good results of SSR molecular markers on the DNA of dry seeds of another 12 B. napus indicating that the DNA extracted from dry seeds was freedom from common contaminating compounds. In conclusion, this method could be widely used in DNA extraction from dry seeds of B. napus.