Optimization of an HPLC Method for Determining the Genomic Methylation Levels of Taxus Cells.

An HPLC method for quantifying total DNA methylation in Taxus chinensis cells is described. Optimal conditions for the method were established as follows: DNA was hydrolyzed with DNA degradase at 37°C for 3 h. The mobile phase was a mixture of Solvent A [50 mM potassium dihydrogen phosphate/triethylamine (100:0.2, v/v)] and Solvent B (methanol); the gradient was 10% (v/v) solvent B. The calibration curves for deoxycytidine monophosphate (dCMP) and methylated dCMP were linear within 1.0-160.0 µg mL(-1), with correlation coefficients of 0.9996 and 0.9998. The limits of detection for dCMP and 5-mdCMP were 0.482 and 0.301 ng mL(-1), respectively, and the limits of quantification were 1.6 and 1.0 ng mL(-1), respectively. The method has been validated according to the current International Conference Harmonization guidelines. The method was able to quantify the content of dCMP and methylated dCMP specifically, accurately and precisely. The global DNA methylation level in different Taxus cells was measured using as little as 3 µg of DNA according to the optimized procedure. In addition, degradation of 5-methylcytosine was prevented.

Proteomic and comparative genomic analysis of two Brassica napus lines differing in oil content.

Ultrastructural observations, combined with proteomic and comparative genomic analyses, were applied to interpret the differences in protein composition and oil-body characteristics of mature seed of two Brassica napus lines with high and low oil contents of 55.19% and 36.49%, respectively. The results showed that oil bodies were arranged much closer in the high than in the low oil content line, and differences in cell size and thickness of cell walls were also observed. There were 119 and 32 differentially expressed proteins (DEPs) of total and oil-body proteins identified. The 119 DEPs of total protein were mainly involved in the oil-related, dehydration-related, storage and defense/disease, and some of these may be related to oil formation. The DEPs involved with dehydration-related were both detected in total and oil-body proteins for high and low oil lines and may be correlated with the number and size of oil bodies in the different lines. Some genes that corresponded to DEPs were confirmed by quantitative trait loci (QTL) mapping analysis for oil content. The results revealed that some candidate genes deduced from DEPs were located in the confidence intervals of QTL for oil content. Finally, the function of one gene that coded storage protein was verified by using a collection of Arabidopsis lines that can conditionally express the full length cDNA from developing seeds of B. napus.

Improvement of arachidonic acid and eicosapentaenoic acid production by increasing the copy number of the genes encoding fatty acid desaturase and elongase into Pichia pastoris.

Genes encoding Delta6 desaturase, Delta6 fatty acid elongase, and Delta5 desaturase from the alga, Phaeodactylum tricornutum, were co-expressed in Pichia pastoris to produce arachidonic acid (ARA; 20:4 Delta(5, 8, 11, 14)) and eicosapentaenoic acid (EPA; 20:5 Delta(5, 8, 11, 14, 17)). A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. ARA and EPA accumulated up to 0.3 and 0.1% of total fatty acids, respectively, in the recombinant P. pastoris carrying with double copies of these three heterologous genes, as compared to 0.1 and 0.05%, respectively, in the recombinant P. pastoris with single copy.

Cloning and expression of Brassica napus beta-carbonic anhydrase cDNA.

A new full-length beta-carbonic anhydrase cDNA was obtained from Brassica napus by homologous cloning. The cDNA has an open-reading frame of 996 nucleotides, encoding 331 amino acids with a calculated molecular weight of 35,692 Da and an estimated pI value of 5.459. The deduced amino acid sequence of beta-carbonic anhydrase from Brassica napus shared significant identity with beta-carbonic anhydrases from Brassica carinata, Arabidopsis thaliana, and Thlaspi caerulescens (97.9%, 94%, and 93.5% identity, respectively). This cDNA was expressed in Escherichia coli BL21 (DE3) using the expression vector pET-32a(+). The expression band corresponded to the calculated mass plus the N-terminal fusion protein derived from the vector.

[The heredity of flower colors and the discovery of flower color chimera in chrysanthemum species].

The reciprocal crosses of yellow colored chrysanthemum x red colored chrysanthemum and white colored chrysanthemum x red colored chrysanthemum were conducted in order to analyze the heredity of flower colors. The results revealed that the heredity of flower colors was very complicated, and mainly exhibited matroclinous characteristics when red colored materials was used as maternal parent but not in the combinations when the yellow or white colored materials were used as maternal parents. The incomplete dominance and mosaic dominance also existed in the heredity of chrysanthemum flower colors. The flower-color chimeras with two kinds of flower buds were discovered in the cross of 3501 x 3509, i.e. one side of the flower buds was completely in red color, which was same as the parental material of 3509, and another side was generally in yellow color with red spots on them. Cytological analysis showed that two sides were both with 36 chromosomes, indicating that the formation of chimera was not resulted from the changes of chromosome numbers, but from the destruction of pigment synthesis genes by the insert of transposable element.

[Development of relationship between A, B and C genomes in Brassica genera].

The study on genetic relationship among A, B and C genomes in Brassica genera has gained prodigious development, which revealed that the relationship between A and C genome was more closer than that of A and B, B and C genome. The results of comparative genomics showed that A, B and C genomes were all originated from a common ancestral genome. A lot of chromosome variations were taken place in the evolution of Brassica genomes, such as duplication, deletion and rearrangement, resulting in the difference of genomes. At last, the genetic relationship between Brassica genera and Arabidopsis thaliana was summarized.