Genetic diversity of SAD and FAD genes responsible for the fatty acid composition in flax cultivars and lines.

BACKGROUND: Flax (Linum usitatissimum L.) is grown for fiber and seed in many countries. Flax cultivars differ in the oil composition and, depending on the ratio of fatty acids, are used in pharmaceutical, food, or paint industries. It is known that genes of SAD (stearoyl-ACP desaturase) and FAD (fatty acid desaturase) families play a key role in the synthesis of fatty acids, and some alleles of these genes are associated with a certain composition of flax oil. However, data on genetic polymorphism of these genes are still insufficient. RESULTS: On the basis of the collection of the Institute for Flax (Torzhok, Russia), we formed a representative set of 84 cultivars and lines reflecting the diversity of fatty acid composition of flax oil. An approach for the determination of full-length sequences of SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes using the Illumina platform was developed and deep sequencing of the 6 genes in 84 flax samples was performed on MiSeq. The obtained high coverage (about 400x on average) enabled accurate assessment of polymorphisms in SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes and evaluation of cultivar/line heterogeneity. The highest level of genetic diversity was observed for FAD3A and FAD3B genes - 91 and 62 polymorphisms respectively. Correlation analysis revealed associations between particular variants in SAD and FAD genes and predominantly those fatty acids whose conversion they catalyze: SAD - stearic and oleic acids, FAD2 - oleic and linoleic acids, FAD3 - linoleic and linolenic acids. All except one low-linolenic flax cultivars/lines contained both the substitution of tryptophan to stop codon in the FAD3A gene and histidine to tyrosine substitution in the FAD3B gene, while samples with only one of these polymorphisms had medium content of linolenic acid and cultivars/lines without them were high-linolenic. CONCLUSIONS: Genetic polymorphism of SAD and FAD genes was evaluated in the collection of flax cultivars and lines with diverse oil composition, and associations between particular polymorphisms and the ratio of fatty acids were revealed. The achieved results are the basis for the development of marker-assisted selection and DNA-based certification of flax cultivars.

Preferential DNA photocleavage potency of Zn(II) over Ni(II) derivatives of carboxymethyl tetracationic porphyrin: the role of the mode of binding to DNA.

The porphyrin-based photosensitizers capable of binding to DNA are perspective drug candidates. Here we report the interactions with calf thymus DNA of 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin (P1) and its derivatives containing Zn(II) or Ni(II) in the coordination sphere. These interactions were studied with absorption and circular dichroism spectroscopy. NiP1 and ZnP1 formed different types of complexes with DNA. NiP1 intercalated into the double helix, whereas ZnP1 bound the DNA groove. Compound P1 displayed both binding modes. The ZnP1-DNA binding constant was approximately three times smaller than the respective values for P1-DNA and NiP1-DNA complexes. Light induced degradation of the reactive oxygen species (ROS) trap 1,3-diphenylisobenzofuran in the presence of P1 and its metal derivatives revealed that NiP1 was a weaker photooxidative agent, whereas P1 and ZnP1 generated ROS to similar extents. Nevertheless, the DNA photodamaging effect of ZnP1 was the most pronounced. Illumination of the supercoiled plasmid caused single-strand DNA photocleavage in the presence of P1 and ZnP1; double strand breaks were detectable with micromolar concentrations of ZnP1. The concentration of ZnP1 required for plasmid photonicking was two times smaller than that of P1 and ~20 times lower than that for NiP1. Thus, the modes of P1, NiP1 and ZnP1 binding to DNA determine the differential photodamaging potency of these porphyrins. A greater accessibility to the solvent of the groove binder ZnP1, compared to the shielded intercalator NiP1 and the intercalated P1 molecules, allows for an efficient local generation of ROS followed by DNA photocleavage.

The role of carboxymethyl substituents in the interaction of tetracationic porphyrins with DNA.

Cationic porphyrin-based compounds capable of interacting with DNA are currently under extensive investigation as prospective anticancer and anti-infective drugs. One of the approaches to enhancing the DNA-binding affinity of these ligands is chemical modification of functional groups of the porphyrin macrocycle. We analyzed the interaction with DNA of novel derivatives containing carboxymethyl and ethoxycarbonylmethyl substituents at quaternary nitrogen atoms of pyridinium groups at the periphery of the porphyrin macrocycle. The parameters of binding of 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin (P1) and 5,10,15,20-tetrakis(N-ethoxycarbonylmethyl-4-pyridinium)porphyrin (P2) to double-stranded DNA sequences of different nucleotide content were determined using optical spectroscopy. The association constant of P1 interaction with calf thymus DNA (K = 3.4 × 10(6) M(-1)) was greater than that of P2 (K = 2.8 × 10(5) M(-1)). Preferential binding of P1 to GC- rather than AT-rich oligonucleotides was detected. In contrast, P2 showed no preference for particular nucleotide content. Modes of binding of P1 and P2 to GC and AT duplexes were verified using the induced circular dichroism spectra. Molecular modeling confirmed an intercalative mode of interaction of P1 and P2 with CpG islands. The carboxyl groups of the peripheral substituent in P1 determine the specific interactions with GC-rich DNA regions, whereas ethoxycarbonylmethyl substituents disfavor binding to DNA. This study contributes to the understanding of the impact of peripheral substituents on the DNA-binding affinity of cationic porphyrins, which is important for the design of DNA-targeting drugs.