Choline Dehydrogenase Contributes to Salt Tolerance in Dunaliella through Betaine Synthesis.

In Dunaliella tertiolecta, a microalga renowned for its extraordinary tolerance to high salinity levels up to 4.5 M NaCl, the mechanisms underlying its stress response have largely remained a mystery. In a groundbreaking discovery, this study identifies a choline dehydrogenase enzyme, termed DtCHDH, capable of converting choline to betaine aldehyde. Remarkably, this is the first identification of such an enzyme not just in D. tertiolecta but across the entire Chlorophyta. A 3D model of DtCHDH was constructed, and molecular docking with choline was performed, revealing a potential binding site for the substrate. The enzyme was heterologously expressed in E. coli Rosetta (DE3) and subsequently purified, achieving enzyme activity of 672.2 U/mg. To elucidate the role of DtCHDH in the salt tolerance of D. tertiolecta, RNAi was employed to knock down DtCHDH gene expression. The results indicated that the Ri-12 strain exhibited compromised growth under both high and low salt conditions, along with consistent levels of DtCHDH gene expression and betaine content. Additionally, fatty acid analysis indicated that DtCHDH might also be a FAPs enzyme, catalyzing reactions with decarboxylase activity. This study not only illuminates the role of choline metabolism in D. tertiolecta's adaptation to high salinity but also identifies a novel target for enhancing the NaCl tolerance of microalgae in biotechnological applications.

Functional Characterization of a CruP-Like Isomerase in Dunaliella.

Dunaliella bardawil is a marine unicellular green algal that produces large amounts of ß-carotene and is a model organism for studying the carotenoid synthesis pathway. However, there are still many mysteries about the enzymes of the D. bardawil lycopene synthesis pathway that have not been revealed. Here, we have identified a CruP-like lycopene isomerase, named DbLyISO, and successfully cloned its gene from D. bardawil. DbLyISO showed a high homology with CruPs. We constructed a 3D model of DbLyISO and performed molecular docking with lycopene, as well as molecular dynamics testing, to identify the functional characteristics of DbLyISO. Functional activity of DbLyISO was also performed by overexpressing gene in both E. coli and D. bardawil. Results revealed that DbLyISO acted at the C-5 and C-13 positions of lycopene, catalyzing its cis-trans isomerization to produce a more stable trans structure. These results provide new ideas for the development of a carotenoid series from engineered bacteria, algae, and plants.

[Identification of Bupleurum chinense and B. longiradiatum based on ITS2 barcode].

In this study, ITS2 barcode was used to identify Bupleurum chinense and B. longiradiatum. The ITS2 regions of 48 samples were amplified and sequenced. The sequences obtained above were aligned and the K2P distances were calculated. We used three methods, BLAST1, nearest distance and phylogenetic tree (NJ-tree), to test the identification ability. The results showed that the maximum intraspecific genetic distance of B. chinense was 0.013, and the minimum interspecific genetic distance between B. chinense and B. longiradiatum was 0.049. The NJ-tree can easily identify B. chinense and B. longiradiatum. Therefore, the ITS2 barcode is suitable to identify B. chinense and B. longiradiatum.

[Identification of albiziae cortex, albiziae flos and their adulterants using ITS2 barcoding].

The ITS2 barcode was used to accurately identify Albiziae Cortex, Albiziae Flos and their adulterants in this study. A total of46 samples from Albiziae Cortex, Albiziae Flos and their adulterants were collected. The ITS2 regions were amplified and sequenced. Sequences were assembled using the CodonCode Aligner. The genetic distances of ITS2 region were calculated using MEGA 5.0. BLAST1, nearest distance and phylogenetic tree (NJ-tree) methods were used to assess the identification efficiency of the ITS2 barcode. The results revealed that the intraspecific genetic distances of Albizia julibrissin were lower than the interspecific genetic distances between A. julibrissin and its adulterants. The identification efficiency of ITS2 barcode using BLAST1 was 100%. The NJ-tree showed that A. julibrissin and their adulterants can be easily differentiated according to their monophyly. The ITS2 barcode is suitable to be as a barcode to identify Albiziae Cortex, Albiziae Flos and their adulterants.

Molecular identification of Corni Fructus and its adulterants by ITS/ITS2 sequences.

UNLABELLED: The DNA barcoding method was used to accurately and rapidly identify Corni Fructus and its adulterants. METHODS: Genomic DNA extracted from Corni Fructus and its adulterants were used as templates. The ITS (internal trascribed spacer) regions were amplified using polymerase chain reaction. Sequence assembly was performed using CodonCode Aligner V 3.5.4. Genetic distances were computed using MEGA V 5.0. Species identification was conducted using neighbor-joining (NJ) trees. RESULTS: The ITS sequence length of Corni Fructus was 659 bp. The average intra-specific genetic distance of Corni Fructus was 0.005, markedly lower than the inter-specific genetic distance between Corni Fructus and its adulterants (0.357). The ITS2 sequence length of Corni Fructus was 250 bp. No variation was found among the different samples. The interspecific genetic distance of ITS2 between Corni Fructus and its adulterants was 0.571. NJ trees and BLAST results indicated that Corni Fructus and its adulterants can be easily differentiated with monophyly. CONCLUSION: ITS/ITS2 regions can accurately and efficiently distinguish Corni Fructus and its adulterants. In addition, the results not only established the foundation for the clinical safety in the utilization of Corni Fructus, but also provided reference for molecular identification of other Chinese herbal medicine and Chinese herbal pieces.

[Principles for molecular identification of traditional Chinese materia medica using DNA barcoding].

Since the research of molecular identification of Chinese Materia Medica (CMM) using DNA barcode is rapidly developing and popularizing, the principle of this method is approved to be listed in the Supplement of the Pharmacopoeia of the People's Republic of China. Based on the study on comprehensive samples, the DNA barcoding systems have been established to identify CMM, i.e. ITS2 as a core barcode and psbA-trnH as a complementary locus for identification of planta medica, and COI as a core barcode and ITS2 as a complementary locus for identification of animal medica. This article introduced the principle of molecular identification of CMM using DNA barcoding and its drafting instructions. Furthermore, its application perspective was discussed.

[Discussion on the botanical origin of Isatidis radix and Isatidis folium based on DNA barcoding].

This paper aimed to investigate the botanical origins of Isatidis Radix and Isatidis Folium, and clarify the confusion of its classification. The second internal transcribed spacer (ITS2) of ribosomal DNA, the chloroplast matK gene of 22 samples from some major production areas were amplified and sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. Phylogenetic study was performed using MEGA 4.0 software in accordance with the Kimura 2-Parameter (K2P) model, and the phylogenetic tree was constructed using the neighbor-joining methods. The results showed that the length of ITS2 sequence of the botanical origins of Isatidis Radix and Isatidis Folium was 191 bp. The sequence showed that some samples had several SNP sites, and some samples had heterozygosis sites. In the NJ tree, based on ITS2 sequence, the studied samples were separated into two groups, and one of them was gathered with Isatis tinctoria L. The studied samples also were divided into two groups obviously based on the chloroplast matK gene. In conclusion, our results support that the botanical origins of Isatidis Radix and Isatidis Folium are Isatis indigotica Fortune, and Isatis indigotica and Isatis tinctoria are two distinct species. This study doesn't support the opinion about the combination of these two species in Flora of China.

Species identification of medicinal pteridophytes by a DNA barcode marker, the chloroplast psbA-trnH intergenic region.

Medicinal pteridophytes are an important group used in traditional Chinese medicine; however, there is no simple and universal way to differentiate various species of this group by morphological traits. A novel technology termed "DNA barcoding" could discriminate species by a standard DNA sequence with universal primers and sufficient variation. To determine whether DNA barcoding would be effective for differentiating pteridophyte species, we first analyzed five DNA sequence markers (psbA-trnH intergenic region, rbcL, rpoB, rpoC1, and matK) using six chloroplast genomic sequences from GeneBank and found psbA-trnH intergenic region the best candidate for availability of universal primers. Next, we amplified the psbA-trnH region from 79 samples of medicinal pteridophyte plants. These samples represented 51 species from 24 families, including all the authentic pteridophyte species listed in the Chinese pharmacopoeia (2005 version) and some commonly used adulterants. We found that the sequence of the psbA-trnH intergenic region can be determined with both high polymerase chain reaction (PCR) amplification efficiency (94.1%) and high direct sequencing success rate (81.3%). Combined with GeneBank data (54 species cross 12 pteridophyte families), species discriminative power analysis showed that 90.2% of species could be separated/identified successfully by the TaxonGap method in conjunction with the Basic Local Alignment Search Tool 1 (BLAST1) method. The TaxonGap method results further showed that, for 37 out of 39 separable species with at least two samples each, between-species variation was higher than the relevant within-species variation. Thus, the psbA-trnH intergenic region is a suitable DNA marker for species identification in medicinal pteridophytes.