Medium-Sized Ring Expansion Strategies: Enhancing Small-Molecule Library Development.

The construction of a small molecule library that includes compounds with medium-sized rings is increasingly essential in drug discovery. These compounds are essential for identifying novel therapeutic agents capable of targeting "undruggable" targets through high-throughput and high-content screening, given their structural complexity and diversity. However, synthesizing medium-sized rings presents notable challenges, particularly with direct cyclization methods, due to issues such as transannular strain and reduced degrees of freedom. This review presents an overview of current strategies in synthesizing medium-sized rings, emphasizing innovative approaches like ring-expansion reactions. It highlights the challenges of synthesis and the potential of these compounds to diversify the chemical space for drug discovery, underscoring the importance of medium-sized rings in developing new bioactive compounds.

Cost-Efficient Transcriptomic-Based Drug Screening.

Transcriptomics allows to obtain comprehensive insights into cellular programs and their responses to perturbations. Despite a significant decrease in the costs of library production and sequencing in the last decade, applying these technologies at the scale necessary for drug screening remains prohibitively expensive, obstructing the immense potential of these methods. Our study presents a cost-effective system for transcriptome-based drug screening, combining miniaturized perturbation cultures with mini-bulk transcriptomics. The optimized mini-bulk protocol provides informative biological signals at cost-effective sequencing depth, enabling extensive screening of known drugs and new molecules. Depending on the chosen treatment and incubation time, this protocol will result in sequencing libraries within approximately 2 days. Due to several stopping points within this protocol, the library preparation, as well as the sequencing, can be performed time-independently. Processing simultaneously a high number of samples is possible; measurement of up to 384 samples was tested without loss of data quality. There are also no known limitations to the number of conditions and/or drugs, despite considering variability in optimal drug incubation times.

In Silico Discovery of Potential Inhibitors Targeting the RNA Binding Loop of ADAR2 and 5-HT2CR from Traditional Chinese Natural Compounds.

Adenosine deaminase acting on RNA 2 (ADAR2) is an important enzyme involved in RNA editing processes, particularly in the conversion of adenosine to inosine in RNA molecules. Dysregulation of ADAR2 activity has been implicated in various diseases, including neurological disorders (including schizophrenia), inflammatory disorders, viral infections, and cancers. Therefore, targeting ADAR2 with small molecules presents a promising therapeutic strategy for modulating RNA editing and potentially treating associated pathologies. However, there are limited compounds that effectively inhibit ADAR2 reactions. This study therefore employed computational approaches to virtually screen natural compounds from the traditional Chinese medicine (TCM) library. The shortlisted compounds demonstrated a stronger binding affinity to the ADAR2 (<-9.5 kcal/mol) than the known inhibitor, 8-azanebularine (-6.8 kcal/mol). The topmost compounds were also observed to possess high binding affinity towards 5-HT2CR with binding energies ranging from -7.8 to -12.9 kcal/mol. Further subjecting the top ADAR2-ligand complexes to molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations revealed that five potential hit compounds comprising ZINC000014637370, ZINC000085593577, ZINC000042890265, ZINC000039183320, and ZINC000101100339 had favorable binding free energies of -174.911, -137.369, -117.236, -67.023, and -64.913 kJ/mol, respectively, with the human ADAR2 protein. Residues Lys350, Cys377, Glu396, Cys451, Arg455, Ser486, Gln488, and Arg510 were also predicted to be crucial in ligand recognition and binding. This finding will provide valuable insights into the molecular interactions between ADAR2 and small molecules, aiding in the design of future ADAR2 inhibitors with potential therapeutic applications. The potential lead compounds were also profiled to have insignificant toxicities. A structural similarity search via DrugBank revealed that ZINC000039183320 and ZINC000014637370 were similar to naringin and naringenin, which are known adenosine deaminase (ADA) inhibitors. These potential novel ADAR2 inhibitors identified herein may be beneficial in treating several neurological disorders, cancers, viral infections, and inflammatory disorders caused by ADAR2 after experimental validation.

RNA-seq analysis reveals an early defense response to tomato leaf curl New Delhi virus in potato cultivar Kufri Bahar.

Potatoes in India are very susceptible to apical leaf curl disease, which causes severe symptoms and greater yield losses. Because the majority of potato cultivars are susceptible to the virus, it is crucial to discover sources of resistance and investigate the mechanism of resistance/susceptibility in potato cultivars. In this study, the gene expression profile of two potato cultivars, Kufri Bahar (resistant) and Kufri Pukhraj (susceptible), varying in their level of resistance to ToLCNDV, was analyzed using RNA-Seq. The Ion ProtonTM system was used to sequence eight RiboMinus RNA libraries from inoculated and uninoculated potato plants at 15 and 20 days after inoculation (DAI). The findings indicated that the majority of differentially expressed genes (DEGs) were cultivar-or time-specific. These DEGs included genes for proteins that interact with viruses, genes linked with the cell cycle, genes for proteins involved in defense, transcription and translation initiation factors, and plant hormone signaling pathway genes. Interestingly, defense responses were generated early in Kufri Bahar, at 15 DAI, which may have impeded the replication and spread of ToLCNDV. This research provides a genome-wide transcriptional analysis of two potato cultivars with variable levels of ToLCNDV resistance. At an early stage, we observed suppression of genes that interact with viral proteins, induction of genes associated with restriction of cell division, genes encoding defense proteins, AP2/ERF transcription factors, and altered expression of zinc finger protein genes, HSPs, JA, and SA pathway-related genes. Our findings add to a greater comprehension of the molecular basis of potato resistance to ToLCNDV and may aid in the development of more effective disease management techniques.

Advances in receptor chromatography for drug discovery and drug-receptor interaction studies.

Receptor chromatography involves high-throughput separation and accurate drug screening based on specific drug-receptor recognition and affinity, which has been widely used to screen active compounds in complex samples. This review summarizes the immobilization methods for receptors from three aspects: random covalent immobilization methods, site-specific covalent immobilization methods and dual-target receptor chromatography. Meanwhile, it focuses on its applications from three angles: screening active compounds in natural products, in natural-product-derived DNA-encoded compound libraries and drug-receptor interactions. This review provides new insights for the design and application of receptor chromatography, high-throughput and accurate drug screening, drug-receptor interactions and more.

Development of a DNA barcode library of plants in the Thai Herbal Pharmacopoeia and Monographs for authentication of herbal products.

Traditional herbal medicine has long been practiced as a method of health care in many countries worldwide. The usage of herbal products has been increasing and is expected to continue to do so in the future. However, admixture and adulteration are concerns regarding the quality of herbal medicine, including its safety and efficacy. We aimed to develop a reference DNA barcode library of plants listed in the Thai Herbal Pharmacopoeia (THP) and Monographs of Selected Thai Materia Medica (TMM) (n = 101 plant species) using four core barcode regions, namely, the ITS2, matK, rbcL and trnH-psbA intergenic spacer regions, for authentication of the plant origin of raw materials and herbal products. Checking sequences from samples obtained from local markets and the Thai Food and Drug Administration (Thai FDA) against our digital reference DNA barcode system revealed the authenticity of eighteen out of twenty tested samples as claimed on their labels. Two samples, no. 3 and 13, were not Cyanthillium cinereum (L.) H.Rob. and Pueraria candollei Wall. ex Benth. as claimed, respectively. They were recognized as Emilia sonchifolia (L.) DC. and Butea superba (Roxb.), respectively. Hence, it is important for the Thai FDA or regulatory agencies to immediately initiate strict enforcement for the development of pharmacopoeial standards as well as revisions or modifications of available regulatory guidelines and to implement close monitoring for the quality control of herbal products in terms of authentication before they enter the herbal market. The centralized digital reference DNA barcode database developed here could play a very important role in monitoring or checking the authenticity of medicinal plants.

Identification of miRNAs and their target genes in genic male sterility lines in Brassica napus by small RNA sequencing.

BACKGROUND: Brassica napus is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been used successfully for rapeseed hybrid production in China. MicroRNAs (miRNAs) play crucial regulatory roles in various plant growth, development, and stress response processes. However, reports on miRNAs that regulate the pollen development of GMS lines in B. napus are few. RESULTS: In this study, 12 small RNA and transcriptome libraries were constructed and sequenced for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines, namely, "6251AB" and "6284AB". At the same time, 12 small RNA and transcriptome libraries were also constructed and sequenced for the flower buds from the fertile and sterile lines of two dominant GMS (DGMS) lines, namely, "4001AB" and "4006AB". Based on the results, 46 known miRNAs, 27 novel miRNAs on the other arm of known pre-miRNAs, and 44 new conserved miRNAs were identified. Thirty-five pairs of novel miRNA-3p/miRNA-5p were found. Among all the identified miRNAs, fifteen differentially expressed miRNAs with over 1.5-fold change between flower buds of sterile and fertile lines were identified, including six differentially expressed miRNAs between "4001A" and "4001B", two differentially expressed miRNAs between "4006A" and "4006B", four differentially expressed miRNAs between "6251A" and "6251B", and ten differentially expressed miRNAs between "6284A" and "6284B". The correlation analysis of small RNA and transcriptome sequencing was conducted. And 257 candidate target genes were predicted for the 15 differentially expressed miRNAs. The results of 5' modified RACE indicated that BnaA09g48720D, BnaA09g11120D, and BnaCnng51960D were cleaved by bna-miR398a-3p, bna-miR158-3p and bna-miR159a, respectively. Among the differentially expressed miRNAs, miR159 was chosen to analyze its function. Overexpression of bna-miR159 in Arabidopsis resulted in decreased seed setting rate, and shortened siliques, illustrating that miR159 may regulate the fertility and silique development in rapeseed. CONCLUSIONS: Our findings provide an overview of miRNAs that are potentially involved in GMS and pollen development. New information on miRNAs and their related target genes are provided to exploit the GMS mechanism and reveal the miRNA networks in B. napus.

Study of Anti-Inflammatory and Analgesic Activity of Scorpion Toxins DKK-SP1/2 from Scorpion Buthus martensii Karsch (BmK).

Buthus martensii Karsch (BmK), is a kind of traditional Chinese medicine, which has been used for a long history for the treatment of many diseases, such as inflammation, pain and cancer. In this study, DKK-SP1/2/3 genes were screened and extracted from the cDNA library of BmK. The DKK-SP1/2/3 were expressed by using plasmid pSYPU-1b in E. coli BL21, and recombinant proteins were obtained by column chromatography. In the xylene-induced mouse ear swelling and carrageenan-induced rat paw swelling model, DKK-SP1 exerted a significant anti-inflammatory effect by inhibiting the expression of Nav1.8 channel. Meanwhile, the release of pro-inflammatory cytokines (COX-2, IL-6) was decreased significantly and the release of anti-inflammatory cytokines (IL-10) were elevated significantly. Moreover, DKK-SP1 could significantly decrease the Nav1.8 current in acutely isolated rat DRG neurons. In the acetic acid-writhing and ION-CCI model, DKK-SP2 displayed significant analgesic activity by inhibiting the expression of the Nav1.7 channel. Moreover, DKK-SP2 could significantly inhibit the Nav1.7 current in the hNav1.7-CHO cells.

The RNA content of human sperm reflects prior events in spermatogenesis and potential post-fertilization effects.

Transcriptome analyses using high-throughput methodologies allow a deeper understanding of biological functions in different cell types/tissues. The present study provides an RNA-seq profiling of human sperm mRNAs and lncRNAs (messenger and long non-coding RNAs) in a well-characterized population of fertile individuals. Sperm RNA was extracted from twelve ejaculate samples under strict quality controls. Poly(A)-transcripts were sequenced and aligned to the human genome. mRNAs and lncRNAs were classified according to their mean expression values (FPKM: Fragments Per Kilobase of transcript per Million mapped reads) and integrity. Gene Ontology analysis of the Expressed and Highly Expressed mRNAs showed an involvement in diverse reproduction processes, while the Ubiquitously Expressed and Highly Stable mRNAs were mainly involved in spermatogenesis. Transcription factor enrichment analyses revealed that the Highly Expressed and Ubiquitously Expressed sperm mRNAs were primarily regulated by zinc-fingers and spermatogenesis-related proteins. Regarding the Expressed lncRNAs, only one-third of their potential targets corresponded to Expressed mRNAs and were enriched in cell-cycle regulation processes. The remaining two-thirds were absent in sperm and were enriched in embryogenesis-related processes. A significant amount of post-testicular sperm mRNAs and lncRNAs was also detected. Even though our study is solely directed to the poly-A fraction of sperm transcripts, results indicate that both sperm mRNAs and lncRNAs constitute a footprint of previous spermatogenesis events and are configured to affect the first stages of embryo development.

The development of highly potent and selective small molecule correctors of Z α1-antitrypsin misfolding.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.

De Novo Transcriptome Assembly of Two Microsorum Fern Species Identifies Enzymes Required for Two Upstream Pathways of Phytoecdysteroids.

Microsorum species produce a high amount of phytoecdysteroids (PEs), which are widely used in traditional medicine in the Pacific islands. The PEs in two different Microsorum species, M. punctatum (MP) and M. scolopendria (MS), were examined using high-performance liquid chromatography (HPLC). In particular, MS produces a high amount of 20-hydroxyecdysone, which is the main active compound in PEs. To identify genes for PE biosynthesis, we generated reference transcriptomes from sterile frond tissues using the NovaSeq 6000 system. De novo transcriptome assembly after deleting contaminants resulted in 57,252 and 54,618 clean transcripts for MP and MS, respectively. The clean Microsorum transcripts for each species were annotated according to gene ontology terms, UniProt pathways, and the clusters of the orthologous group protein database using the MEGAN6 and Sma3s programs. In total, 1852 and 1980 transcription factors were identified for MP and MS, respectively. We obtained transcripts encoding for 38 and 32 enzymes for MP and MS, respectively, potentially involved in mevalonate and sterol biosynthetic pathways, which produce precursors for PE biosynthesis. Phylogenetic analyses revealed many redundant and unique enzymes between the two species. Overall, this study provides two Microsorum reference transcriptomes that might be useful for further studies regarding PE biosynthesis in Microsorum species.

Effective approaches to discover new microbial metabolites in a large strain library.

Natural products have provided many molecules to treat and prevent illnesses in humans, animals and plants. While only a small fraction of the existing microbial diversity has been explored for bioactive metabolites, tens of thousands of molecules have been reported in the literature over the past 80 years. Thus, the main challenge in microbial metabolite screening is to avoid the re-discovery of known metabolites in a cost-effective manner. In this perspective, we report and discuss different approaches used in our laboratory over the past few years, ranging from bioactivity-based screening to looking for metabolic rarity in different datasets to deeply investigating a single Streptomyces strain. Our results show that it is possible to find novel chemistry through a limited screening effort, provided that appropriate selection criteria are in place.

Integrated miRNA-mRNA analysis reveals the roles of miRNAs in the replanting benefit of Achyranthes bidentata roots.

The yield and quality of the medicinal plant Achyranthes bidentata can be increased when it is replanted into a field cultivated previously with the same crop, however, fundamental aspects of its biology (so-called "replanting benefit") still remain to be elucidated. miRNAs are sRNA molecules involved in the post-transcriptional regulation of gene expression in plant biological processes. Here, 267 conserved and 36 novel miRNAs were identified in A. bidentata roots. We compared the miRNA content of the roots (R1) from first-year planting with that of the roots (R2) of second-year replanting, and screened 21 differentially expressed (DE) miRNAs. Based on in silico functional analysis, integrated miRNA-mRNA datasets allowed the identification of 10 miRNA-target family modules, which might participate in the benefit. The expression profiles of the miRNA-target modules were potentially correlated with the presence of the replanting benefit. The indication was that the miRNA-responsive continuous monoculture could reprogram miRNA-mRNA expression patterns, which possibly promote the root growth and development, enhance its transport activity and strengthen its tolerance to various stresses, thereby improving A. bidentata productivity as observed in the replanting benefit. Our study provides basic data for further research on the molecular mechanisms of the benefit in A. bidentata.

Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides.

Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets.

Overexpression of polygalacturonase-inhibiting protein (PGIP) gene from Hypericum perforatum alters expression of multiple defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens in tobacco.

Hypericum perforatum L is a remarkable source of high-value secondary metabolites with increasing applications in pharmaceutical industry. However, improvement in the production of secondary metabolites through genetic engineering is a demanding task, as H. perforatum is not amenable to Agrobacterium tumefaciens-mediated transformation. In this study, we identified a Polygalacturonase-inhibiting protein (PGIP) gene from a subtractive cDNA library of A. tumefaciens-treated H. perforatum suspension cells. The role of HpPGIP in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum overexpressing HpPGIP alone or fused at the N-terminus to Phenolic oxidative coupling protein (Hyp-1), a gene that positively modulates resistance to A. tumefaciens. Furthermore, virus-induced gene silencing was employed to knock down the expression of the PGIP homologous in N. benthamiana. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in both HpPGIP and Hyp-1-PGIP transgenic plants, as assessed by GUS staining assays. However, silencing of PGIP in N. benthamiana increased the resistance to A. tumefaciens rather than susceptibility, which correlated with induction of pathogenesis-related proteins (PRs). The expression of core genes involved in several defense pathways was also analyzed in transgenic tobacco plants. Overexpression of HpPGIP led to up-regulation of key genes involved in hormone signaling, microRNA-based gene silencing, homeostasis of reactive oxygen species, and the phenylpropanoid pathway. Overexpression of Hyp-1-PGIP seemed to enhance the effect of PGIP on the expression of most genes analyzed. Moreover, HpPGIP was detected in the cytoplasm, nucleus and the plasma membrane or cell wall by confocal microscopy. Overall, our findings suggest HpPGIP modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

2'-fluoro-modified pyrimidines enhance affinity of RNA oligonucleotides to HIV-1 reverse transcriptase.

Nucleic acid aptamers can be chemically modified to enhance function, but modifying previously selected aptamers can have nontrivial structural and functional consequences. We present a reselection strategy to evaluate the impact of several modifications on preexisting aptamer pools. RNA aptamer libraries with affinity to HIV-1 reverse transcriptase (RT) were retranscribed with 2'-F, 2'-OMe, or 2'-NH2 pyrimidines and subjected to three additional selection cycles. RT inhibition was observed for representative aptamers from several structural families identified by high-throughput sequencing when transcribed with their corresponding modifications. Thus, reselection identified specialized subsets of aptamers that tolerated chemical modifications from unmodified preenriched libraries. Inhibition was the strongest with the 2'-F-pyrimidine (2'-FY) RNAs, as compared to inhibition by the 2'-OMeY and 2'-NH2Y RNAs. Unexpectedly, a diverse panel of retroviral RTs were strongly inhibited by all 2'-FY-modified transcripts, including sequences that do not inhibit those RTs as unmodified RNA. The magnitude of promiscuous RT inhibition was proportional to mole fraction 2'-FY in the transcript. RT binding affinity by 2'-FY transcripts was more sensitive to salt concentration than binding by unmodified transcripts, indicating that interaction with retroviral RTs is more ionic in character for 2'-FY RNA than for unmodified 2'-OH RNA. These surprising features of 2'-FY-modified RNA may have general implications for applied aptamer technologies.

[Screening and verification of proteins of Salvia miltiorrhiza polyphenol oxidase interaction].

Polyphenol oxidase(PPO) is an important antioxidant enzyme in plants. It has the functions of scavenging active oxygen and synthesizing phenols, lignin, and plant protection factors, and can enhance the plant's resistance to stress and resistance to pests and diseases. Our previous research found that Salvia miltiorrhiza PPO gene can positively regulate salvianolic acid B synthesis. In order to further explore the mechanism, a pGBKT7-PPO bait vector was constructed using the cloned S. miltiorrhiza polyphenol oxidase gene(SmPPO, GenBank accession number: KF712274.1), and verified that it had no self-activation and no toxicity. The titer of S. miltiorrhiza cDNA library constructed by our laboratory was 4.75 × 107 cfu·mL~(-1), which met the requirements for library construction. Through yeast two-hybrid test, 22 proteins that could interact with SmPPO were screened. Only yeast PAL1 and TAT interacted with SmPPO through yeast co-transformation verification. Further verification was performed by bimolecular fluorescence complementary detection(BiFC). Only TAT and SmPPO interacted, so it meant that TAT and SmPPO interacted. TAT and SmPPO were truncated according to the domain, respectively. The first 126 amino acids of SmPPO and tyrosine amino transferase(TAT) were obtained to interact on the cell membrane and chloroplast. SmPPO was obtained by subcellular localization test, which was mainly loca-lized on the nucleus and cell membrane; TAT was localized on the cell membrane. Real-time quantitative PCR results showed that the SmPPO gene was mainly expressed in roots and stems; the TAT gene was expressed in roots, and the expression level in stems and flowers was low. This article lays a solid foundation for the in-depth study of the molecular mechanism of the interaction of S. miltiorrhiza SmPPO and TAT to regulate the synthesis of phenolic substances.

Novel target capture DNA library preparation method using CircLigase-mediated hook ligation.

Target capture DNA library preparation methods primarily use the biotin-streptavidin interaction to enrich target DNA, requiring complex operations and a time-consuming workflow with a series of wash steps at regulated temperatures. Here, a new method is presented termed 'hook ligation' for target DNA library preparation, using CircLigase to capture target DNA. The concept was validated by library preparation, sequencing, and acquisition of target DNA fragment information through bioinformatics analysis. An efficient reference point for single-strand DNA ligation to a hook sequence using CircLigase is also provided and it is shown that the efficiency can be influenced by the length and the position of the complementary area on the target DNA.

In silico guided structural and functional analysis of genes with potential involvement in resistance to coffee leaf rust: A functional marker based approach.

Physiology-based differentiation of SH genes and Hemileia vastatrix races is the principal method employed for the characterization of coffee leaf rust resistance. Based on the gene-for-gene theory, nine major rust resistance genes (SH1-9) have been proposed. However, these genes have not been characterized at the molecular level. Consequently, the lack of molecular data regarding rust resistance genes or candidates is a major bottleneck in coffee breeding. To address this issue, we screened a BAC library with resistance gene analogs (RGAs), identified RGAs, characterized and explored for any SH related candidate genes. Herein, we report the identification and characterization of a gene (gene 11), which shares conserved sequences with other SH genes and displays a characteristic polymorphic allele conferring different resistance phenotypes. Furthermore, comparative analysis of the two RGAs belonging to CC-NBS-LRR revealed more intense diversifying selection in tomato and grape genomes than in coffee. For the first time, the present study has unveiled novel insights into the molecular nature of the SH genes, thereby opening new avenues for coffee rust resistance molecular breeding. The characterized candidate RGA is of particular importance for further biological function analysis in coffee.

Identification and expression of DoCCaMK during Sebacina sp. symbiosis of Dendrobium officinale.

Dendrobium officinale Kimura et Migo is a famous precious medicinal plant in China. Seed and seedling were cultivated with the mycorrhizal fungus Sebacina sp. CCaMK was initially cloned from D. officinale based on a SSH cDNA library of symbiotically germinated seeds with Sebacina sp. Phylogenetic analysis was performed among DoCCaMK and other CCaMKs. The particle bombardment technique was used to visualize DoCCaMK-GFP. qRT-PCR and western blot analysis were conducted to determine the tissue expression patterns of DoCCaMK with (SGS) and without (UGS) Sebacina sp. Furthermore, the effect of KN-93 on CCaMK expression was also examined. Using NMT the net Ca2+ fluxes and the CCaMK concentration were measured during D. officinale seed germination. DoCCaMK had the highest homology with Lilium longiflorum CCaMK. The DoCCaMK-GFP protein localized in the nucleus and cell membrane. CCaMK expression was significantly upregulated after symbiosis with Sebacina sp. KN-93 could be used as an inhibitor of CCaMK to inhibit D. officinale seed germination. Ca2+ influx and the concentration of the CCaMK in the SGS group was significantly more than that of the UGS group. The characterization of CCaMK provides certain genetic evidence for the involvement of this gene during seed germination and mycorrhizal cultivation in D. officinale.