Genomic, transcriptomic, and epigenomic analysis of a medicinal snake, Bungarus multicinctus, to provides insights into the origin of Elapidae neurotoxins

The many-banded krait, Bungarus multicinctus, has been recorded as the animal resource of JinQianBaiHuaShe in the Chinese Pharmacopoeia. Characterization of its venoms classified chief phyla of modern animal neurotoxins. However, the evolutionary origin and diversification of its neurotoxins as well as biosynthesis of its active compounds remain largely unknown due to the lack of its high-quality genome. Here, we present the 1.58 Gbp genome of B. multicinctus assembled into 18 chromosomes with contig/scaffold N50 of 7.53 Mbp/149.8 Mbp. Major bungarotoxin-coding genes were clustered within genome by family and found to be associated with ancient local duplications. The truncation of glycosylphosphatidylinositol anchor in the 3'-terminal of a LY6E paralog released modern three-finger toxins (3FTxs) from membrane tethering before the Colubroidea divergence. Subsequent expansion and mutations diversified and recruited these 3FTxs. After the cobra/krait divergence, the modern unit-B of β-bungarotoxin emerged with an extra cysteine residue. A subsequent point substitution in unit-A enabled the β-bungarotoxin covalent linkage. The B. multicinctus gene expression, chromatin topological organization, and histone modification characteristics were featured by transcriptome, proteome, chromatin conformation capture sequencing, and ChIP-seq. The results highlighted that venom production was under a sophisticated regulation. Our findings provide new insights into snake neurotoxin research, meanwhile will facilitate antivenom development, toxin-driven drug discovery and the quality control of JinQianBaiHuaShe.

Genome-wide identification and transcriptional profiling analysis of PIN/PILS auxin transporter gene families in Panax ginseng / 中草药·英文版

Objective: Plant hormones act as chemical messengers in the regulation of plant development and metabolism. The production of ginsenosides in Panax hybrid is promoted by auxins that are transported and accumulated by PIN-FORMED (PIN) and PIN-LIKES (PILS) auxin transporters. However, genome-wide studies of PIN/PILS of ginseng are still scarce. In current study, identification and transcriptional profiling of PIN/PILS gene families, as well as their potential relationship with ginsenoside biosynthesis in Panax ginseng were investigated. Methods: PIN/PILS genes in P. ginseng was identified via in silico genome-wide analysis, followed by phylogenetic relationships, gene structure, and protein profiles investigation. Moreover, previously reported RNA-sequence data from various tissues and roots after infection were utilized for PIN/PILS genes expression pattern analysis. The Pearson's correlation analysis of specific PIN/PILS genes expression level and main ginsenoside contents were taken to reveal the potential relationship between auxin transports and ginsenoside biosynthesis in P. ginseng. Results: A genome-wide search of P. ginseng genome for homologous auxin transporter genes identified a total of 17 PIN and 11 PILS genes. Sequence alignment, putative motif organization, and sub-cellular localization indicated redundant and complementary biological functions of these PIN/PILS genes. Most PIN/PILS genes were differentially expressed in a tissue-specific manner, and showed significant correlations with ginsenoside content correspondingly. Eight auxin transporter genes, including both PIN and PILS subfamily members, were positively correlated with ginsenoside content (cor > 0.60; P-value <0.05). The expression levels of eleven auxin transporter genes were increased dramatically in the early stage (0–0.5 DPI) after Cylindrocarpon destructans infection, accompanied with various overall expression patterns, implying the dynamic auxin transport in response to biotic stress. Conclusion: Based on the results, we speculate that the accumulation or depletion in temporal or spatial manner of auxin by PIN/PILS transporters involved in the regulation of HMGR activity and subsequent ginsenoside biosynthesis.

Genome-wide characterization and analysis of bHLH transcription factors in

Ginseng ( C.A. Meyer) is one of the best-selling herbal medicines, with ginsenosides as its main pharmacologically active constituents. Although extensive chemical and pharmaceutical studies of these compounds have been performed, genome-wide studies of the basic helix-loop-helix (bHLH) transcription factors of ginseng are still limited. The bHLH transcription factor family is one of the largest transcription factor families found in eukaryotic organisms, and these proteins are involved in a myriad of regulatory processes. In our study, 169 bHLH transcription factor genes were identified in the genome of , and phylogenetic analysis indicated that these PGbHLHs could be classified into 24 subfamilies. A total of 21 RNA-seq data sets, including two sequencing libraries for jasmonate (JA)-responsive and 19 reported libraries for organ-specific expression analyses were constructed. Through a combination of gene-specific expression patterns and chemical contents, 6 PGbHLH genes from 4 subfamilies were revealed to be potentially involved in the regulation of ginsenoside biosynthesis. These 6 PGbHLHs, which had distinct target genes, were further divided into two groups depending on the absence of MYC-N structure. Our results would provide a foundation for understanding the molecular basis and regulatory mechanisms of bHLH transcription factor action in .

Estimation of Genome Size of Ginseng Based on Herbgenomics by Flow Cytometric Analysis And High-throughput Sequence / 世界科学技术-中医药现代化

Ginseng is the dried root and rhizome of Panax ginseng.The lack of genomic data has restricted the development of ginseng industry and basic research.The genome size of P.ginseng was estimated to be 3.42 Gb by using the genome data of Oryza sativa ssp.Nipponbare and Glycine max (L.) Merrill as the reference and the flow cytometric analysis.Meanwhile,shotgun libraries with the insert size of 250 bp and 500 bp were constructed,and sequenced for double terminal PE 150 by using Illumina Hiseq X Ten platform.Totally,183.82 Gb high quality data was obtained after filtering the raw data.The genome size of P.ginseng was 3.35 Gb and the sequencing depth was 54.87 X by K-mer analysis.In this study,flow cytometry and K-mer analysis were used to identify the genome size of ginseng,which provided basic data for the further whole genome sequencing and herbgenomics studies.

Comparative optical genome analysis of two pangolin species: Manis pentadactyla and Manis javanica.

Background: The pangolin is a Pholidota mammal with large keratin scales protecting its skin. Two pangolin species ( Manis pentadactyla and Manis javanica ) have been recorded as critically endangered on the International Union for Conservation of Nature Red List of Threatened Species. Optical mapping constructs high-resolution restriction maps from single DNA molecules for genome analysis at the megabase scale and to assist genome assembly. Here, we constructed restriction maps of M. pentadactyla and M. javanica using optical mapping to assist with genome assembly and analysis of these species. Findings: Genomic DNA was nicked with Nt.BspQI and then labeled using fluorescently labeled bases that were detected by the Irys optical mapping system. In total, 3,313,734 DNA molecules (517.847 Gb) for M. pentadactyla and 3,439,885 DNA molecules (504.743 Gb) for M. javanica were obtained, which corresponded to approximately 178X and 177X genome coverage, respectively. Qualified molecules (≥150 kb with a label density of >6 sites per 100 kb) were analyzed using the de novo assembly program embedded in the IrysView pipeline. We obtained two maps that were 2.91 Gb and 2.85 Gb in size with N50s of 1.88 Mb and 1.97 Mb, respectively. Conclusions: Optical mapping reveals large-scale structural information that is especially important for non-model genomes that lack a good reference. The approach has the potential to guide de novo assembly of genomes sequenced using next-generation sequencing. Our data provide a resource for Manidae genome analysis and references for de novo assembly. This note also provides new insights into Manidae evolutionary analysis at the genome structure level.

Cloning and analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase genes HsHDR1 and HsHDR2 in Huperzia serrate

We cloned and analyzed the two genes of the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) gene family from Huperzia serrate. The two transcripts coding HDR, named HsHDR1 and HsHDR2, were discovered in the transcriptome dataset of H. serrate and were cloned by reverse transcription-polymerase chain reaction (RT-PCR). The physicochemical properties, protein domains, protein secondary structure, and 3D structure of the putative HsHDR1 and HsHDR2 proteins were analyzed. The full-length cDNA of the HsHDR1 gene contained 1431 bp encoding a putative protein with 476 amino acids, whereas the HsHDR2 gene contained 1428 bp encoding a putative protein of 475 amino acids. These two proteins contained the conserved domain of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (PF02401), but without the transmembrane region and signal peptide. The most abundant expression of HsHDR1 and HsHDR2 was detected in H. serrate roots, followed by the stems and leaves. Our results provide a foundation for exploring the function of HsHDR1 and HsHDR2 in terpenoid and sterol biosynthesis in Huperziaceae plants.

A Rapid SNP-based Identification Method for Distinguishing the Three Origins of Fructus Amomi / 世界科学技术-中医药现代化

Medicinal plants of the Fructus Amomi containing three species (A momum villosum, A momum longiligu-lare, Amomum villosum var. xanthioides)are well-known, which are widely used as traditional medicines. The mor-phological characteristics of the three origins are very similar, especially in the form of seed. In this study, 60 sam-ples of Fructus Amomi were co llected, and 34 sequences of the Fructus Amomi and their adulterants from GenBank were analyzed. Single nucleotide polymorphisms (SNPs) were detected. All the ITS2 sequences here (including our ex-periments and GenBank data)were examined for SNPs at the interspecies level. Results from the study revealed that two stable bases at position 135 bp and 199 bp were found, which could be used as a unique marker to distinguish the three origins of Fructus Amomi. The two SNPs in the ITS2 were found to exist stably between the three species, and all the GenBank sequences of the Fructus Amomi. Our findings indicated that SNP-based DNA barcoding could be used as an efficient method for the rapid and accurate identification of the three origins of Fructus Amomi.

Identification of Rosa laevigata Michx and Its Adulterants based on ITS2 DNA Barcode / 世界科学技术-中医药现代化

In order to verify the sstability and accuracy of DNA barcode technique, we chose Rosa laevigata Michx as study object. Genomic DNAs of 10 samples were extracted by modified CTAB method. ITS2 sequences were obtained by direct PCR sequencing; the other 6 sequences were obtained from GenBank. The sequences were assembled using the CodonCode Aligner. All of the 16 ITS2 sequences were aligned through Clustal-W and the genetic distances were computed using MEGA 5.0 in accordance with the Kimura 2-parameter (K-2-P) model. Results indicated that the lengths of ITS2 regions of R. laevigata ranged from 219 to 221 bp with two Poly C structure in it. The intra-specific genetic distances were smaller than inter-specific ones in ITS2 regions of R. laevigata. The NJ tree showed that R. laevigata and adulterants were divided into two clades, with 99%bootstrap value, showing good monophyly. So, ITS2 was considered a good marker to identify R. laevigata and its adulterants.