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Objective: The complete chloroplast genome of medicinal plant Tetrastigma hemsleyanum sequenced by high-throughput technologies was assembled for the sequence analysis to provide evidence for its population genetics and diversity studies. Methods: HiSeq X Ten was used to sequence DNA of T. hemsleyanum, and the chloroplast genome was assembled by NOVOPlasty. Sequence analysis was performed based on gene annotation results. Results: The complete chloroplast genome of T. hemsleyanum was 160 189 bp in length with a GC content of 37.5%. The chloroplast genome exhibited a typical quadripartite structure, including a large single copy region, a pair of inverted repeats, and a small single copy, and the sequence lengths were 88 184 bp, 26 519 bp, and 18 967 bp, respectively. The chloroplast genome harbored 133 genes, including 88 protein-coding genes, eight rRNA genes, and 37 tRNA genes. The ycf1 gene located at the border of IR/SSC was proved to be a pseudogene, with its 3’ end truncated. Conclusion: Sequence assembly and analysis of T. hemsleyanum chloroplast genome provide new insights into future studies on both population genetics and genetic diversity.
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Objective: The content of 16 kinds of organochlorine pesticides including As, Hg, Pb, Cd, and Cu in Tetrastigma hemsleyanum from different places were determined. Methods: The content of As, Hg, Pb, Cd, and Cu in T. hemsleyanum from different places were determined by ICP-MS, and organochlorine pesticides were determined by GC. Results: The contents of heavy metals in T. hemsleyanum were Pb≤4.167 7 mg/kg, Cd≤0.194 6 mg/kg, As≤0.455 0 mg/kg, Hg≤0.042 4 mg/kg, Cu≤7.892 5 mg/kg. Organochlorine preticides were lower in T. hemsleyanum. Conclusion: The method is simple, efficient and accurate, which can be used for the safety evaluation of T. hemsleyanum.
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Objective In this paper, the genetic diversity of 64 samples of Tetrastigma hemsleyanum germplasm resources in Chinese was analyzed. Methods ISSR-PCR was firstly used to amplify, and then POPGENE 32 software and NTSYS software was used to analyze the genetic diversity and phylogenetic relationship of 64 samples of T. hemsleyanum germplasm resources, and phylogenetic tree was constructed according to the UPGMA method. Results Ten primers with clear and reproducible bands were screened from 30 primers and used for genomic DNA amplification of 64 sample materials. A total of 83 polymorphic locis were amplified, whose polymorphic percentages were 71.43%-100% and average polymorphism percentage was 94.31%. The amplification polymorphic locis of primer S17 were the most (11) and the amplification polymorphic locis of primer P6 were the least (5), the average amplified polymorphic locis of 10 primers were 8.3. Genetic diversity analysis showed that the average number of alleles (Na) of 64 samples was 1.943 1, the average effective allele number (Ne) was 1.381 08, the average Nei’s gene diversity index (H) was 0.242 98, and the average Shannon diversity index (I) was 0.385 83. The variation range of the genetic similarity coefficient of the 64 samples was 0.431 8-0.988 6. A total of 64 samples were divided to six groups by UPGMA clustering method according to the similarity coefficient matrix when the genetic similarity coefficient was 0.715 5, which showed the abundant genetic diversity and relative gene stability of T. hemsleyanum germplasm resources. In addition, amplification figures by primers ISSR20, UBC857, and S17 were screened based on amplification result of 10 ISSR primers, and DNA fingerprinting was constructed, which can be used to identify 64 samples of T. hemsleyanum tested. Conclusion There are abundant genetic diversity and relative gene stability in T. hemsleyanum germplasm esources in China. ISSR analysis can reveal the genetic relationship among T. hemsleyanum germplasm resources in China, and provide certain reference for the evaluation, identification and new variety breeding of T. hemsleyanum germplasm resources in China.
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Objective To understand the genetic diversity and genetic relationship of 64 samples of Tetrastigma hemsleyanum germplasm resources. Methods Fluorescently labeled SSR were used for PCR amplification, POPGENE32 software was used to analyze genetic diversity and genetic relationship of 64 samples of T. hemsleyanum germplasm resources, UPGMA method was used to construct their genetic tree map, NTSYS software was used to construct their two-dimensional principal component analysis map and three-dimensional scatter map. Result Eight pairs of primers with clear and reproducible bands were screened from 14 pairs of primers and used for genomic DNA amplification of 64 sample materials. The observed number of alleles (Na) ranged from 3 to 13, and the mean value was 7.875 0; The effective number of alleles (Ne) ranged from 1.424 9 to 6.087 4, and the mean value was 3.605 2; The Shannon information index (I) ranged from 0.689 5 to 2.082 4, and the mean value was 1.424 0; The observed heterozyghosity (Ho) ranged from 0.206 3 to 0.734 4, and the mean value was 0.524 7; The expected heterozygosity (He) ranged from 0.300 6 to 0.842 4, and the mean value was 0.658 4; The Nei’s gene diversity (H) ranged from 0.298 2 to 0.835 7, and the mean value was 0.653 2; The polymorphism information content (PIC) ranged from 0.288 0 to 0.817 5, and the mean value was 0.614 5; The genetic similarity coefficient ranged from 0.115 4 to 0.954 5; The genetic distance ranged from 0.000 0 to 3.218 1. It was indicated that the genetic relationship of 64 T. hemsleyanum germplasm resources was far, and the degree of genetic differentiation was high. At the genetic distance of 1.018 9, 64 T. hemsleyanum germplasm resources could be divided into five groups. Conclusion There was no necessary connection between geographical difference and genetic difference of germplasm. Themsleyanum germplasm resources are rich in genetic diversity. The results of fluorescently labeled SSR analysis can provide some references for the utilization and variety breeding of T. hemsleyanum germplasm resources.
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Objective To obtain the transcriptome database and differentially expressed genes of Tetrastigma hemsleyanum Diels et Gilg. by Illumina HiSeq 4000; To provide important molecular information for its molecular biology research. Methods Leaves and roots of Tetrastigma hemsleyanum Diels et Gilg. were chosen as experimental materials to conduct transcriptome sequencing. Then bioinformatics analysis of gene function annotations, metabolic pathways, and microsatellites was performed on the test data. Results 24.13 Gb Clean Data were assembled. Afer assembly steps, 84 433 of T. hemsleyanum Unigene were obtained, and then they were compared in the 7 gene database, and 47 766 annotated information of Unigene was obtained. There were 27 790 annotations in the GO database. The number of differentially expressed genes in the roots, stems and leaves was 4989, of which 3511 were up-regulated and 1478 were down-regulated. The COG database obtained 16 152 homologous sequences of Unigene, which were divided into 25 categories. In the KEGG database, there were 14 511 Unigene obtained the corresponding Ko number, which could be divided into 130 branches of signal metabolism, among which the number of Unigene in the ribosome synthesis pathway was the most, with 1042, and there was only 1 Unigene in the biosynthetic pathway of isoflavones. Conclusion A large number of transcripts of the transcriptome were obtained through splicing, assembling and functional annotation of Tetrastigma hemsleyanum Diels et Gilg., which can provide genomic database resources for molecular biology research of Tetrastigma hemsleyanum Diels et Gilg.
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Objective: To establish a quick method of ultra-performance liquid chromatography-quadrupole time-of-fight mass spectrometry (UPLC-Triple-TOF/MS) for the analysis of compounds in Tetrastigma hemsleyanum. Methods: The separation was performed on the chromatographic column of Agilent Eclipse Plus-C18 (100 mm × 4.6 mm, 1.8 μm), and the mobile phase was methanol (0.1% formic acid)-0.1% formic acid solution, with a gradient elution at a flow rate of 1 mL/min. The column temperature was 30℃, and negative ion mode was used for TOF-MS. Results: Fifty-one compounds were identified or tentatively characterized based on the retention time and MS spectra. They are flavonoids, phenolic acids, phospholipids, glycolipids, benzenesulfoic acid, etc. And the preliminary fragmentation rules of phospholipid, glycolipids, and benzenesulfoic acid were summarized. Conclusion: The results demonstrate that UPLC-Triple-TOF/MS method is novel, quick, and efficient for the identification of the compounds in T. hemsleyanum.
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Objective: To study the chemical constituents of endophytic fungus Phomopsis sp. YK-7 isolated from Tetrastigma hemsleyanum. Methods: The compounds were isolated and purified by chromatographic techniques and their structures were identified on the basis of spectral features. Results: Fifteent known compounds, including 13 arylbenzofuran derivatives and two steroids, named wittifuran X (1), 4-hydroxy-2-(4'-hydroxy-2'-methoxyphenyl)-6-methoxybenzofuran-3-carbaldehyde (2), erypoegin J (3), moracin S (4), moracin T (5), moracin C (6), wiffifuran E (7), iteafuranal A (8), burttinol D (9), 7-methoxy-2-(4-methoxyphenyl)-3- methyl-5-(3-prenyl)-benzofuran (10), moracin N (11), moracin P (12), moracin M (13), ergosterol peroxide (14), and 25-hydroxy- ergosta-4,6,8(14),22-tetraen-3-one (15) were isolated from the fermentation products of Phomopsis sp. YK-7. Conclusion: Compounds 1-9 are obtained from genus Phomopsis for the first time.
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Objective: To clone the sequence and characterize gene expression of an expansin gene from Tetrastigma hemsleyanum, and to predict its probable function. Methods: According to the conservative region sequences of expansin gene in GenBank database, one pair of degenerate primers were designed for RT-PCR. The conservative region fragment was first amplified by RT-PCR from cDNA template of T. hemsleyanum. The full-length sequence of expansin gene cDNA (named as Th-exp) was extended by RACE (rapid-amplification of cDNA ends). Th-exp expression levels in different organs were conducted by semi-quantitative RT-PCR and fluorescence quantitative PCR. Results: The full-length cDNA sequences of Th-exp gene were obtained in 782 bp. Th-exp gene containsed a 630 bp open reading frame, which encoded a protein of 209 amino acids (GenBank accession number KP693606). The protein encoded by Th-exp habours a typical expansin structure, including eight cysteine domains in N terminal region, four conserved tryptophan domain in C terminal region, and histidine function domain in intermediate region. Blast alignment showed that Th-exp was similar to expansin genes of Vitis vinifera and Tarenaya hassleriana. Semi-quantitative RT-PCR and fluorescence quantitative PCR indicated that Th-exp could express in the leaves, stems, ordinary fine roots, and calabash-shaped roots, while the expression level in calabash-shaped roots was higher than those in other organs. Conclusion: The expansin gene (Th-exp) is cloned from T. hemsleyanum, and it could be involved in the development of root tubers in T. hemsleyanum.
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OBJECTIVE: To study the chemical constituents and antitumor activity of Tetrastigma hemsleyanum Diels et Gilg. METHODS: The compounds were isolated by various column chromatography. Their structures were identified by spectroscopic methods including 1H-NMR, 13C-NMR, MS, HR-MS, etc. The antiproliferative activity of the purified flavonoids compounds were evaluated by MTT assay against HepG2, HCT-8, and A549 tumor cells. RESULTS: Thirteen compounds were isolated and identified as β-sitosterol (1), palmitic acid (2), heptadecanoic acid (3), apigenin (4), quercitrin (5), kaempferitrin (6), kaempferol-3-O-neohes-peridoside (7), apigenin-6-C-β-D-glucopyranoside (8), apigenin-8-C-α-L-rhamnopyranosyl-(1-2)-β-D-glucopyranoside (9), apige-nin-8-C-β-D-glucopyranoside (10), apigenin-8-C-β-D-glucopyranoside-(1-4)-β-D-glucopyranoside (11), orientin (12), and isoorientin (13). CONCLUSION: Compounds 4-6 and 8-13 were isolated from T. hemsleyanum for the first time. The flavonoids of T. hemsleyanum showed inhibitory activity on HepG2, HCT-8, and A549 tumor cells.
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Objective: To study the chemical compositions from the root tuber of Tetrastigma hemsleyanum and their anti-oxidative activities. Methods: The compounds were isolated by various column chromatography. Their structures were identified by spectroscopic methods including 1H-NMR, 13C-NMR, MS, HR-MS, etc. The anti-oxidative activities were evaluated by DPPH radical scavenging assay. Results: Fourteen compounds were isolated from the root tuber of T. hemsleyanum, they were identified as kaempferol (1), quercetin (2), salicylic acid (3), benzoic acid (4), oxyresveratrol (5), catechin (6), L-epicatechin (7), epigallocatechin (8), procyanidin B2 (9), procyanidin B1 (10), 3-O-caffeoylquinic acid (11), protocatechualdehyde (12), p-hydroxybenzoic acid (13), and protocatechuic acid (14). The anti-oxidative IC50 values of compounds 2, 8, 9, 10, and 12 were 14.15, 14.19, 15.99, 12.4, and 15.98 μmol/L, respectively, better than the positive control Vit C (IC50 value was 23.0 μmol/L). Conclusion: Compounds 4-12 are isolated from T. hemsleyanum for the first time. It has been demonstrated that the root tuber contain flavonoids and also is rich in organic acids. Moreover, compounds 2, 8, 9, 10, and 12 have the certain prospects in the development of natural anti-oxidative agents.
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Objective: Taking annual Tetrastigma hemsleyanum (Tetrastigma Radix) from three habitats, Taizhou, Minhou, and Shunchang, as materials to study the effects of different light intensity stress on the stomatal shape and leaf transpiration. Methods: The stomata, leaf transpiration rate, net photosynthetic rate, and stomal structure of T. hemsleyanum were observed using a scanning [0-2000 μmol/(m2·s)] to explore the optimal light intensity. Results: The stomata with surround and parallel distribution types were only distributed in the lower epidermis in the leaves of T. hemsleyanum; The transpiration rate, stomatal conductance, and net photosynthetic rate of T. hemsleyanum from the two habitat regions (Minhou and Taizhou) by rising after falling with the 30℃ stress, which suggested the stress could make the light intensity heavier. At the same time, the transpiration rate, stomatal conductance, and net photosynthetic rate of T. hemsleyanum from the habitat region Shunchang were rising. The stomatal type of leaves was 600 μmol/(m2·s) in Minhou, and 1900 μmol/(m2·s) in Shunchang, when the water utilization ratio of T. hemsleyanum leaves from the three habitats was higher. Conclusion: Different habitats and light intensity are the important factors of leaves transpiration in natural world, which is more beneficial to the growth of the root crops and suitable for the accumulation of flavonoids in plants.