Identification and analysis of R1-MYB gene family in Rheum palmatum L. based on full-length transcriptome sequencing / 药学学报

As one kind of v-myb avian myeloblastosis viral oncogene homolog (MYB) transcription factors, R1-MYB (MYB-related) family plays an important role in plant growth and development, as well as environmental stress and hormone signal transduction. In this study, R1-MYB family genes in Rheum palmatum L. were systematically screened based on full-length transcriptome sequencing analysis. Firstly, the physicochemical, protein domain and molecular evolution characteristics of the coding proteins were analyzed. Furthermore, the tissue expression levels of R1-MYB genes were analyzed by RNA-seq. We also investigated the expression pattern of RpMYB24 in response to various hormones and abiotic stresses. The results showed that a total of 49 R1-MYB genes were identified, which mainly encoded thermally stable hydrophilic proteins. Most of the deduced proteins were predicted to locate in nucleus. Each protein had a large proportion of random curl and α helix, and also had the W-type conserved amino acids which were the signature of MYB. R1-MYB family members were distributed in five subgroups, including circadian clock associated 1 (CCA1)-like, I-box (GATAAG)-like, CAPRICE (CPC)-like, telomere repeat binding factor (TRF)-like and TATA binding protein (TBP)-like, and the number of CCA1-like was the majority. RNA-seq revealed that 49 R1-MYB genes were differentially expressed in roots, rhizomes and leaves of R. palmatum, and the expression levels of 15 and 23 genes in roots and rhizomes were higher than those in leaves, respectively. RpMYB24 transcript was induced by abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA) treatment, and could also significantly respond to injury, low temperature and high temperature stresses except drought stress. This study systematically identified the R1-MYB family genes and their molecular characteristics, better for further gene functional validation, and then provide a scientific basis for the transcriptional regulation mechanism research into rhubarb quality formation.

Characterization of the complete chloroplast genome of Polygonatum franchetii Hua, a Chinese endemic medicinal species, and phylogenetic relationships of Polygonatum / 药学学报

italic>Polygonatum franchetii Hua is a medicinal plant endemic to China from Polygonatum Mill. The chloroplast genomes of two P. franchetii individuals sampled from two different habitats were sequenced by using the DNBSEQ-T7 high-throughput sequencing platform. After assembly and annotation, the two complete chloroplast genomes were characterized, and then comparative and phylogenetic analyses were performed with other published chloroplast genome sequences from Polygonatum. The whole chloroplast genomes of the two P. franchetii individuals were 155 942 and 155 962 bp in length, with a large single copy region (LSC, 84 670 and 84 722 bp), a small single copy region (SSC, 18 564 and 18 566 bp) and a pair of reverse repeats (IRa/IRb, 26 354 and 26 337 bp), respectively. Both of them contained 113 genes, including 79 protein-coding genes (PCGs), 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. Comparative analyses showed that the genome length, the guanine and cytosine (GC) content, genes content and order were highly conserved between the two P. franchetii individuals and among different Polygonatum species. The detected repeat sequences, including dispersed repeats, tandem repeats and simple sequence repeats (SSRs), were also relatively similar in types and positions, though showing a slightly difference in number. No significant expansion or contraction of the inverted repeat regions was found. Sequences variation between the two P. franchetii individuals was lower than that among different Polygonatum species. Besides, coding sequences (CDS) showed less divergence than noncoding sequences, and sequence divergence of IRs regions was lower than that of the LSC and SSC regions, both intraspecifically and interspecifically. Eight sequences with high nucleotide diversity among different species were screened, all of which were found located in the LSC and SSC regions. Phylogenetic inference showed that all Polygonatum species clustered into a monophyletic clade with a 100% bootstrap value, within which, species in section Verticillata formed a distinct group, section Sibirica and section Polygonatum were sister groups. The two P. franchetii individuals grouped together and showed the closest phylogenetic affinity to P. stenophyllum Maxim., belonging to the section Verticillata. The chloroplast genome of P. franchetii and its phylogenetic position in Polygonatum were comprehensively investigated and clearly elucidated in this study, the results may lay a foundation for the resource development and utilization of P. franchetii, as well as further molecular identification and phylogenetic studies of medicinal Polygonatum species.

Perifosine inhibits biofilm formation of Pseudomonas aeruginosa by interacting with PqsE protein / 中华预防医学杂志

To explore the biofilm inhibitory efficacy of perifosine against Pseudomonas aeruginosa (P. aeruginos) and its mechanisms. Twenty-fourwell plate was used to form biofilms at the bottom and crystal violet staining was used to determine the biofilm inhibitory effects of perifosine against P. aeruginosa, the wells without perifosine was set as control group. Glass tubes combined with crystal violet staining was used to detect the gas-liqud interface related bioiflm inhibitory effects of perifosine, the wells without perifosine was set as control group. Time-growth curved was used to detect the effects of perifosine on the bacteial planktonic cells growth of P. aeruginosa, the wells without perifosine was set as control group. The interaction model between perifosine and PqsE was assessed by molecular docking assay. The inhibitory effects of perifosine on the catalytic activity of PqsE was determined by detection the production of thiols, the wells without perifosine was set as control group. Binding affinity between perifosine and PqsE was detected by plasma surface resonance. The biofims at the bottom of the microplates and air-liquid interface were effectively inhibited by perifosine at the concentration of 4-8 μg/ml. There was no influence of perifosine on the cells growth of P. aeruginosa. The resuts of molecular docking assay indicates that perifosine could interacted with PqsE with the docking score of -10.67 kcal/mol. Perifosine could inhibit the catalytic activity of PqsE in a dose-dependent manner. The binding affinity between perifosine and PqsE was comfirmed by plasma surface resonance with KD of 6.65×10-5mol/L. Perifosine could inhibited the biofilm formation of P. aeruginosa by interacting with PqsE.

Cloning and expression characteristics of a transcription factor gene RpMYB4 in Rheum palmatum L / 药学学报

MYB transcription factors play many important regulatory roles in plant growth and development, secondary metabolism, and stress adaptation processes. In this work, an MYB gene containing a complete open reading frame (ORF) was selected from the transcriptome database of R. palmatum L. RpMYB4 ORF and cloned, encoding a polypeptide of 245 amino acids with a molecular weight of 26.99 kDa. RpMYB4 lacks a signal peptide or transmembrane domain but contains two conserved DNA binding domains (HTH-MYB) of the R2R3-MYB subfamily at the N-terminus. Multiple-sequence alignment demonstrated that RpMYB4 shared as high as 61% identity with many MYB proteins from other species. Phylogenetic analysis showed that RpMYB4 had the closest relationship with FtMYB8 and was clustered in the S4 subfamily. Subcellular localization by confocal microscopy showed that an RpMYB4-GFP-fusion protein localized to the nucleus in tobacco. Real-time fluorescence quantitative PCR analyses revealed that RpMYB4 was differentially expressed in various tissues, with the highest expression in leaves, followed by petioles, rhizome, and roots, and with the lowest level in mature seeds. After treatment of R. palmatum L. seedlings with 200 μmol·L-1 MeJA, the expression of RpMYB4 in leaves was down-regulated within 24 h, and significantly up-regulated after 200 μmol·L-1 SA treatment at 12 h and 24 h. However, gene expression did not change with 200 μmol·L-1 ABA treatment. The transcripts of RpMYB4 under drought, high temperature, and mechanical injury stresses reached a peak at 24 h, 24 h, and at 3 h, respectively, while RpMYB4 expression was inhibited by low temperature stress, reaching its lowest value at 6 h. The gene showed no significant response to salt stress. Overall, RpMYB4 was cloned from R. palmatum L. for the first time, showed high expression in leaves, and was responsive to SA and various abiotic stress treatments including drought, high temperature, and mechanical injury. The results will be useful for further analysis of secondary metabolism and stress adaptations in R. palmatum L.

Complete plastid genomes of Bupleurum chinense DC. and B. boissieuanum H. Wolff, with comparative and phylogenetic analyses of medicinal Bupleurum species / 药学学报

italic>Bupleurum L. (Apiaceae) is an economically important genus, in which many species are of medicinal value. In this study, the complete plastid genomes (plastomes) of B. chinense DC. and B. boissieuanum H. Wolff were sequenced and their characteristics were investigated. Comparative and phylogenetic analyses were conducted with other published Bupleurum plastomes. The complete plastomes of B. chinense and B. boissieuanum were 155 458 and 155 800 bp in length, and both exhibited the typical quadripartite circular structure consisting of a large single copy region (LSC, 85 343 and 85 804 bp), a small single copy region (SSC, 17 495 and 17 410 bp), and a pair of inverted repeat regions (IRa/b, 26 310 and 26 293 bp), respectively. A total of 129 genes, including 84 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes were identified from each of the two plastomes. Repeat sequences detected were similar in types and distribution patterns, but the numbers were slightly different. Comparative analyses revealed that the Bupleurum plastomes were highly conserved in length, structure, the guanine and cytosine (GC) content, and gene content and order, both intraspecifically and interspecifically, and no obvious expansion or contraction of the inverted repeat regions occurred. Sequence variation was lower within the same species than among different species, noncoding sequences (including intergenic regions and introns) showed a higher divergence than the protein-coding sequences, and sequences in the LSC and SSC regions were more divergent than those in the IR regions. In addition, 11 sequences with higher nucleotide diversity among species were detected in the LSC and SSC regions. All studied Bupleurum species were inferred forming a monophyletic group with a 100% bootstrap value. Bupleurum chinense and B. boissieuanum were phylogenetically closest to B. commelynoideum and B. falcatum, separately, with all three B. chinense accessions clustered into a distinct clade. These results provide genetic information for further species identification, phylogenetic resolution, and will assist in exploration and utilization of medicinal Bupleurum species.

Anatomical study on reconstruction of calcaneus fibula ligament / 中国骨伤

OBJECTIVE@#To measure anatomical data of calcaneofibular ligament (CFL), relevant data of CFL attachment to provide an anatomical basis for CFL reconstruction.@*METHODS@#Twenty-seven adult ankle specimens were selected, including 11 males and 16 females, aged from 22 to 71 years old with an average of (41.6±17.2) years old;9 cases on the left side and 18 cases on the right side. The specimens reserved at least 20 cm above ankle joint and a complete foot, and exclude deformities, fractures, incomplete development and degenerative lesions. CFL was performed detailed anatomical observation, morphological parameters of CFL was measured, and coordinates of fibula side and calcaneal side of CFL in the coordinate axis were measured. The distance between fibula insertion of CFL and fibula tip, distance between calcaneal insertion of CFL and lateral calcaneal nodule, and Angle between CFL and long axis of fibula were also measured.@*RESULTS@#In these 27 specimens, CFL cases were all single bundles and the length of CFL was (32.83 ± 8.19) mm. The center point of fibula attachment in CFL was(2.87± 1.21) mm proximal with a coefficient of variation of 42.16% and (2.08±1.34) mm anteriorly with a coefficient of variation of 64.42%. The center point of calcaneal attachment region of CFL was located on coordinate axis on the distal end (15.32±5.33) mm, with a coefficient of variation of 34.79%, and the posterior part (6.38±2.15) mm, with a coefficient of variation of 33.86%. The distance between center point of fibula attachment and fibula tip was (4.81±0.82) mm. The distance between center point of calcaneal attachment area of CFL and lateral calcaneal nodules was(17.25±3.12) mm. Angle between CFL and fibula axis is (43 ±18)° .@*CONCLUSION@#According to anatomical studies, we could locate the fibula and calcaneal attachment of CFL by anatomical markers around ankle joint. However, the location of CFL attachment has a large variation, and the anatomical characteristics need to be considered in anatomical reconstruction.

Clinical and immunological characteristics of myositis complicated with thromboembolism / 北京大学学报(医学版)

OBJECTIVE@#To investigate and analyse the clinical and immunological features of patients with myositis complicated with thromboembolism.@*METHODS@#We identified a cohort of 390 myositis patients diagnosed with myositis admitted to People's Hospital of Peking University from 2003 to 2019. The patients were retrospectively enrolled in this investigation. According to the outcome of the color Doppler ultrasound, CT pulmonary angiography, pulmonary ventilation and perfusion scan patients were divided into myositis with and without thromboembolism group. Demographic, clinical (heliotrope rash, Gottron's sign/papules, periungual erythema, skin ulceration, subcutaneous calcinosis, Mechanic's hands, myalgia, interstitial lung disease, pulmonary arterial hypertension), laboratory, immunological [anti-autoantibodies including melanoma differentiation associated gene 5 (anti-MDA5), anti-Mi-2, anti-transcription intermediary factor-1γ (anti-TIF-1γ, anti-nuclear matrix protein 2 (anti-NXP2), anti-small ubiquitin-like modifier activating enzyme (anti-SAE), anti-synthetase], imaging and therapeutic status data of the patients at the diagnosis of myositis with and without thromboembolism were collected and the differences in these data were analyzed. Logistic regressive analysis was used to identify the risk factors of thromboembolism.@*RESULTS@#In the retrospective study, 390 myositis patients were investigated. The mean age of onset was (49.6±13.4) years, male to female ratio was 0.31 :1. Thromboembolism was identified in 4.62% (18/390) of the myositis patients, which was lower than the published reports. Out of 18 patients with thromboembolism, 55.6% (10/18) of them were deep venous thrombosis, followed by cerebral infarction (22.2%, 4/18), pulmonary embolism (11.1%, 2/18), renal artery embolism (5.6%, 1/18) and embolism of upper extremity (5.6%, 1/18). Fifty percent of thromboembolism events occurred 6 months after the diagnosis of myositis, 38.9% of thromboembolism events occurred 6 months within the diagnosis of myositis, 11.1% of thromboembolism events occurred 6 months before the diagnosis of myositis. As compared with the myositis patients without thromboembolism, the myositis patients complicated with thromboembolism were older [(58.3±11.7) years vs. (49.3±13.4) years, P=0.006]. C-reaction protein (CRP) (12.2 mg/L vs. 4.1 mg/L, P < 0.001), ferritin (20 085.5 μg/L vs. 216.6 μg/L, P < 0.001) and D-dimer (529.0 μg/L vs. 268.0 μg/L, P=0.002) were significantly higher in thromboembolism group. Diabetes (44.4% vs. 16.4%, P=0.006), coronary heart disease (22.2% vs. 3.0%, P=0.003) and surgery (16.7% vs. 3.5%, P=0.032) were observed more common in thromboembolism group than those without thromboembolism. Activated partial thromboplastin time (APTT) (26.9 s vs. 28.7 s, P=0.049) and albumin (32.4 g/L vs. 36.5 g/L, P=0.002) was lower in thromboembolism group. The risk factors of thromboembolism in the myositis patients were low level of albumin (OR=0.831, 95%CI: 0.736-0.939, P=0.003), diabetes (OR=4.468, 95%CI: 1.382-14.448, P=0.012), and coronary heart disease (OR=22.079, 95%CI: 3.589-135.837, P=0.001) were independent significant risk factors for thromboembolism in the patients with myositis. There was no significant difference in clinical manifestations, myositis-specific antibodies or myositis-associated antibodies between the two groups.@*CONCLUSION@#Thromboembolism is a complication of myositis. Lower levels of albumin, diabetes, and coronary heart disease might be risk factors of thromboembolism in myositis patients.

Molecular Characterization of A Protein Phosphatase 2C Gene of Subgroup A in Dendrobium officinale / 中国药学杂志

OBJECTIVE: To characterize a protein phosphatase (PP) encoding gene DoPP2C2 in a rare endangered medicinal orchid species Dendrobium officinale, followed by bioinformatics analysis and expression profiling. METHODSE: RACE and RT-PCR were used to isolate the full length cDNA of DoPP2C2. The physiochemical properties, conserved domains and subcellular localization of the deduced DoPP2C2 protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 7.0 and MEGA 6.0 softwares, respectively. Quantitative PCR was used for gene expression analysis. RESULTS: The full length cDNA of DoPP2C2 (GenBank accession KT957553) was 1 624 bp in length, and encoded a 387-aa protein with a molecular weight of 42.76×103 and an isoelectric point of 7.03. The deduced DoPP2C2 protein sequence had two PP2C domains (91-152, 216-376), which are all conserved among the PP2Cs. The protein without a signal peptide or a transmembrane region, was predicted to locate in nucleus with hydropathicity. DoPP2C2 had high identities (47.8%-73.4%) with various PP2C proteins from several plants. DoPP2C2 protein belonged to the subgroup A of Arabidopsis and rice PP2C evolutionary tree, and was closely related to OsPP2C30; DoPP2C2 was differentially expressed in the three included organs. The transcripts were less in the roots and stems, being 0.76 and 0.59 folds, respectively, of that in the leaves. CONCLUSION: The molecular characteristics of a subgroup protein phosphatase encoding gene DoPP2C2 of the full length cDNA in D. officinale was obtained. The results will be useful for further functional determination of the gene involving in the growth and development of D. officinale.

HPLC Fingerprint Analysis and Chemistry Pattern Recognition of Wild and Cultivated Notholirion bulbuliferum from Different Regions / 中国实验方剂学杂志

Objective: To establish the HPLC fingerprint of wild and cultivated Notholirion bulbuliferum,and recognize them according to the chemical pattern, in the expectation of providing the basis for the quality control and domestication cultivation of N. bulbuliferum of origins. Method: Twenty samples of wild and cultivated N. bulbuliferum collected from different origins were detected by HPLC, and a common mode of fingerprint was established. The Similarity Evaluation System for Chromatographic Fingerprint of TCM (2012A edition) was used to evaluate the similarity of the samples. The differences among the samples were identified by chemical pattern recognition methods, including principal component analysis (PCA),cluster analysis (HCA) and partial least squares discriminate analysis (PLS-DA). Result: The HPLC fingerprint of N. bulbuliferum was obtained,and 26 common peaks were found in the chromatograph. Similarities of all samples were over 0.9,PCA,and HCA and PLS-DA results demonstrated obvious distinctions between wild and cultivated N. bulbuliferum. Eight constituents,such as pcoumaric acid were identified as biomarkers,representing major differences between the two varieties. Conclusion: The HPLC chromatogram of N. bulbuliferum developed in this paper has strong characteristics and repeatability. After being combined with the pattern recognition mode, it can be used as an effective method for evaluating the quality of N. bulbuliferum and distinguishing wild and cultivated N. bulbuliferum,and provide a reference for the quality control and domestication introduction of N. bulbuliferum.

Sub-chronic Toxicity of Defoamer Used in Seawater Desalination / 生物医学与环境科学（英文）

OBJECTIVE@#To explore the possible long-term health effects of the defoamer used in seawater desalination by sub-chronic toxicity testing.@*METHODS@#Blood analysis, internal organ assessment, and histopathological examination were carried out in rats exposed to low, medium, and high (0.5, 1.0, and 2.0 g/kg BW, respectively) doses of defoamer for 90 days through oral administration.@*RESULTS@#The high dose group showed decreased blood alanine aminotransferase and aspartate aminotransferase (P < 0.05). All doses resulted in a significant increase in albumin and decrease in globulin (P < 0.05). The direct bilirubin and indirect bilirubin were decreased in the medium and high dose groups (P < 0.05). All dose groups showed significant induction of alkaline phosphatase (P < 0.05). Pathological examination revealed a case of liver mononuclear cell infiltration in the medium dose group and three cases of liver congestion, steatosis of hepatic cells around the central vein, and punctate necrosis with multiple focal mononuclear cell infiltration in male rats administered the high dose. The No Observed Adverse Effect Level was 0.5 g/kg BW in rats, with albumin and total bilirubin as health effect indices.@*CONCLUSION@#Long-term defoamer exposure may cause liver injury but has no significant impact on renal function in rats. The effect on blood cells in female rats was more prominent than that in male rats.

Role of TGF-β1/Smads signaling pathway in tissue remodeling of cultured nasal polyps in vitro / 上海交通大学学报（医学版）

Objective: To further explore the role of TGF-β1/Smads signaling pathway in the tissue remodeling process of cultured nasal polyps in vitro. Methods: Fifteen cases of chronic rhinosinusitis with nasal polyps (CRSwNP) and 15 cases of chronic rhinosinusitis without nasal polyps (CRSsNP) were screened out, and the control group was 10 patients with deviation of nasal septum. The location and expression of proteins in tissues were analyzed by immunohistochemistry. The expression and distribution of collagen were detected by using the method of Masson trichrome staining. The levels of mRNA and protein expression were detected by quantitative realtime polymerase chain reaction (qRT-PCR) and Western blotting respectively. Nasal polyps were treated ex vivo by TGF-β1 (n=15). The levels of mRNA and protein expression in culture pellets and collagen expression in culture supernatant were analyzed by qRT-PCR, Western blotting and ELISA respectively. Results: The TGF-β1, Smad2, Smad3 mRNA and protein expression levels were significantly decreased in the CRSwNP group compared with the CRSsNP group (all P<0.05). TGF-β1 and pSmad2/3 protein level showed positive correlation in CRS (r=0.991, P<0.01), so was TGF-β1 and Smad2, Smad3 mRNA levels (r=0.581, r=0.658, both P<0.01). TGF-β1 had positive correlation with collagen expression in CRS (r=0.982, P<0.01). Compared with the controls ex vivo, the levels of Smad2, Smad3, pSmad2/3 and collagen were markedly increased after TGF-β1 treatment. Conclusion: TGF-β1/Smads signaling pathway may play an important role in tissue remodeling of CRSwNP, and cause collagen reduction and edematous mucous membrane in nasal polyps.

High-throughput transcriptomic sequencing of Rheum palmatum L. seedlings and elucidation of genes in anthraquinone biosynthesis / 药学学报

Anthraquinones are not only the main active constituents but also the index components for the quality control of Rhei Radix et Rhizoma. To study the anthraquinone biosynthesis, Rheum palmatum L. seedlings were subjected to a high-throughput transcriptomic sequencing analysis by Illumina HiSeqTM 2000 150PE. The Illumina sequencing generated a total of 11.04 G clean data resulting in 736 309 74 clean reads, deposited in the sequence read archive (SRA accession SRP160030). Trinity do novo assembly yielded 93 646 unigenes, with an average of 1 108 nt. Functional annotation revealed that all unigenes were successfully annotated in the NR, NT, Swiss-port, PFAM, and KOG databases. GO enrichments showed that 57 subgroups were involved in biological process, cellular component, and molecular function. KEGG analysis indicated that 1 107 unigenes were implicated in 19 standard secondary metabolic pathways. 172 unigenes were analyzed to encode 28 key enzymes during the MVA, MEP, shikimic acid, and polyketide pathways related to anthraquinone biosynthesis. 125 CYP450 and 73 UGTs unigenes were related the modification of secondary metabolites in R. palmatum L. Furthermore, seven unigenes with full length cDNAs were successfully verified by RT-PCR and sequencing analyses. Then, MISA prediction produced a number of 18 885 simple sequence repeats (SSRs). Herein, the transcriptomic gene expression profiles of R. palmatum L. and candidate genes during the anthraquinone biosynthesis pathway were obtained for the first time. The results provided basic information for subsequent gene function characterization, secondary metabolic pathway analysis, and anthraquinone biosynthesis and regulation elucidation in R. palmatum L.

Identification and expression analysis of four CIPKs genes from a rare and medicinal plant, Dendrobium officinale / 药学学报

The calcineurin B-like protein (CBL)-interacting protein kinase (CIPK) plays a vital role in the growth, development, and stresses adaptation in plants by interaction with the calcium signaling. In this study, four full length cDNAs of CIPKs genes, namely DoCIPK1, DoCIPK2, DoCIPK3 and DoCIPK4 (GenBank accession No. KT957557, KT957558, KT957559 and KT957560, respectively) were cloned from the rear and medicinal plant, Dendrobium officinale, by rapid amplification of cDNA ends (RACE) for the first time. The corresponding encoded proteins, consisting of 473, 449, 451 and 440 amino acids (aa), respectively, with a molecular weight of 53.50, 50.93, 51.50 and 50.16 kDa, and an isoelectric point (pI) of 7.99, 9.25, 8.81 and 9.11, respectively, shared 70%-90%, 69%-80%, 78%-93%, and 66%-82% identities CIPKs with various plants. Each deduced protein contained a conserved protein kinase domain (respectively at 21 -275, 14-268, 16-271 and 12-266 aa position), a CIPKs family characteristic NAF/FISL domain (respectively at 335-391, 313-370, 310-369 and 305-362 aa position) and some functional motifs. The four DoCIPK proteins, without signal peptide or transmembrane region, were located in the plasma membrane and endoplasmic reticulum at the subcellular level. The three dimensional structure of the proteins were similar to that of Arabidopsis AtCIPK24. DoCIPK1 and DoCIPK3 were respectively clustered in the group E and A of the Arabidopsis and rice CIPK evolutionary tree, while DoCIPK2 and DoCIPK4 belonged to group C. The relative expression of DoCIPK1 showed no significant difference in the leaves and stems, and its transcripts in the roots was 0.35 fold over that in the leaves. The abundance of DoCIPK3 transcripts in the stems and the roots were 3.36 fold and 3.47 fold higher, respectively, than those in the leaves. DoCIPK2 exhibited similar expression pattern to DoCIPK4. Their relative expression in the leaves and the stems had no apparent difference, and the transcript levels were higher in the roots than that in the leaves, with 2.08 fold and 7.86 fold, respectively. Cloning, bioinformatics analyses, and expression patterns of the four DoCIPK genes provide a basis for functional elucidation of these genes further during the physiological responses in D. officinale.

Isolation and expression analysis of a hexose transporter gene DoHT1 in Dendrobium officinale / 中国中药杂志

In this study, RACE technology was employed to isolate the full length cDNA of DoHT1 in Dendrobium officinale, followed by bioinformatics analysis of the sequence characteristics. And the expression pattern of the gene was also analyzed by quantitative PCR. The full length cDNA of DoHT1 was 1 586 bp in length, containing a 1 536 bp ORF, which encoded a 511-aa protein with molecular weight of 56.18 kD and isoelectric point of 9.08. The deduced DoHT1 protein had the major facilitator superfamily conserved domain (22-483), SUGAR₋TRANSPORT₋1 (139-164), and SUGAR₋TRANSPORT₋2 (338-355), typical for sugar transporter; DoHT1, without a single peptide had 11 transmembrane regions, and was predicted to locate in the plasma membrane; DoHT1 had high identities (54.7%-80.7%) with HTs proteins from various plants. DoHT1 belonged to the MST (monosaccharide transporter) group of the evolutionary tree, and was closely related to the Phalaenopsis equestris. DoHT1 was differentially expressed in the three included organs. The transcripts were significantly the most abundant in the leaves with 19.36 fold than roots, then 1.82 fold in the stems than the roots. The identification and molecular characterization of the full length DoHT1 will be essential for further function study of the gene during the regulation of sugar metabolism of D. officinale.

Transcriptomic data analyses of rhizome of Souliea vaginata via Illumina high-throughput sequencing technology / 中草药

Objective To obtain the transcriptome dataset of rhizome of Souliea vaginata Methods Using the high-throughput illumina sequencing platform Illumina HiSeqTM 2000 150PE, a rhizome transcriptome dataset of S. vaginata was obtained, followed by systemic bioinformatics analyses. Results The transcriptome sequencing analyses produced to a great number of 63 322 086 high quality clean reads. Trinity de novo assembling resulted in a total of 52 575 unigenes with an average length of 909 nt. BLAST analysis indicated that 28 842 (accounting for 54.86% of the total unigenes), 10 712 (20.37%), 9 245 (17.58%), and 11 559 (21.99%) unigenes were successfully annotated in the NR, Swiss-port, KOG, and KEGG databases, respectively. GO classification contained the basic three major groups, including biological process, cellular component, and molecular function, and 45 subgroups. Among them126 KEGG standard pathways were designated, of which 17 were defied as the secondary metabolism. Of all unigenes, 2 215 with protein coding sequences were predicted, and 55 families of plant transcription factors were also identified. MISA prediction yielded a number of 4 609 simple sequence repeats (SSRs), among which the tri-nucleotide SSRs were abundant with 2 106 (45.7%), whereas the penta-nucleotide SSRs were relatively less, accounting for 2.9%. Conclusion The transcriptomic characteristics of S.vaginata rhizome were revealed by the high-throughput Illumina sequencing technology along with bioinformatics analyses, which would be of great importance for the functional gene characterization, secondary metabolism pathway dissections, and their regulatory mechanisms in S. vaginata.

High-throughput transcriptome sequencing of roots of Dictamnus dasycarpus and data analyses / 中草药

Objective To obtain the transcriptome dataset of roots of Dictamnus dasycarpus. Methods The root transcriptome dataset of D. dasycarpus was obtained using the high-throughput sequencing platform Illumina HiSeqTM 2000 150PE, followed by systemic bioinformatics analyses. Results A great number of 69 643 286 high quality clean reads were obtained by the transcriptome sequencing analyses. Using Trinity de novo assembling, a total of 49 050 unigenes were finally obtained, with an average length of 841 nt. BLAST analysis indicated that 31 636 (accounting 64.49% of the total unigenes), 22 367 (45.60%), 19 246 (39.23%), 12 595 (25.68%) unigenes were successfully annotated in the NR, Swiss-port, KOG, and KEGG databases, respectively. And GO classification contained the basic three major groups, including biological process, cellular component, and molecular function with 42 subgroups. A total of 132 KEGG standard metabolic pathways were designated, 18 of which were defied as the secondary metabolism. Further analysis revealed that a total of 90 unigenes were involved in the biosynthesis of various alkaloids. Of all unigenes, 1 908 were predicted to have CDS, and 55 families of plant transcription factors were also identified. Using MISA prediction, 4 579 simple sequence repeats (SSRs) were obtained, among which the tri-nucleotide SSRs were abundant with 2 021 (44.1%), whereas the penta-nucleotide SSRs accounted for 3.5%. Conclusion The root transcriptome of D. dasycarpus revealed by the high-throughput sequencing technology will be important for gene functional characterization, secondary metabolism pathway exploration, and regulation mechanism research in this species.

Transcriptomic analyses of Chloranthus japonicus by RNA-seq / 中草药

Objective To obtain the transcriptome dataset of Chloranthus japonicas. Methods Using the Illumina HiSeqTM 2000 150PE, a rhizome transcriptome of C. japonicus was generated, followed by systemic bioinformatics analyses. Results A total of 68 458 750 high quality clean reads were produced by the transcriptome sequencing. Trinity de novo assembling resulted in a total of 56 096 unigenes with an average length of 801 nt. BLAST analysis indicated that 25 773 (45.94%), 17 801 (31.73%), 16 082 (28.67%), and 9 649 (17.20%) unigenes were successfully annotated in the NR, Swiss-port, KOG, and KEGG databases, respectively. All unigenes were classified into three major groups by GO, including biological process, cellular component, and molecular function, and then, grouped into 40 subgroups. And 131 KEGG standard pathways were designated, 16 of which were defied as the secondary metabolism. Further analysis revealed that a total of 170 unigenes were involved in the biosynthesis of mono-, di-, sesqui-, or triterpene. Meantime, 1 887 unigenes were predicted to contain protein coding sequences. Totally 54 families of transcription factors of higher plant were identified. Using MISA prediction, 8 987 simple sequence repeats (SSRs) were obtained, among which the di-nucleotide SSRs were abundant with 5 948 (66.2%), whereas the penta-nucleotide SSRs were relatively less, accounting for 1.3%. Conclusion The transcriptome of C. japonicus rhizome was generated by RNA-seq along with the identification of unigenes implicated in various terpenes biosynthesis, which will provide a fundamental basis for secondary metabolism pathway dissections and their regulatory mechanisms in this plant species.

Molecular cloning and expression analysis of two genes encoding F family ATP-binding cassette transporters in Dendrobium officinale / 中草药

Objective: To clone and characterize the F family ATP-binding cassette (ABC) transporter genes in Dendrobium officinale. Methods: RACE was used to isolate ABC transporter genes from the leaf cDNA of D. officinale. Characteristics including molecular weight, theoretical pI (isoelectric point), conserved domain, transmembrane structure, signal peptide, and subcellular localization of the deduced protein were analyzed by serials of bioinformatics algorithms. The analyses of multiple alignment and phylogenetic tree were respectively performed by DNASTAR and MEGA6. Tissue specific expression patterns were determined by real-time quantitative PCR (qPCR) analyses. Results: Two full length genes DoABCF1 and DoABCF2 (GenBank accessions KU160474 and KU160475), 2 104 and 2 193 bp in length, respectively, were obtained. DoABCF1 was deduced to a 600 aa (amino acid) protein with a molecular weight of 67 030 and pI of 6.20, while DoABCF2 encoded a 659 aa protein with a molecular weight of 74 140 and pI of 5.71. The two deduced DoABCF1 and DoABCF2 proteins both had two conserved ABC domains (74-314 and 385-600 for the former, 65-323 and 392-607 for the latter) and several functional motifs. The proteins did not contain any signal peptide or transmembrane domain, and were predicted to locate in the chloroplast. The two genes were highly identical to the plant F family ABC transporter genes with more than 80% similarity, and were mostly close to monocots ABC F family members from maize and rice. DoABCF1 and DoABCF2 showed different expression among the three organs and both had relatively highest expression levels in the leaves, whereas no significant difference could be observed in the roots and stems. Taken the stem as the calibrator sample, DoABCF1 transcripts were 1.74 fold over that in the stems and DoABCF1 transcripts were 3.44 fold, respectively. Conclusion: Two F family DoABCF1 and DoABCF2 genes with full length cDNAs are successfully cloned in this study. The highest expression levels of the two ABC genes in the leaves of D. officinale suggest that they should play an important role during the growth and development of D. officinale.

Analysis of Applying Chinese Medical Clinical Pathway for Treating Attention-deficit Hyperactivity Disorder / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To evaluate the application effect of Chinese medical clinical pathway for treating attention-deficit hyperactivity disorder (ADHD), and to provide evidence for further improving clinical pathways.</p><p><b>METHODS</b>Totally 270 ADHD children patients were recruited and treated at pediatrics clinics of 9 cooperative hospitals from December 2011 to December 2012. The treatment course for all was 3 months. Scores of attention deficit and hyperactivity rating scale, scores of behavior, Conners index of hyperactivity (CIH), and Chinese medical syndrome scores were compared between before and after treatment. The efficacy difference in various sexes, ages, and disease courses were evaluated by judging standards for Chinese medical syndrome and ADHD.</p><p><b>RESULTS</b>Fifteen children patients who entered clinical pathway dropped out, and the rest 255 completed this trial. Compared with before treatment, total scores of attention deficit and hyperactivity rating scale, scores of attention deficit and hyperactivity rating scale, CIH, and Chinese medical syndrome scores obviously decreased (all P < 0.01). The total effective rate in disease efficacy was 87.8% (224/255 cases), and the total effective rate in Chinese medical syndrome curative effect was 87.5% (223/255 cases). The clinical curative effect was not influenced by age, gender, or course of disease when statistically analyzed from judging standards for Chinese medical syndrome or for disease efficacy.</p><p><b>CONCLUSION</b>Intervention by Chinese medical clinical pathway could improve ADHD patients' symptoms, and its efficacy was not influenced by sex, age, or course of disease.</p>

Cloning and expression analysis of a zinc-regulated transporters (ZRT), iron-regulated transporter (IRT)-like protein encoding gene in Dendrobium officinale / 中国中药杂志

The zinc-regulated transporters (ZRT), iron-regulated transporter (IRT)-like protein (ZIP) plays an important role in the growth and development of plant. In this study, a full length cDNA of ZIP encoding gene, designed as DoZIP1 (GenBank accession KJ946203), was identified from Dendrobium officinale using RT-PCR and RACE. Bioinformatics analysis showed that DoZIP1 consisted of a 1,056 bp open reading frame (ORF) encoded a 351-aa protein with a molecular weight of 37.57 kDa and an isoelectric point (pI) of 6.09. The deduced DoZIP1 protein contained the conserved ZIP domain, and its secondary structure was composed of 50.71% alpha helix, 11.11% extended strand, 36.18% random coil, and beta turn 1.99%. DoZIP1 protein exhibited a signal peptide and eight transmembrane domains, presumably locating in cell membrane. The amino acid sequence had high homology with ZIP proteins from Arabidopsis, alfalfa and rice. A phylogenetic tree analysis demonstrated that DoZIP1 was closely related to AtZIP10 and OsZIP3, and they were clustered into one clade. Real time quantitative PCR analysis demonstrated that the transcription level of DoZIP1 in D. officinale roots was the highest (4.19 fold higher than that of stems), followed by that of leaves (1.12 fold). Molecular characters of DoZIP1 will be useful for further functional determination of the gene involving in the growth and development of D. officinale.