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In order to reveal the molecular mechanism of the small heat shock proteins (sHSPs) involved in stress resistance and active ingredients accumulation in Salvia miltiorrhiza, a small heat shock protein gene was cloned from Salvia miltiorrhiza by reverse transcription PCR according to the transcriptome data of orange root Salvia miltiorrhiza. The gene is named SmHSP21.8 based on the molecular weight of the protein, and it contains an open reading frame of 585 bp, which encodes 194 amino acids. The results of phylogenetic analysis and amino acid sequence alignment showed that SmHSP21.8 protein belongs to the endoplasmic reticulum (ER) subfamily, and contains a conserved endoplasmic reticulum-specific DPFR-I/V-LE-H/Q-x-P motif at N-terminus. The prokaryotic expression vector pMAL-c2X-SmHSP21.8 was constructed and transformed into E. coli BL21 competent cells. The recombinant protein was successfully expressed after inducted. Temporal and spatial expression analysis showed that SmHSP21.8 gene was the highest expressed in flowers and had significant tissue specificity. The relative expression of the gene was significantly increased in seedlings after induction by 38 ℃, PEG6000, abscisic acid(ABA), and indole-3-acetic acid (IAA), indicating that SmHSP21.8 gene may be involved in abiotic stress such as high temperature and drought, as well as the response to exogenous hormones ABA and IAA. These results lay the foundation for further research on the molecular mechanism of small heat shock proteins involved in adversity stress.
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3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the first rate-limiting enzyme of terpenoid biosynthesis in the mevalonic acid pathway (MVA) pathway. It is an important regulatory site in terpenoids metabolism pathway in the cytoplasm. According to the transcriptome database of Cinnamomum camphora, two HMGRs named CcHMGR1 (GenBank: MN163055) and CcHMGR2 (GenBank: MN163056) were cloned by cDNA from C. camphora. The ORF of CcHMGR1 and CcHMGR2 is composed of 1 689 bp and 1 683 bp, respectively, encoding 562 and 560 amino acids. The bioinformatics analysis of CcHMGR1 and CcHMGR2 indicated that the molecular weight of the encoded protein is 59.819 kDa and 59.397 kDa, with a theoretically isoelectric point of 8.20 and 8.61, respectively. There are 2 transmembrane structures without signal peptide existing in the encoded amino acid of CcHMGRs. The analysis of sequence alignment and phylogenetic tree showed that theCcHMGRs belonged to the HMGR family. The camphor is divided into five chemitypes, according to the main chemical compoundsin C. camphora. The results of the real time PCR indicated that the expression level of CcHMGRs in Cineol type was higher than that in Linalool type, iso-nerolidol type, Camphor type and Borneol type. CcHMGRs expressed highest in roots and lowest in branches. In this study, the cDNA full length of CcHMGRs were cloned from C. camphora for the first time. Our results revealed that the expression level of CcHMGRs were different among five chemical types and different plant tissues, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.
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Objective@#The study is to explore daily violence exposure and its association with campus bullying, to provide theoretical basis for positive development of middle school students.@*Methods@#Questionnaire survey was conducted by using Violence Exposure Scale, Normative Beliefs about Aggression Scale, Middle School Students’ Self-control Scale, and Middle School Students’ Campus Bullying Scale. During Aug. to Oct. 2019, 1 372 middle school students were selected by the convenient sampling method as subjects of study from 2 junior high schools and 3senior high schools in Xinxiang.@*Results@#The total score in daily violence exposure was (34.22±12.09). The scores of violence exposure, traditional bullying and cyberbullying in female were lower than in male(t=-2.60--6.32, P<0.05). The scores of violence exposure, traditional bullying and cyberbullying in junior high school students were higher than senior high school students(t=4.59-7.50, P<0.05). The relationship between violence exposure and normative beliefs about aggression, traditional bullying, cyberbullying were positive (r=0.20, 0.44, 0.51, P<0.01). The relationship between violence exposure and self-control was negative (r=-0.29, P<0.01) . The relationship between normative beliefs about aggression and traditional bullying, cyberbullying were positive (r=0.28, 0.22, P<0.01). The relationship between normative beliefs about aggression and self-control was negative (r=-0.38, P<0.01). Violence exposure indirectly affects traditional bullying/cyberbullying through normative beliefs about aggression. The effect of normative beliefs about aggression on the traditional bullying/cyberbullying of middle school students is reduced with the increase of self-control.@*Conclusion@#Normative beliefs about aggression plays an intermediary role in violence exposure and traditional bully/cyberbullying, and self-control regulates the relationship between them.
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Objective To investigate the value of methyl thiazolyl tetrazolium assay ( MTT) in predicting drug sensitivity of breast cancer cells in vitro. Methods From January 2010 to July 2016,one hundred and ninety-two patients with breast cancer who underwent modified radical mastectomy or breast conserving surgery (no preoperative radiotherapy or chemotherapy) in the Shanghai Fengxian District Central Hospital were selected. MTT method was used to determine the inhibitory level and sensitivity of 12 drugs and 3 chemotherapy regimens to primary cultured cancer cells of 192 patients with breast cancer. Results (1) The sensitivity of breast cancer cells to 12 drugs were in sequence from high to low as follows: Paclitaxel (PTX)＞ Epirubicin ( EPI )＞ Cisplatin ( DDP )＞ 5-Fluorouracil ( 5-FU )＞ Mitoxantrone ( MIT )＞Vincristine ( VCR )＞ Pirarubicin ( THP )＞ Isosophosphamide ( IFO )＞ Carboplatin ( CBP )＞Cyclophosphamide ( CTX)＞ Methotrexate ( MTX)＞ Changchun Rui bin ( NVB) . The sensitivity of chemotherapy regimens in the three groups from high to low was docetaxel/doxorubicin/cyclophosphamide (TAC )＞cyclophosphamide/epirubicin/fluorouracil ( CEF )＞cyclophosphamide/methotrexate/fluorouracil (CMF). The sensitivity rates of PTX,EPI and DDP were 54%(104/192),42%(81/192) and 37%(71/192) respectively. (2) The average inhibitory rates of DDP,CBP and MIT in stage III breast cancer was higher than those in stage I and II breast cancer,and the differences were statistically significant ( F＝11. 14,4. 303,3. 182,P＜0. 05). (3) HR-breast cancer is more sensitive than HR+breast cancer,PTX, EPI,THP,MIT in HER-2(+) breast cancer is more sensitive than in HER-2(－) breast cancer. Conclusion As a widely used drug sensitivity test method, MTT assay has a certain reference value for screening sensitive drugs and selecting clinical chemotherapy regimens in neoadjuvant chemotherapy of breast cancer. PTX,EPI and DDP are more sensitive to other breast cancer cells than other drugs. Chemotherapy based on in vitro susceptibility results improves the efficiency of chemotherapy and decreases the proportion of changes in chemotherapy schemes due to inefficiency.
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Quality marker(Q-marker) is a new concept and pattern for quality control of traditional Chinese medicine(TCM),which will lead the development direction for quality control of TCM.Among them,how to characterize the overall quality attribute of TCM and its biological effect,is a critical scientific problem in the study of Q-marker.In this paper,integrated pharmacology is utilized to screen out and confirm the Q-marker from the complex system of TCM,so as to solve the critical scientific problem.System biology in vivo is firstly applied to establish the correlation of chemical fingerprints of TCM,their metabolic fingerprints,network targets,biological effects and efficacy of TCM,which is used to preliminary screen out Q-marker of TCM.Following that,a pharmacological method in vitro,including intestinal absorption in vitro coupled with bioactivity assessment,is employed to simultaneously determine the absorbed doses of TCM and evaluate their biological activity.Furthermore,data mining is utilized to establish the exact quantitative mathematic model between Q-marker of TCM and bioactivity.Meanwhile,two representative examples,including Yuanhu Zhitong tablets,Xinsuning capsules,are introduced to identify Q-marker of TCM and establish their quality standards related with bioactivity,which will be beneficial to improve the level of quality control of TCM and ensure the effectiveness and safety of clinical applications.
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Objective To investigate the clinical effect of therapeutic cervical cerclage on short cervix syndrome for anti-premature birth in the second trimester. Methods Totally 44 singleton pregnant patients were diagnosed as short cervix syndrome, which was cervical length ≤2.5 cm without cervical dilatation,and received treatment from January 2008 and July 2015 in Peking University Third Hospital were collected. Among them, 30 patients who received therapeutic cervical cerclage were defined as cerclage group and another 14 cases who received conservative treatment were defined as un-cerclage group. The days of conservative treatment, delivery rate of different gestational weeks, birth weight of newborns, neonatal survival rate within 7 days of birth were analyzed between the two groups. Results There were no significant differences between the two groups in days of pregnancy conservative treatment [103(84-141)vs 105(85-114)days], delivery weeks [38.0(35.5-39.4)vs 38.5(37.3-39.5)weeks], birth weight of newborns [3120(2750-3400)vs 3130(2760-3545)g], and survival rate of newborns [100％(30/30)vs 13/14]. The fetuses of both groups were all delivered after 28 weeks. There was no significant difference in accumulated delivery rate between the two groups after 32 weeks, 34 weeks, and 37 weeks, respectively(all P>0.05). Conclusions The treatment of cervical cerclage is not superior to conservative means in single pregnancy of cervical length ≤2.5 cm without cervical dilatation. For such patients with short cervix syndrome, the treatment of cervical cerclage may not be necessary, but dynamic monitoring and search for the causing factors and prompt treatment are more important.
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Objective To investigate the clinical efficacy of implementation of standardized enteral nutrition (EN) and its effects on prognosis in patients with severe traumatic brain injury (sTBI) undergoing mechanical ventilation (MV). Methods Eighty-eight patients with sTBI undergoing MV admitted to the Department of Critical Care Medicine of Yuyao People's Hospital from January 2016 to December 2017 were enrolled, they were divided into a control group (42 cases) and an experiment group (46 cases) depending on the demarcation timing of January 1, 2017, the beginning time of implementing standardized EN. All the patients received early EN and conventional treatment in the two groups. Additionally, the procedure of standardized EN was implemented in the experiment group. The differences in starting time of EN, the first defecation time, the rates of EN therapeutic energy and protein supply reaching their respective targets, duration of MV and ICU stay and 28-day mortality were compared between the two groups. Results The starting time of EN (hours: 25.61±8.74 vs. 32.79±8.63) and first defecation time (days: 3.03±0.79 vs. 3.61±0.89) were significantly earlier in the experiment group than those in the control group (both P < 0.05); the rates of energy and protein supply reaching the respective targets on the 5th day and 7th day after receiving EN were all significantly higher in the experiment group than those in the control group [rates of energy supply reaching target on the 5th day: (44.83±13.99)% vs. 37.59±10.88, and on the 7th day: (68.07±10.68)% vs. (62.69±9.87)%; rate of protein supply reaching target on the 5th day: (31.93±9.49)% vs. (27.06±8.08)%, and on the 7th day: (62.09±9.91)% vs. (54.55±11.27) %, all P < 0.05]; the durations of MV (hours: 9.24±2.91 vs. 10.67±3.41) and ICU stay (days: 12.09±3.37 vs. 13.93±4.98) in the experiment group were significantly shorter than those in the control group (all P < 0.05). No statistical significant difference in the 28-day mortality was observed between the experiment group and control group [21.74% (10/46) vs. 19.05% (8/42), P > 0.05]. Conclusion The efficacy of implementation of standardized EN in patients with sTBI undergoing MV is very significant, as it can significantly improve the rate of reaching EN target, and shorten the duration of MV and ICU stay.
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Jasmonates, as a plant endogenous hormone, can induce the biosynthesis of terpenoids, alkaloids and flavonoids and other medicinal active ingredients, and play an important role in the plant secondary metabolic process. The tanscription factors can activate the expression of multiple genes in the biosynthesis of plant secondary metabolites by binding the cis-elements of the target genes. Then, it effectively activates or inhibits the activities of the enzymes on the biosynthesis of plant secondary metabolites, further regulate specific biosynthesis and accumulation of secondary metabolites. Here, We review recent major progresses regarding the regulation of secondary metabolites by JAs-responsive transcription factors (TFs) (including AP2/ERFs, bHLH, MYB and WRKY). That provides suggestions for further analysis of jasmonic acid signaling pathway and regulation of secondary metabolism, and explores the potential value of transcription factor in improving the medicinal active ingredients.
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Objective:To construct the expression vector of the fusion protein of human serum albumin (HSA) and thymopentin (TP5) and to express it in Pichia pastoris,and to elucidate the biological activity of fusion protein.Methods:The HSA-TP5 fusion gene was constructed by gene recombination and transfected into Pichia pastoris to construct the eukaryotic expression system of HSA-TP5.The recombinant eukaryotic expression plasmid of PPICZα-HSA-TP5 was obtained by agarose gel electrophoresis and purification reagent.The two step fermentation method was used to ferment gene engineering bacteria of HSA-TP5 in high density,and the fermentation supernatant protein was precipitated and concentrated;the purified fusion protein was obtained by cation exchange chromatography and hydrophobic chromatography and analyzed by SDS-polyacrylamide gel electrophoresis.The effect of the fusion protein on the proliferation of lymphocytes was detected by MTT assay.Results:The HSA target gene fragment with length of 1 845 bp was achieved by PCR method.The HSA-TP5-pPICZαC fusion plasmid was identified by restriction endonuclease digestion,and the fragment length was 707 bp.The sequence analysis showed that the HSA and TP5 sequences of the target genes were identical with the gene sequences reported in GenBank and were fused by forward fusion.PCR method confirmed that the eukaryotic recombinant plasmid PPICZ αC-HSA-TP5 was integrated into the yeast genome,and compared with control group,the target gene PCR product length was found to be 1 860 bp.SDS-PAGE analysis showed that the expression level of HSA-TP5 fusion protein was gradually increased with the induction time within 72 h.HSA-TP5 fusion protein was purified by cation exchange chromatography and AKTA multifunctional protein purification system.The MTT assay results showed that HSA-TP5 fusion protein was consistent with TP5 protein in promoting lymphocyte proliferation activity.Conclusion:HSA-TP5 fusion protein can be obtained by constructing the eukaryotic expression system of Pichia pastoris and owns the biological activity.
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Objective To investigate the effect of blood pressure (BP) control level on perinatal outcomes in women with mild-moderate gestational hypertension (GHp). Methods Totally,344 women diagnosed initially as mild-moderate GHp who delivered in Peking University Third Hospital from January 2012 to December 2016 were recruited. They were divided into four groups according to the stabilized level of BP during pregnancy. (1) Group A:BP<130/80 mmHg(1 mmHg=0.133 kPa);(2) Group B:BP(130-139)/(80-89) mmHg; (3) Group C: BP (140-149)/(90-99) mmHg; (4) Group D: BP (150-159)/(100-109)mmHg. The clinical profile and incidence of severe GHp, pre-eclampsia with proteinuria (PE+Upro), severe pre-eclampsia (sPE), small-for-gestational age (SGA) were compared among the four groups. Student t-test was preformed to normal distributive data and Kruskal-Wallis test was used to non-normally distributed variables. Chi-square test was used in count data. Logistic regression analysis was adopted for multiple-factor analysis. Results (1) The incidence of severe GHp in group A was lower than group B (P<0.05). The incidences of severe GHp and sPE in the group B was lower than those in group C (P<0.05). While there was no difference in the incidence of PE+Upro and SGA among the four groups (P>0.05). And the incidence of severe GHp in group D had no difference with group A, B, C (P>0.05). (2) In the 48 patients who used medications to control BP, the occurence of severe GHp in those whose initial BP was(140-149)/(90-99)mmHg was lower than those of≥160/110 mmHg (P<0.05). But the incidence of severe GHp had no significant difference between patients whose initial BP was(140-149)/(90-99)mmHg and patients whose initial BP was(150-159)/(100-109)mmHg (P>0.05). The initial BP level had no impact on the incidence of PE+Upro, sPE and SGA (P>0.05). (3) Multivariate logistic regression analysis showed that the BP level before using medications (OR=3.566, 95%CI:1.080-11.771, P=0.037) and the BP level maintained (OR=4.787, 95%CI:1.115-20.551,P=0.035) were independent factor that affected the incidence of severe GHp. Edema (OR=2.651, 95%CI:1.628-4.316 P=0.000), fetal growth restriction(FGR;OR=1.103, 95%CI:1.427-5.914,P=0.002)and the onset gestational age of GHp (OR=0.755, 95%CI:0.578-0.985,P=0.038) were independent factors that affected the incidence of PE+Upro. The tendency of FGR (OR=17.787, 95%CI:1.833-40.396 P=0.000), history of PE (OR=5.294, 95%CI:1.086-25.800,P=0.039) and the BP level during pregnancy (OR=2.109, 95%CI:1.274-3.491,P=0.004) were independent factors affecting the incidence of sPE. FGR tendency was independent factor affecting the incidence of SGA (OR=25.622, 95%CI:2.596-252.864,P=0.005). Conclusion A satisfied control of BP is helpful to reduce severe GHp and sPE, but the incidence of SGA does not affected.
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Objective:To construct the expression vector of the fusion protein of human serum albumin (HSA) and thymopentin (TP5) and to express it in Pichia pastoris,and to elucidate the biological activity of fusion protein.Methods:The HSA-TP5 fusion gene was constructed by gene recombination and transfected into Pichia pastoris to construct the eukaryotic expression system of HSA-TP5.The recombinant eukaryotic expression plasmid of PPICZα-HSA-TP5 was obtained by agarose gel electrophoresis and purification reagent.The two step fermentation method was used to ferment gene engineering bacteria of HSA-TP5 in high density,and the fermentation supernatant protein was precipitated and concentrated;the purified fusion protein was obtained by cation exchange chromatography and hydrophobic chromatography and analyzed by SDS-polyacrylamide gel electrophoresis.The effect of the fusion protein on the proliferation of lymphocytes was detected by MTT assay.Results:The HSA target gene fragment with length of 1 845 bp was achieved by PCR method.The HSA-TP5-pPICZαC fusion plasmid was identified by restriction endonuclease digestion,and the fragment length was 707 bp.The sequence analysis showed that the HSA and TP5 sequences of the target genes were identical with the gene sequences reported in GenBank and were fused by forward fusion.PCR method confirmed that the eukaryotic recombinant plasmid PPICZ αC-HSA-TP5 was integrated into the yeast genome,and compared with control group,the target gene PCR product length was found to be 1 860 bp.SDS-PAGE analysis showed that the expression level of HSA-TP5 fusion protein was gradually increased with the induction time within 72 h.HSA-TP5 fusion protein was purified by cation exchange chromatography and AKTA multifunctional protein purification system.The MTT assay results showed that HSA-TP5 fusion protein was consistent with TP5 protein in promoting lymphocyte proliferation activity.Conclusion:HSA-TP5 fusion protein can be obtained by constructing the eukaryotic expression system of Pichia pastoris and owns the biological activity.
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Objective To investigate the effect of blood pressure (BP) control level on perinatal outcomes in women with mild-moderate gestational hypertension (GHp). Methods Totally,344 women diagnosed initially as mild-moderate GHp who delivered in Peking University Third Hospital from January 2012 to December 2016 were recruited. They were divided into four groups according to the stabilized level of BP during pregnancy. (1) Group A:BP<130/80 mmHg(1 mmHg=0.133 kPa);(2) Group B:BP(130-139)/(80-89) mmHg; (3) Group C: BP (140-149)/(90-99) mmHg; (4) Group D: BP (150-159)/(100-109)mmHg. The clinical profile and incidence of severe GHp, pre-eclampsia with proteinuria (PE+Upro), severe pre-eclampsia (sPE), small-for-gestational age (SGA) were compared among the four groups. Student t-test was preformed to normal distributive data and Kruskal-Wallis test was used to non-normally distributed variables. Chi-square test was used in count data. Logistic regression analysis was adopted for multiple-factor analysis. Results (1) The incidence of severe GHp in group A was lower than group B (P<0.05). The incidences of severe GHp and sPE in the group B was lower than those in group C (P<0.05). While there was no difference in the incidence of PE+Upro and SGA among the four groups (P>0.05). And the incidence of severe GHp in group D had no difference with group A, B, C (P>0.05). (2) In the 48 patients who used medications to control BP, the occurence of severe GHp in those whose initial BP was(140-149)/(90-99)mmHg was lower than those of≥160/110 mmHg (P<0.05). But the incidence of severe GHp had no significant difference between patients whose initial BP was(140-149)/(90-99)mmHg and patients whose initial BP was(150-159)/(100-109)mmHg (P>0.05). The initial BP level had no impact on the incidence of PE+Upro, sPE and SGA (P>0.05). (3) Multivariate logistic regression analysis showed that the BP level before using medications (OR=3.566, 95%CI:1.080-11.771, P=0.037) and the BP level maintained (OR=4.787, 95%CI:1.115-20.551,P=0.035) were independent factor that affected the incidence of severe GHp. Edema (OR=2.651, 95%CI:1.628-4.316 P=0.000), fetal growth restriction(FGR;OR=1.103, 95%CI:1.427-5.914,P=0.002)and the onset gestational age of GHp (OR=0.755, 95%CI:0.578-0.985,P=0.038) were independent factors that affected the incidence of PE+Upro. The tendency of FGR (OR=17.787, 95%CI:1.833-40.396 P=0.000), history of PE (OR=5.294, 95%CI:1.086-25.800,P=0.039) and the BP level during pregnancy (OR=2.109, 95%CI:1.274-3.491,P=0.004) were independent factors affecting the incidence of sPE. FGR tendency was independent factor affecting the incidence of SGA (OR=25.622, 95%CI:2.596-252.864,P=0.005). Conclusion A satisfied control of BP is helpful to reduce severe GHp and sPE, but the incidence of SGA does not affected.
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Geranylgeranyl pyrophosphate synthase enzyme is one of the key enzymes in the synthesis pathway of diterpenoid. Nine Lamiaceae genus GGPS synthase in Genebank was analyzed in this article. GGPS synthase the nucleic acid sequences and amino acid sequences, physicochemical properties, the signal peptide, leader peptides, transmembrane topological structure, hydrophobic, hydrophilic, subcellular localization, secondary structure, function domain, tertiary structure and evolutional relationship were predicted by using bioinformatics methods.Phylogenetic tree was constructed for the geranylgeranyl pyrophosphate synthase enzyme protein family. The results showed that GGPS amino acid sequence of the physical and chemical properties were basically identical, mainly hydrophilic protein, there existed chloroplast transit peptide, and no signal peptide and membrane structure domain, which mainly located in the chloroplast, the minor part located in mitochondria. The main secondary structures of the proteins are alpha helix and random coil. All these proteins have catalytic residues, aspartate-rich region, active site lid residues, substrate-Mg2+ binding site. The results provide theoretical reference for study on both the enzymatic characteristics of GGPS and the biosynthesis pathway of diterpenoid.
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Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medicinal constituent. To investigate the underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata leaves with MeJA treatment was performed. In A. paniculata transcriptomic data, the expression pattern of one member of NAC transcription factor family (ApNAC1) matched with andrographolide accumulation. The coding sequence of ApNAC1 was cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene predominantly expresses in leaves. Compared with control sample, its expression abundance sharply increased with methyl jasmonate treatment. Based on its expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis. ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein was purified by Ni-NTA agarose. Further study will help us to understand the function of ApNAC1 in andrographolide biosynthesis.
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1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is the second rate-limiting enzyme of terpenoid biosynthesis in the methylerythritol-4-phosphate pathway. According to the transcriptome database of Cinnamomum camphora, the DXR cDNA was cloned by rapid amplification of cDNA ends (RACE) from C. camphora, and was named CcDXR1 (GenBank number:KU886266). The ORF of CcDXR1 is composed of 1 413 bp, and it encodes 470 amino acids. The bioinformatics analysis suggests that the molecular weight of the encoded protein is 51.1 kD and the theoretically isoelectric point is 6.62, and there is no signal peptide and transmembrane structure in putative protein. The analysis of sequence alignment and phylogenetic tree showed that the CcDXR1 belonged to the DXR family. The results of the realtime PCR indicated that expression level of CcDXR1 in mature leaves was higher than tender leaves, which in roots was similar to leaves and the lowest in branches. The camphor is divided into five chemotypes, according to the main chemical compounds in C. camphora. It also showed that the expression level of CcDXR1 in borneol C. camphora was highest than that in cineol, iso-nerolidol, camphor and linalool. Our results revealed that the expression level of CcDXR1 exhibits diversity among plant tissues, growth periods and five chemical types, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.
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Jasmonic acid carboxyl methyltransferase (JMT), a key enzyme for jasmonate (JA) biosynthesis, catalyzes the methylation of JA to form MeJA. To characterize the function of JMT, a plasmid pGEX-4T-SmJMT1 harboring JMT1 (SmJMT1) gene from Salvia miltiorrhiza was successfully transformed into E.coli BL21 (DE3) for protein expression. The recombination SmJMT1 was separated using SDS-PAGE and the size of expressed SmJMT1 protein was consistent with the prediction. The bacterial growth conditions were determined for optimal expression, which include growth temperature, incubation time, IPTG concentrations and culture density. The optimal growth conditions for SmJMT1 were that the bacterial cultures were grown to an A600 of 0.8, and induced with IPTG at a final concentration of 0.4 mmol·L-1, and then incubated for 8 h at 20℃. The expression of SmJMT1 in E.coli was confirmed by Western blotting, and mass spectrometry analysis of methyltransferase family. The successful expression and purification of JMT in this study provide the basis for more study of JA biosynthetic pathway and JA-regulated secondary metabolism of medicinal plants.
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Herb residue is post-decoction material that can be used as organic fertilizer. Unfortunately, it is currently disposed of as solid waste. This method of disposal is a waste of this resource and a source of environmental pollution. For this case,we studied effects of six different herb residues compost on growth and phenols of Perilla frutescens by pot experiment. Our results show that all six herb residues can improve the growth of P. frutescens. The order of their efficiencies was as follows： Salviae Miltiorrhizae Radix residue>Hordei Fructus germinates residue>Forsythia fructus residue>Atractylodis Macrocephalae Rhizome residue＞Sophorae Flavescentis Redix residue and Moutan cortex residue. Effects of Sophorae Flavescentis Radix residue and Moutan Cortex residue weren't significantly different from CK. Six herb residue all improve root system architecture and leaf area. To phenols of P. frutescens, six herb residues all increased the rosmarinic acid and caffeic acid content of root, and accumulation of four phenols. All the analysis showed herb residues compost can improve the growth and four phenols accumulation of P. frutescens, and Salviae Miltiorrhizae Radix residue had the most pronounced effect on P. frutescens.
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The 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase was the fourth key enzymes in plant terpenoid biosynthesis pathway of methyl erythritol phosphate pathway(MEP). According to the study of Cinnamomum camphora transcriptome data,we abtained the 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase gene using RT-PCR,and named CcCMK1,then deposited it in GeneBank(Accession number： Ku376098).Bioinformatics analysis showed the open reading frame (ORF) of the CcCMK1 was 1 212 bp.The putative protein encoded 403 amino acids,and its molecular weight was 44.46 kDa and theoretically isoelectric point was 4.99.Transmembrane structure analysis showed that there was no transmembrane structure. Signal peptide analysis showed that it was a non secretory protein, and there was no signal peptide. The subcellular localization showed that the chloroplast was located in the chloroplast.Analysis of the expression of CcCMK1 gene in five chemotypes of C. camphora using Real-time PCR showed its expression level was highest in C. longepaniculatum, and the lowest in Borneol camphor.This research provided a basis for characterizing the key enzyme genes of terpenoid biosynthetic pathway in C. camphora.
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Traditional Chinese medicine industry product a lot of herb residue. Herb residue was treated as household waste. This treatment leads to environmental pollution and resource waste. For this case, we study the effect of different herb residues on the growth and active ingredient content of Licorice by random control experiment. Our results showed that the effects of different herb residues were difference. Atractylodes macrocephala residue and Forsythia suspense residue had the stronger effect and the effect of A. macrocephala residue was inferior to the effect of F. suspense residue. A.macrocephala residue significantly improved the shoot biomass banch number, leaf number, root diameter and root biomass by 0.53-1.81 fold. A. macrocephala residue also increased the glycyrrhizic acid content of root by 1.54 fold. F. suspense residue significantly improved the shoot biomass,branch number, root diameter and root biomass by 0.43-1.13 fold. Four kind herb residues all improved the shoot biomass by 0.43-1.81fold. So, the authors recommand to considered that we can apply A. macrocephala residue and F. suspense residue in Licorice cultivation.
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Objective To investigate the effect of lung protective and ventilatory management strategies for brain death donors on eligibility and availability of lungs for transplantation.Method The clinical data of two brain dead patients who accepted lung protective ventilatory management strategies from Feb.2015 to Mar.2015 were retrospectively analyzed.Two cases of brain-dead patients,due to severe cerebral trauma,accepted the aggressive lung protective ventilatory management strategies and airway management for 9 days and 4 days respectively.PaO2/FiO2,chest imaging manifestations,surface of the lung harvested and pulmonary rehabilitation of recipients after operation were observed.Result Two lung recipients were liberated from ventilation and pulmonary function improved significantly after double lung transplantation.Conclusion The application of lung protective ventilatory strategies in potential organ donors with brain death can increase the number of eligible and harvested lungs.