ABSTRACT
The rol genes on pRiA4 plasmid of Agrobacterium rhizogenes are potent genes that promote secondary metabolism. Molecular breeding of Atropa belladonna can be conducted by introducing rol genes to increase tropane alkaloids (TAs) content in A. belladonna. In this study, the rolB gene was overexpressed in A. belladonna plants to study the effect of rolB gene on the biosynthesis of TAs. The phenotype, TAs content and expression levels of key enzyme genes in the pathway of TAs biosynthesis of transgenic A. belladonna were analyzed. The results showed that transgenic A. belladonna had developed root system, enlarged leaves, increased leaf fresh weight, deepened leaf color, enlarged flowers, changed flower shape, reduced pistil height and decreased pollen vitality. The content of TAs in the stems of transgenic A. belladonna was significantly higher than that of the control, and the contents of scopolamine, anisodamine, hyoscyamine can reach 2.11-2.91, 1.23-2.37 and 4.88-5.20 times of the control, respectively. Compared with the control group, the expressions of key enzymes putrescine N-methyltransferase (PMT), type III polyketide synthase (PYKS), tropinone reductase I (TRI), aromatic amino acid aminotransferase 4 (ArAT4), UDP-glycosyltransferase 1 (UGT1) and hyoscyamine 6-β-hydroxylase (H6H) in the TAs biosynthesis pathway were up-regulated, and the expression of tropinone reductase II (TRII) as a metabolic shunting gene was down-regulated. The results indicated that rolB gene enhanced TAs synthesis ability in roots and accumulation in stems of A. belladonna by enhancing metabolic flow of TAs synthesis pathway and weakening the metabolic shunt of competing pathway. This study laid a foundation for molecular breeding of A. belladonna with high-yield TAs content using rolB gene.
ABSTRACT
On the basis of the Uncaria transcriptome, specific primers were designed for UrSTR. The full-length cDNA of UrSTR (GeneBank: OL310251) was 1 541 bp, encoding 345 amino acid residues, and the promoter region sequence of UrSTR (GeneBank: OL310252) was 1 179 bp. Phylogenetic tree is revealed that UrSTR had a closest relationship with STR from Ophiorrhiza pumila and Ophiorrhiza japonica. Localization of UrSTR protein is revealed located in the vacuole membrane. Plant-care analysis indicated that the promoter region sequence of UrSTR, covering multiple light, stress and hormone-response cis-regulatory elements, and verified transcriptional activity. The results of SDS-PAGE show that pET-28a-UrSTR recombinant protein was successfully expressed, and the size was anticipated. The UrSTR prokaryotic expression system needs to be optimized in the later stage. The research lays the foundation for further purification to study its structure and functional characterization of the UrSTR protein.
ABSTRACT
Hair roots induced by Agrobacterium rhizogene produce higher levels of secondary metabolites than non-induced plants, and the enhanced metabolic capacity is driven by the rol gene. We hypothesized that rol genes can be utilized to improve the biosynthesis of tropane alkaloids (TAs) in Atropa belladonna. In this study, the rolC gene from Agrobacterium rhizogene pRiA4 plasmid, driven by a CaMV35S promoter, was overexpressed in A. belladonna. The phenotypes, TAs content and transcriptional expression of key genes in TAs biosynthesis were analyzed in transgenic A. belladonna plants. Results show that transgenic A. belladonna exhibited a well-developed root system, male sterility, higher stamen column length than pistil, early flowering, internode shortening, smaller but more flowers, increased axillary buds and lateral buds, decreased apical dominance, and long and narrow leaves as compared to wild-type plants. Transgenic A. belladonna produced more TAs than wild-type plants, with the content of hyoscyamine, anisodamine and scopolamine reaching 2.58, 3.59 and 15.77-fold that of the control group, respectively. The gene expression of putrescine N-methyltransferase (PMT), tropinone reductase I (TRⅠ) and hyoscyamine 6-β-hydroxylase (H6H), key enzymes in TAs biosynthesis, were up-regulated compared with the control group. The above results indicate that the rolC gene enhances TAs biosynthesis in A. belladonna by up-regulating the expression of key enzymes in the TAs biosynthesis pathway, laying a foundation for genetic manipulation of A. belladonna to increase TAs content by increasing rolC gene expression.
ABSTRACT
Objective To investigate the clinical effect of improved portable mirabilite bag by abdominal application in the treatment of severe acute pancreatitis. Methods A total of 50 patients with severe acute pancreatitis admitted to our hospital from May2015 to January 2016 were enrolled as the control group, which was treated with traditional self-made towel bag filled with mirabilite for the abdominal application. Another 50 patients with severe acute pancreas hospitalized in our hospital from February 2016 to April 201750 patients as the experimental group, which was treated with improved portable mirabilite bag by abdominal application. The two groups were compared in terms of contamination by clothing, shedding, dislocation, aggregation, abdominal pain, bloating, and recovery time of gastrointestinal function. Results The incidences of shedding and displacement of mirabilite bag, mirabilite accumulation and contamination in the experimental group were lower than those of the control group. The abdominal distension time and bowel sound recovery time were significantly shorter than that in the control group (P ＜0.05, P ＜0.001). Conclusions By the improved portable mirabilite bag, the mirabilite can be evenly applied to the external abdomen of the patients. It is simple to make the bag and convenient for the clinical use, worthy of clinical application.
ABSTRACT
Objective To study the incidence of osteopenia in patients with initial systemic lupus erythematosus(SLE). Investigate the levels of the vitamin D (VitD) endocrine system in peripheral blood of SLE patients and its relation to bone mineral density (BMD). Analyse the relationship between the estrogen receptor (ER) and BMD and evaluate the role of ER in the pathogenesis osteopenia. Methods Serum levels of 25-OH VitD_3 and 1,25-(OH)_2 VitD_3 were detected by enzyme linked immunosorbent assay. The gene expression levels of VitD receptor (VDR) and ER were determined by real-time PCR. BMD measurements in the lumbar spine (L1-L4) and left proximal femur (femoral neck) were performed using dual X-ray absorptiometry before treatment. Results The initial SLE patients had significantly lower BMD values, and higher frequency of bone loss at both sites of measurement compared with normal controls (P < 0. 05). The levels of 25-OH VitD_3 and 1,25-(OH)_2 VitD3 were lower in the initial SLE patients than normal controls(P<0.01 both). There is no difference in the levels of 25-OH VitD_3 and 1,25-(OH)_2 VitD_3 between the osteopenia SLE group and the normal BMD SLE group (P > 0. 05, P > 0. 05). There are no correlations between the Vitd and BMD in initial SLE patients (P>0.05 both). The expressions of VDR gene were significantly increased in the initial SLE patients compared with the normal controls(P<0.01). There was no difference in VDR gene expression between osteopenia SLE group and normal BMD SLE group (P>0.05). The VDR gene expression does not correlate with the bone mass (P>0.05). The levels of ER-β gene expression are higher in the initial SLE group than the normal controls (P<0.01).Conclusions The incipient SLE patients may have lower BMD than expected. SLE patients present abnormal VitD endocrine system and higher ER-β mRNA expression than those in normal controls, but these weren't concerned with osteopenia.
ABSTRACT
Objective To investigate the prevalence of glomerular microthrombosis in lupus nephritis (LN) and the significance of antibodies to anti-coagulation related factors and anti-phospholipid antibodies in glomerular microthrombosis (GMT).Methods Kidney biopsy specimens and plasma samples were obtained consecutively from 124 patients with LN. Kidney biopsy specimens were examined for the presence of glomerular microthrombi.Plasma samples from 25 LN patients with GMT (LN-GMT group) and 99 LN patients without GMT (LN-non-GMT group) were tested for lupus anticoagnlant (LA) and antibodies to cardiolipin (ACL),β2 glycoprotein I (β2GP I ),plasmin,thrombin,tissue plasminogen activator (t-PA) and Annexin A II.Results The prevalence of GMT in LN patients was about 20.2%.Compared to LN-non-GMT group,LN-GMT group had elevated SLE disease activity indices (SLEDAI),elevated activity and chronicity indices of kidney tissue injury,and elevated serum creatinine,blood urea nitrogen and proteinuria levels,and also had a higher frequency of hypertension (P＜0.01).The positive rates of LA,IgG class anti-β2GP I and anti-thrombin antibodies were higher in LN-GMT group than in LN-non-GMT group (P＜0.05).The positive rates of IgG class antibodies to ACL,plasmin,t-PA and Annexin A II in LN-GMT group were not statistically different from those in LN-non-GMT group (P＞0.05).No difference was found in the positive rate of any IgM class antibody between the two groups (P＞0.05).Conclusion This study has shown that GMT occurs approximately in 20.2% of the LN patients.Patients with GMT have more severe kidney tissue injury and more poor renal outcomes than patients without GMT.LA and antibodies to β2GP I and thrombin play a role in glomerular microthrombosis in lupus nephritis.
ABSTRACT
Objective This study has explored the role of antibody against annexin A2 in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). Methods Using purified recombinant annexin A2, IgG anti-annexin A2 antibody was measured by ELISA in 101 APS patients, 41 SLE patients with thrombosis, 124 SLE patients without thrombosis and 120 healthy controls. Results The positive rate of IgG anti-annexin A2 antibody in APS patients and SLE patients with thrombosis was 21.8%, 26.8%, respectively, they were all significantly higher than in SLE patients without thrombosis (6.5%). IgG anti-annexin A2 antibody was associated with thrombosis and/or pregnancy morbidity (P<0.01). Conclusion Anti-annexin A2 antibody is associated with thrombosis and/or pregnancy mnrbidity. It suggests that anti-annexin A2 antibody may be helpful in identifying in some potential AIRS.
ABSTRACT
Objective ① To investigate the level of the vitamin D endocrine system in peripheral relationships with bone mineral density (BMD) and the disease activity respectively. Methods The level of the 25-hydroxylate vitamin D3 (25OHD3) and 1,25-dihydroxyvitamin D3 [1,25(OH)D3] in plasma from 43 SLE patients and 44 normal controls were detected by enzyme linked immunosorbent assay. Vitamin D receptor (VDR) gene expression was determinied by real-time PCR in peripheral blood. BMD measurements in the lumbar spine (L1-4) and left proximal femur (femoral neck) were performed using dual X-ray absorptiometry before treatment. The relationship between the vitamin D endocrine system and the bone mass were studied. We also discussed the relationship between the vitamin D endocrine system and the disease activity. Results The levels of 25OHD3 and 1,25 (OH)2D3 were lower in the initial SLE patients than normal controls (P<0.01, P<0.01). The expressions of VDR gene were significantly increased in initial SLE compared with normal controls (P<0.01). The initial SLE patients had significantly lower BMD values, and higher frequency of osteopenia (35%) at both sites of measurement compared with matched healthy controls (P<0.01). The initial SLE patients were divided into two groups by BMD, abnormal group and normal group. There were no differences in 25OHD3, 1,25 (OH)D3 and VDR gene expression (P0.05). There was no correlation between the vitamin D endocrine system and BMD in initial SLE patients. There was no correlation between the vitamin D endocrine system and the disease activity either. Conclusion Vitamin D endocrine system may play an important role in SLE, but the level of VDR gene is not correlated with BMD and disease activity.