A Study on the Functional Identification of Overexpressing Winter Wheat Expansin Gene TaEXPA7-B in Rice under Salt Stress.

Expansin is a cell wall relaxant protein that is common in plants and directly or indirectly participates in the whole process of plant root growth, development and morphogenesis. A well-developed root system helps plants to better absorb water and nutrients from the soil while effectively assisting them in resisting osmotic stress, such as salt stress. In this study, we observed and quantified the morphology of the roots of Arabidopsis overexpressing the TaEXPAs gene obtained by the research group in the early stage of development. We combined the bioinformatics analysis results relating to EXPA genes in five plants and identified TaEXPA7-B, a member of the EXPA family closely related to root development in winter wheat. Subcellular localization analysis of the TaEXPA7-B protein showed that it is located in the plant cell wall. In this study, the TaEXPA7-B gene was overexpressed in rice. The results showed that plant height, root length and the number of lateral roots of rice overexpressing the TaEXPA7-B gene were significantly higher than those of the wild type, and the expression of the TaEXPA7-B gene significantly promoted the growth of lateral root primordium and cortical cells. The plants were treated with 250 mM NaCl solution to simulate salt stress. The results showed that the accumulation of osmotic regulators, cell wall-related substances and the antioxidant enzyme activities of the overexpressed plants were higher than those of the wild type, and they had better salt tolerance. This paper discusses the effects of winter wheat expansins in plant root development and salt stress tolerance and provides a theoretical basis and relevant reference for screening high-quality expansin regulating root development and salt stress resistance in winter wheat and its application in crop molecular breeding.

3-ketoacyl-CoA synthase 7 from Xanthoceras sorbifolium seeds is a crucial regulatory enzyme for nervonic acid biosynthesis.

Nervonic acid (C24:1) is a very-long-chain fatty acid that plays an imperative role in human brain development and other health benefits. In plants, 3-ketoacyl-CoA synthase (KCS) is the key rate-limiting enzyme for C24:1 biosynthesis. Xanthoceras sorbifolium is a valuable oil-producing economic woody species with abundant C24:1 in seed oils, but the key KCS gene responsible for C24:1 accumulation remains unknown. In this work, a correlation analysis between the transcript profiles of KCS and dynamic change of C24:1 content in developing seeds of X. sorbifolium were conducted to screen out three members of KCS, namely XsKCS4, XsKCS7 and XsKCS8, potentially involved in C24:1 biosynthesis. Of which, the XsKCS7 was highly expressed in developing seeds, while XsKCS4 and XsKCS8 displayed the highest expression in fruits and flowers, respectively. Overexpression of XsKCS4, XsKCS7 and XsKCS8 in yeast Saccharomyces cerevisiae and plant Arabidopsis thaliana indicated that only XsKCS7 possessed the ability to facilitate the biosynthesis of C24:1. These findings collectively suggested that XsKCS7 played a crucial role in specific regulation of C24:1 biosynthesis in X. sorbifolium seeds.

Molecular cloning and functional characterization of 2,3-oxidosqualene cyclases from Artemisia argyi.

Artemisia argyi is a traditional medicinal and edible plant, generating various triterpenoids with pharmacological activities, such as anti-virus, anti-cancer, and anti-oxidant. The 2,3-oxidosqualene cyclase family of A. argyi offers novel insights into the triterpenoid pathway, which might contribute to the medicinal value of its tissue extracts. Nevertheless, the biosynthesis of active triterpenoids in Artemisia argyi is still uncertain. In this study, four putative OSC (2,3-oxidosqualene cyclase) genes (AaOSC1-4) were first isolated and identified from A. argyi. Through the yeast heterologous expression system, three AaOSCs were characterized for the biosynthesis of diverse triterpenoids including cycloartenol, ß-amyrin, (3S,13R)-malabarica-14(27),17,21-trien-3ß-ol, and dammara-20,24-dien-3ß-ol. AaOSC1 was a multifunctional dammara-20,24-dien-3ß-ol synthase, which yielded 8 different triterpenoids, including tricyclic, and tetracyclic products. AaOSC2 and AaOSC3 were cycloartenol, and ß-amyrin synthases, respectively. As a result, these findings provide a deeper understanding of the biosynthesis pathway of triterpenes in A. argyi.

Cloning, Expression Characteristics of Farnesyl Pyrophosphate Synthase Gene from Platycodon grandiflorus and Functional Identification in Triterpenoid Synthesis.

Platycodon grandiflorus is a medicinal plant whose main component is platycodins, which have a variety of pharmacological effects and nutritional values. The farnesyl pyrophosphate synthase (FPS) is a key enzyme in the isoprenoid biosynthesis pathway, which catalyzes the synthesis of farnesyl diphosphate (FPP). In this study, we cloned the FPS gene from P. grandiflorus (PgFPS) with an ORF of 1260 bp, encoding 419 amino acids with a deduced molecular weight and theoretical pI of 46,200.98 Da and 6.52, respectively. The squalene content of overexpressed PgFPS in tobacco leaves and yeast cells extract was 1.88-fold and 1.21-fold higher than that of the control group, respectively, and the total saponin content was also increased by 1.15 times in yeast cells extract, which verified the biological function of PgFPS in terpenoid synthesis. After 48 h of MeJA treatment and 6 h of ethephon treatment, the expression of the PgFPS gene in roots and stems reached its peak, showing a 3.125-fold and 3.236-fold increase compared to the untreated group, respectively. Interestingly, the expression of the PgFPS gene in leaves showed a decreasing trend after exogenous elicitors treatment. The discovery of this enzyme will provide a novel perspective for enhancing the efficient synthesis of platycodins.

[Functional characterization of ent-kaurane-type diterpenoid synthases from Stellera chamaejasme].

Sequential catalysis by ent-copalyl diphosphate(CPS) and ent-kaurene synthase(KS) is a critical step for plants to initiate the biosynthesis of gibberellin with geranylgeranyl pyrophosphate(GGPP) as the substrate. This study mined the transcriptome data of Stellera chamaejasme and cloned two key diterpene synthase genes, SchCPS and SchKS, involved in the gibberellin pathway. The two genes had the complete open reading frames of 2 595 bp and 1 701 bp, encoding two hydrophilic proteins composed of 864 and 566 amino acid residues and with the relative molecular mass of 97.9 kDa and 64.6 kDa and the theoretical isoelectric points of 5.61 and 6.12, respectively. Sequence comparison and phylogenetic tree showed that SchCPS contained LHS, PNV, and DxDD motifs conserved in the CPS family and was categorized in the TPS-c subfamily, while SchKS contained DDxxD, NSE/DTE and PIx motifs conserved in the KS family and was categorized in the TPS-e subfamily. Functional validation showed that SchCPS catalyzed the protonation and cyclization of GGPP to ent-CPP, while SchKS acted on ent-CPP dephosphorylation and re-cyclization to ent-kaurene. In this study, the full-length sequences of SchCPS and SchKS were cloned and functionally verified for the first time, which not only enriched the existing CPS and KS gene libraries but also laid a foundation for the cloning and biosynthesis pathway analysis of more genes involved in the synthesis of active components in S. chamaejasme.

Functional Characterization of PmDXR, a Critical Rate-Limiting Enzyme, for Turpentine Biosynthesis in Masson Pine (Pinus massoniana Lamb.).

As one of the largest and most diverse classes of specialized metabolites in plants, terpenoids (oprenoid compounds, a type of bio-based material) are widely used in the fields of medicine and light chemical products. They are the most important secondary metabolites in coniferous species and play an important role in the defense system of conifers. Terpene synthesis can be promoted by regulating the expressions of terpene synthase genes, and the terpene biosynthesis pathway has basically been clarified in Pinus massoniana, in which there are multiple rate-limiting enzymes and the rate-limiting steps are difficult to determine, so the terpene synthase gene regulation mechanism has become a hot spot in research. Herein, we amplified a PmDXR gene (GenBank accession no. MK969119.1) of the MEP pathway (methyl-erythritol 4-phosphate) from Pinus massoniana. The DXR enzyme activity and chlorophyll a, chlorophyll b and carotenoid contents of overexpressed Arabidopsis showed positive regulation. The PmDXR gene promoter was a tissue-specific promoter and can respond to ABA, MeJA and GA stresses to drive the expression of the GUS reporter gene in N. benthamiana. The DXR enzyme was identified as a key rate-limiting enzyme in the MEP pathway and an effective target for terpene synthesis regulation in coniferous species, which can further lay the theoretical foundation for the molecularly assisted selection of high-yielding lipid germplasm of P. massoniana, as well as provide help in the pathogenesis of pine wood nematode disease.

Identification and functional analysis of the LEAFY gene in longan flower induction.

BACKGROUND: Flowering at the right time is a very important factor affecting the stable annual yield of longan. However, a lack of knowledge of the regulatory mechanism and key genes of longan flowering restricts healthy development of the longan industry. Therefore, identifying relevant genes and analysing their regulatory mechanism are essential for scientific research and longan industry development. RESULTS: DlLFY (Dimocarpus longan LEAFY) contains a 1167 bp open reading frame and encodes 388 amino acids. The amino acid sequence has a typical LFY/FLO family domain. DlLFY was expressed in all tissues tested, except for the leaf, pericarp, and pulp, with the highest expression occurring in flower buds. Expression of DlLFY was significantly upregulated at the early flower induction stage in "SX" ("Shixia"). The results of subcellular localization and transactivation analysis showed that DlLFY is a typical transcription factor acting as a transcriptional activator. Moreover, overexpression of DlLFY in Arabidopsis promoted early flowering and restrained growth, resulting in reduced plant height and rosette leaf number and area in transgenic plants. DNA affinity purification sequencing (DAP-Seq) analysis showed that 13 flower-related genes corresponding to five homologous genes of Arabidopsis may have binding sites and be putative target genes. Among these five flower-related genes, only AtTFL1 (terminal flower 1) was strongly inhibited in transgenic lines. CONCLUSION: Taken together, these results indicate that DlLFY plays a pivotal role in controlling longan flowering, possibly by interacting with TFL1.

[Cloning and functional analysis of 3ß-hydroxysteroid dehydrogenase gene involved in biosynthesis of digitoxin].

Digitoxin, an important secondary metabolite of Digitalis purpurea, is a commonly used cardiotonic in clinical practice. 3ß-Hydroxysteroid dehydrogenase(3ßHSD) is a key enzyme involved in the biosynthesis of digitoxin. It belongs to the short-chain dehydrogenase/reductase(SDR) family, playing a role in the biosynthesis of cardiac glycosides by oxidizing and isomerizing the precursor sterol. In this study, two 3ßHSD genes were cloned from D. purpurea. The results showed that the open reading frame(ORF) of Dp3ßHSD1 was 780 bp, encoding 259 amino acid residues. The ORF of Dp3ßHSD2 was 774 bp and encoded 257 residues. Dp3ßHSD1/2 had the cofactor binding site TGxxxA/GxG and the catalytic site YxxxK. In vitro experiments confirmed that Dp3ßHSD1/2 catalyzed the generation of progesterone from pregnenolone, and Dp3ßHSD1 had stronger catalytic capacity than Dp3ßHSD2. The expression level of Dp3ßHSD1 was much higher than that of Dp3ßHSD2 in leaves, and digitoxin was only accumulated in leaves. The results implied that Dp3ßHSD1 played a role in the dehydrogenation of pregnenolone to produce progesterone in the biosynthesis of digitoxin. This study provides a reference for further exploring the biosynthetic pathway of cardiac glycosides in D. purpurea.

Genome-wide analysis of FRF gene family and functional identification of HvFRF9 under drought stress in barley.

FHY3 and its homologous protein FAR1 are the founding members of FRS family. They exhibited diverse and powerful physiological functions during evolution, and participated in the response to multiple abiotic stresses. FRF genes are considered to be truncated FRS family proteins. They competed with FRS for DNA binding sites to regulate gene expression. However, only few studies are available on FRF genes in plants participating in the regulation of abiotic stress. With wide adaptability and high stress-resistance, barley is an excellent candidate for the identification of stress-resistance-related genes. In this study, 22 HvFRFs were detected in barley using bioinformatic analysis from whole genome. According to evolution and conserved motif analysis, the 22 HvFRFs could be divided into subfamilies I and II. Most promoters of subfamily I members contained abscisic acid and methyl jasmonate response elements; however, a large number promoters of subfamily II contained gibberellin and salicylic acid response elements. HvFRF9, one of the members of subfamily II, exhibited a expression advantage in different tissues, and it was most significantly upregulated under drought stress. In-situ PCR revealed that HvFRF9 is mainly expressed in the root epidermal cells, as well as xylem and phloem of roots and leaves, indicating that HvFRF9 may be related to absorption and transportation of water and nutrients. The results of subcellular localization indicated that HvFRF9 was mainly expressed in the nuclei of tobacco epidermal cells and protoplast of arabidopsis. Further, transgenic arabidopsis plants with HvFRF9 overexpression were generated to verify the role of HvFRF9 in drought resistance. Under drought stress, leaf chlorosis and wilting, MDA and O2 - contents were significantly lower, meanwhile, fresh weight, root length, PRO content, and SOD, CAT and POD activities were significantly higher in HvFRF9-overexpressing arabidopsis plants than in wild-type plants. Therefore, overexpression of HvFRF9 could significantly enhance the drought resistance in arabidopsis. These results suggested that HvFRF9 may play a key role in drought resistance in barley by increasing the absorption and transportation of water and the activity of antioxidant enzymes. This study provided a theoretical basis for drought resistance in barley and provided new genes for drought resistance breeding.

Genome-wide identification of the GRF family in sweet orange (Citrus sinensis) and functional analysis of the CsGRF04 in response to multiple abiotic stresses.

BACKGROUND: Citrus is one of the most valuable fruits worldwide and an economic pillar industry in southern China. Nevertheless, it frequently suffers from undesirable environmental stresses during the growth cycle, which severely restricts the growth, development and yield of citrus. In plants, the growth-regulating factor (GRF) family of transcription factors (TF) is extensively distributed and plays an vital part in plant growth and development, hormone response, as well as stress adaptation. However, the systematic identification and functional analysis of GRF TFs in citrus have not been reported. RESULTS: Here, a genome-wide identification of GRF TFs was performed in Citrus sinensis, 9 members of CsGRFs were systematically identified and discovered to be scattered throughout 5 chromosomes. Subsequently, physical and chemical properties, phylogenetic relationships, structural characteristics, gene duplication events, collinearity and cis-elements of promoter were elaborately analyzed. In particular, the expression patterns of the CsGRF genes in response to multiple phytohormone and abiotic stress treatments were investigated. Predicated on this result, CsGRF04, which exhibited the most differential expression pattern under multiple phytohormone and abiotic stress treatments was screened out. Virus-induced gene silencing (VIGS) technology was utilized to obtain gene silenced plants for CsGRF04 successfully. After the three stress treatments of high salinity, low temperature and drought, the CsGRF04-VIGS lines showed significantly reduced resistance to high salinity and low temperature stresses, but extremely increased resistance to drought stress. CONCLUSIONS: Taken together, our findings systematically analyzed the genomic characterization of GRF family in Citrus sinensis, and excavated a CsGRF04 with potential functions under multiple abiotic stresses. Our study lay a foundation for further study on the function of CsGRFs in abiotic stress and hormone signaling response.

Isolation and functional identification of immune cells in hemolymph of blood clams Tegillarca granosa.

Blood clam Tegillarca granosa is a type of economically cultivated bivalve mollusk with red blood, and it primarily relies on hemocytes in its hemolymph for immune defense. However, there are currently no reports on the isolation and identification of immune cells in T. granosa, which hinders our understanding of their immune defense. In this study, we employed single-cell transcriptome sequencing (scRNA-seq) to visualize the molecular profile of hemocytes in T. granosa. Based on differential expression of immune genes and hemoglobin genes, hemocytes can be molecularly classified into immune cells and erythrocytes. In addition, we separated immune cells using density gradient centrifugation and demonstrated their stronger phagocytic capacity compared to erythrocytes, as well as higher levels of ROS and NO. In summary, our experiments involved the isolation and functional identification of immune cells in hemolymph of T. granosa. This study will provide valuable insights into the innate immune system of red-blood mollusks and further deepen the immunological research of mollusks.

Functional Identification of Olfactory Receptors of Cnaphalocrocis medinalis (Lepidoptera: Crambidae) for Plant Odor.

Cnaphalocrocis medinalis (Lepidoptera: Crambidae) is a migratory insect pest on rice crops. The migratory C. medinalis population in a particular location may be immigrants, local populations, emigrants, or a mix of these. Immigrants are strongly attracted to plant odor. We conducted research to identify the olfactory receptors in a floral scent mixture that is strongly attractive to C. medinalis. Through gene cloning, 12 olfactory receptor (OR) genes were amplified and expressed in Xenopus oocytes in vitro, and three of them were found to be responsive to plant foliar and floral volatiles. These were CmedOR31, a specific receptor for geraniol; CmedOR32, a broad-spectrum OR gene that responded to both foliar and floral odors; and CmedOR1, which strongly responded to 10-4 M phenylacetaldehyde. The electrophysiological response to phenylacetaldehyde was extremely high, with a current of 3200 ± 86 nA and an extremely high sensitivity. We compared the phylogenetic tree and sequence similarity of CmedOR genes and found that CmedOR1 belonged to a uniquely conserved OR pedigree in the evolution of Glossata species, and the ORs of this pedigree strongly responded to phenylacetaldehyde. The expression of OR1 was significantly higher in the females than in the males. Localization of CmedOR1 in the antennae of C. medinalis by fluorescence in situ hybridization showed that CmedOR1 was expressed in both males and females. CmedOR1 may be an odor receptor used by females to locate food sources. The function of these ORs and their role in pest monitoring were discussed.

Systematic analysis of the UDP-glucosyltransferase family: discovery of a member involved in rutin biosynthesis in Solanum melongena.

Eggplant (Solanum melongena) is an economically important crop and rich in various nutrients, among which rutin that has positive effects on human health is found in eggplant. Glycosylation mediated by UDP-glycosyltransferases (UGTs) is a key step in rutin biosynthesis. However, the UGT gene has not been reported in eggplant to date. Herein, 195 putative UGT genes were identified in eggplant by genome-wide analysis, and they were divided into 17 subgroups (Group A-P and Group R) according to the phylogenetic evolutionary tree. The members of Groups A, B, D, E and L were related to flavonol biosynthesis, and rutin was the typical flavonol. The expression profile showed that the transcriptional levels of SmUGT genes in Clusters 7-10 were closely related to those of rutin biosynthetic pathway genes. Notably, SmUGT89B2 was classified into Cluster 7 and Group B; its expression was consistent with rutin accumulation in different tissues and different leaf stages of eggplant. SmUGT89B2 was located in the nucleus and cell membrane. Virus-induced gene silencing (VIGS) and transient overexpression assays showed that SmUGT89B2 can promote rutin accumulation in eggplant. These findings provide new insights into the UGT genes in eggplant, indicating that SmUGT89B2 is likely to encode the final enzyme in rutin biosynthesis.

Comparison of catalytic functions and expression patterns of two pinene synthases from Wurfbainia villosa / 中国中药杂志

Wurfbainia villosa fruit is rich in volatile terpenoids, among which pinene is one of the main components and has anti-inflammatory, antibacterial, anti-tumor, and other pharmacological activities. This research group found that W. villosa fruits were rich in α-pinene by GC-MS, and terpene synthase(WvTPS63, formerly known as AvTPS1) with β-pinene as the main product was cloned and identified, but α-pinene synthase had not been identified. In this study, based on the genome data of W. villosa, we screened and found WvTPS66 with highly similar sequences to WvTPS63, identified enzyme functions of WvTPS66 in vitro, and performed a comparative analysis of sequence, catalytic function, expression pattern, and promoter with WvTPS63. Multiple sequence alignment showed that the amino acid sequences of WvTPS63 and WvTPS66 were highly similar and the conservative motif of terpene synthase was almost identical. In vitro enzymatic experiments on catalytic functions showed that both could produce pinene, and the main product of WvTPS63 was β-pinene, while that of WvTPS66 was α-pinene. Expression pattern analysis showed that WvTS63 was highly expressed in flowers, WvTPS66 was expressed in the whole plant, and the highest expression level was found in the pericarp, which indicated that it might be mainly responsible for the synthesis of α-pinene in fruits. In addition, promoter analysis revealed the presence of multiple regulatory elements related to stress response in the promoter regions of both genes. The findings of this study can provide a reference for the functional study of terpene synthase genes and new genetic elements for pinene biosynthesis.

Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius / 中草药·英文版

OBJECTIVE@#Flavonoids are the bioactive compounds in safflower (Carthamus tinctorius), in which chalcone synthase (CHS) is the first limiting enzyme. However, it is unclear that which chalcone synthase genes (CHSs) are participated in flavonoids biosynthesis in C. tinctorius. In this study, the CHSs in the molecular characterization and enzyme activities were investigated.@*METHODS@#Putative chalcone biosynthase genes were screened by the full-length transcriptome sequences data in C. tinctorius. Chalcone biosynthase genes in C. tinctorius (CtCHSs) were cloned from cDNA of flowers of C. tinctorius. The cloned gene sequences were analyzed by bioinformatics, and their expression patterns were analyzed by real-time PCR (RT-PCR). The protein of CtCHS in the development of flowers was detected by polyclonal antibody Western blot. A recombinant vector of CtCHS was constructed. The CtCHS recombinant protein was induced and purified to detect the enzyme reaction (catalyzing the reaction of p-coumaryl-CoA and malonyl-CoA to produce naringin chalcone). The reaction product was detected by HPLC and LC-MS.@*RESULTS@#Two full-length CtCHS genes were successfully cloned from the flowers of safflower (CtCHS1 and CtCHS3), with gene lengths of 1525 bp and 1358 bp, respectively. RT-PCR analysis showed that both genes were highly expressed in the flowers, but the expression of CtCHS1 was higher than that of CtCHS3 at each developmental stage of the flowers. WB analysis showed that only CtCHS1 protein could be detected at each developmental stage of the flowers. HPLC and LC-MS analyses showed that CtCHS1 could catalyze the conversion of p-coumaryl-CoA and malonyl-CoA substrates to naringin chalcone.@*CONCLUSION@#CtCHS1 is involved in the biosynthesis of naringin chalcone in safflower.

Functional characterization of SsNES responsible for nerolidol biosynthesis in Senecio scandens / 中国中药杂志

A short terpene synthase gene was obtained by screening the transcriptome data of Senecio scandens. The phylogenetic tree and sequence alignment putatively identified this gene as a nerolidol synthase gene, named SsNES(GenBank MH518312). Protein homology modeling indicated that SsNES contained a complete conserved domain and folded correctly. SsNES was cloned and successfully expressed in Escherichia coli as soluble protein. The biochemical function of SsNES was characterized by E. coli metabolic engineering, which showed that SsNES catalyzed formation of trans-nerolidol with(E, E)-farnesyl diphosphate as the substrate. Nerolidol was also detected in stems and leaves of S. scandens, indicating that SsNES might act as the nerolidol synthase in plant. RT-PCR analysis indicated that SsNES was mainly expressed in stem, flowers and leaves, and no expression was observed in roots. After the treatment of SA, MeJA or Ala, SsNES was induced significantly at 6 h, indicating involvement in the defense response of S. scandens. The identification of SsNES not only clarified biosynthesis of nerolidol in S. scandens, but also provided diversity of sesquiterpene synthase, as well as theoretical basis for disease and pest defense mediated by the terpene metabolites.

Cloning and functional identification of a new NADPH-cytochrome P450 reductase in Andrographis paniculata / 中国中药杂志

Andrographolide is a main active ingredient in traditional Chinese medicine Andrographis paniculata，with a variety of pharmacological activity，widely used in clinical practice. However its biosynthetic pathway has not been resolved. Cytochrome P450 reductase provides electrons for CYP450 and plays an important role in the CYP450 catalytic process. In this study，the coding sequence of A. paniculata CPR was screened and cloned by homologous alignment，named ApCPR4. The ApCPR4 protein was obtained by prokaryotic expression. After isolation and purification，the enzyme activity was identified . The results showed that ApCPR4 could reduce the cytochrome c and ferricyanide in NADPH-dependent manner. In order to verify its function，ApCPR4 and CYP76AH1 were co-transformed into yeast engineering bacteria. The results showed that ApCPR4 could help CYP76AH1 catalyze the formation of rustols in yeast. Real-time quantitative PCR results showed that the expression of ApCPR4 increased gradually in leaves treated with methyl jasmonate (MeJA). The expression pattern was consistent with the trend of induction and accumulation of andrographolide by MeJA，suggesting that ApCPR4 was associated with biosynthesis of andrographolide.

Prokaryotic expression, functional identification of squalene synthase in Alisma orientale and its immunoassay study / 中国中药杂志

Squalene synthase of Alisma orientale catalyzes farnesyl diphosphate (FPP) to form squalene, which is the key regulatory enzyme of the carbon source flow to protostane triterpenes biosynthesis. For further research on the function and expression of AoSS gene, the open reading frame (ORF) of squalene synthase gene (accession no. JX866770) from A. orientale was subcloned into a prokaryotic expression vector pCzn1 and induced the expression of AoSS gene in Escherichia coli BL21(Roseta). The fusion protein was mainly in the form of inclusion bodies and purified to obtain high purity protein. By verifying its functionality through vitro enzymatic reaction, the results showed that the catalytic protein had the catalytic activity of FPP into squalene. In order to research the expression of AoSS in A. orientale, the purified protein was used to immunized rabbits to prepare polyclonal antibody which was then purified, the titer of the antibody was greater than 1∶51 200 by ELISA detection, and displayed good specificity by Western blotting. The prepared antibody was used for immunoassay of AoSS in different organs of A. orientale, and the results showed that the AoSS expression level was the highest in tubers, followed by leaves, and lowest in root. Successful construction of prokaryotic expression vector, validation of gene functions and establishment of rapid immunoassay lay the foundation for further researches on the function and regulation of AoSS gene, and also provide scientific basis on the application of the protostane triterpenes of A. orientale in the field of synthetic biology.

Culture and functional identification of macrophages from human peripheral blood / 中国免疫学杂志

Objective:To isolate monocytes from human peripheral blood mononuclear cells( PBMC) ,induce macrophages,and identify the function of macrophages.Methods:Monocytes were isolated from PBMC using magnetic activated cell sorting( MACS) anti-CD14 microbead.Sorted CD14+and CD14-cells were checked by flow cytometer to evaluate the efficiency of sorting.The sorted CD14+cells were cultured in IMDM media with 10%human AB serum and 10 ng/ml M-CSF for 7 days to generate macrophages,which were identified by morphological features and phagocytosis function.Results:A high purity of monocytes was obtained by MACS anti-CD14 microbead.The percentage of CD14+cells was 10% and 85.8% before and after sorting, respectively.The macrophages were approximately 40-45 μm in maximum diameter and had the fried egg colony morphological features after 7 days culture.The lymphoma ( Raji) cells were efficiently engulfed by macrophages.Conclusion: The high purity of CD14+monocytes is isolated from PBMC and monocyte-derived macrophages efficiently engulfed lymphoma cells.

Isolated culture and functional identification of mouse bone marrow derived tolerogenic dendritic cells / 中国免疫学杂志

Objective:To establish the methods of isolated culture and functional identification of mice bone marrow derived tolerogenic dendritic cells (CD11b+F4/80 +TDCs) in vitro.Methods: Mice bone marrow cells were isolated and cultured to obtain iDCs with the simulation of mouse rmGM-CSF and rmIL-4.CD11b+F4/80 +TDCs were purified by fluorescence-activated cell sorting on day 6.The morphological changes of TDCs were observed with the inverted microscope dynamically .The expression of CD11b+F4/80 +TDCs were analyzed by the flow cytometry .Tolerogenic function of CD11b+F4/80 +TDCs was evaluated by the expression of MHCⅡ, CD83, IDO, TLR-2, IL-10 and TGF-β1.The expression of MHCⅡ was analyzed by the flow cytometry , and the expression of CD83, IDO and TLR-2 were analyzed by immune-histochemistry.The levels of IL-10 and TGF-β1 in the supernatant of CD11b+F4/80 +TDC were analyzed by ELISA .Meanwhile mature DCs ( mDCs) induced by LPS were used as control .Results:The fresh isolated bone marrow cells look like round and small under microscope .After two days of culture , cells became big and formed into clusters . Five or six days later, cells clusters increased, and the morphology of cells became irregular .At the same time, more dendrite ap-peared on the surface of cells .The percentage of CD11b+F4/80 +TDCs induced by rmGM-CSF and rmIL-4 was about 23%, and the purity of the purified BM CD11b+F4/80 +iDC was about 99%.Compared with mDCs, CD11b+F4/80 +TDCs expressed low levels of MHCⅡand CD83 and high levels of IDO, TLR-2, IL-10 and TGF-β1.Conclusion:CD11b+F4/80 +TDCs derived from mouse bone marrow could be induced successfully by rmGM-CSF and rmIL-4 in vitro.CD11b+F4/80 +TDCs showed tolerogenic function by the expressions of IL-10, TGF-β1, IDO and TLR-2.