Isolation and characterization of promoter of ADS from Artemisia annua / 中国中药杂志

<p><b>OBJECTIVE</b>To try to find the ways to enhance the expression of ADS gene encoding amorpha-4,11-diene synthase, a key enzyme in artemisinin biosynthesis pathway catalyzing the formation of amorpha-4,11-diene from farnesyl diphosphate, and accelerate the artemisinin synthesis, the promoter of ADS was isolated and characterized.</p><p><b>METHOD</b>5' untranslated regions of ADS were isolated from Artemisia annua with PCR. For functional characterization, the isolated fragment was fused with GUS reporter gene and introduced into Nicotiana tabacum by Agrobacterium-mediated transformation. The GUS expression regulated by 5' untranslated regions of ADS in transgenic N. tabacum under the normal or stressed conditions were detected by histochemical staining and quantitative spectrophotometry assay.</p><p><b>RESULT</b>The 2 448 bp DNA fragment upstream of ADS coding sequence was isolated from A. annua and introduced into N. tabacum. Histochemical staining showed that the isolated fragment conferred stable GUS expression in transgenic plants. The quantitative results showed that the GUS activity in transgenic tobacco plants treated by low-temperature (4 degrees C) and ultraviolet irradiation were 1. 6 and 2.2 folds higher than that in the controls.</p><p><b>CONCLUSION</b>It was suggested that the isolated fragment had promoter activity and maybe responsive to adverse environmental stresses.</p>

Cloning and Sequencing of Artemisia annua Squalene Synthase Gene and Its cDNA / 广州中医药大学学报

【Objective】 To clone the squalene synthase gene of Artemisia annua L. for improving the quality and production of Artemisia annua L. by genetic engineering. 【Methods】 PCR amplification, RT-PCR amplification, ligation of the target fragment with a T-vector and sequence analysis of the interested gene were performed. 【Results】 An expected 3590 bp fragment was amplified by PCR and an expected 1257 bp fragment was amplified by RT-PCR. The two cloned fragments were identified by PCR and restriction enzyme digestion respectively. The preliminary sequence data indicated that the results obtained were similar to that from GenBank, and the difference was only found in several base pairs. 【Conclusion】 The squalene synthase gene and cDNA of Artemisia annua L. were successfully cloned and sequenced.

Studies on Artemisinin Sensitizing Compound in Inhibiting Tumor Cell Proliferation and Suppressing Transplanted Tumor Growth in Tumor-bearing Nude Mice / 广州中医药大学学报

Objective To enhance the anti-tumor efficiency of artemisinin and to explore the anti-tumor and sensitivity-enhancing mechanism of Artemisinin Sensitizing Compound(ASC).Methods The growth-inhibition rate of HepG2 cells was determined by methyl thiazolyl tetrazolium(MTT) method.Tumor-bearing nude mice were given inoculation of HepG2 cells subcutaneously.After treatment for 15 days,the tumor-inhibition rate was calculated according to the tumor mass weight.Results The growth-inhibition rate of tumor cells in ASC group(artesunate combined with diethyl maleate,mercaptosuccinic acid and aminotriazole),was increased 43.84%,and the tumor-inhibition rate was three times as much as that in artesunate alone group with the same titer.The amount of antioxidant agents and the activity of antioxidant enzymes were higher in the control group than those in the artesunate alone group and ASC group,while were lower in ASC group than those in the artesunate alone group.Conclusion ASC has anti-tumor and sensitivity-enhancing effect through decreasing the amount of anti-oxidant agents and inhibiting activities of anti-oxidant enzymes.

Tissue Culture and Plant Regeneration of Aristolochia contorta Bge / 广州中医药大学学报

[Objective] To explore the culture conditions of inducing callus from tissue of Aristolochia contorta Bge (ACB). [Methods] The leaves of ACB were used as the explants. Basic medium (including 1/2 MS medium, MS medium and modified MS medium) containing corresponding phyto-hormones was applied for the induction of ACB callus. The influerices of different culture conditions such as three kinds of basic medium, different pH values and addition of different kinds of phyto-hormones at different concentrations into the basic medium respectively or simultaneously, on the callus induction of ACB were observed. [Results] (1) After the three kinds of basic medium were added with the phytohormones of 0.1mg/L kinetin (KT) and 0.2mg/L ?-naphthalene acetic acid (NAA) , a large amount of ACB calluses were induced in the MS medium and modified MS medium, while ACB calluses did not occur in the 1/2 MS medium. (2) When the pH value of the culture medium composed of modified MS medium and 0.1mg/L KT and 0.2mg/L NAA was at 5.8, 7.0 and 8.0, a large amount of ACB calluses were induced in the medium with pH value being 7.0 and 8.0 while a few calluses occurred in the medium with pH value being 5.8. (3) ACB calluses were induced in modified MS medium with NAA added , but calluses did not occur in modified MS medium with KT added. When the modified MS medium was added with different kinds of phyto-hormones at different concentrations, ACB calluses were loose in the medium with high-concentration NAA and this did not benefit to the differentiation of buds for too fast cell division, and the callus induction rate was low in the medium with low-concentration NAA. The optimized culture medium was modified MS medium with 1mg/L KT and 0.5mg/L NAA added. [Conclusion] The optimum culture conditions of inducing callus from ACB tissues are: MS medium or modified MS medium with 1mg/L KT and 0.5 mg/L NAA added being the culture medium, and pH value being 7.0-8.0.

Cloning of Squalene Synthase Gene from Saccharomyces cerevisiae and Construction of Yeast Expression Vector / 广州中医药大学学报

Objective To lay the foundation for studying the synthesis of artemisinin in microorganism,squalene synthase(SS) gene,a key enzyme gene from Saccharomyces cerevisiae,was cloned and a yeast expression vector was constructed.Methods After amplification of SS gene by polymerase chain reaction(PCR),ligation to T-vector and analysis of the cloned sequence,enzyme digestion and reconfirmation of the target gene,the antisense yeast expression vector was constructed by inverted insertion of the target gene into a yeast expression vector,pGAPZ?A,and digested with two restriction enzymes for vertifying the recombinant.Results The length of SS gene was 1335bp.The preliminary sequence data indicated that SS gene obtained from the experiment had a high sequence homology with that from GenBank,except for a few base pairs.The antisense yeast expression vector has been constructed and vertified by digesting with two enzymes.Conclusion SS gene from Saccharomyces cerevisiae has been successfully cloned and sequenced.An antisense yeast expression vector has been also constructed.

Evaluation on expression of recombinant S gene of human hepatitis B virus in vitro and in vivo / 中华传染病杂志

Objective To construct a DNA vaccine capable of expressing S gene of hepatitis B virus(HBV) and evaluate the expression of recombinant S gene in vitro and in vivo. Methods A cloned S X gene fragment was inserted into a eukaryote expression vector to construct a recombinant plasmid. The S gene was transcribed in vitro and expressed in a transfected cell line, and the efficiency of HBsAg in eliciting anti HBs was evaluated in mice. Results The expression of S gene was confirmed by Northern blotting, Western blotting, and ELISA(for both antigen and antibody detection). Conclusions The recombination and expression of S gene is achieved successfully in vitro.

Cloning, Sequencing and Homologous Analysis of Tyrosine Decarboxylase Gene from Aristolochia contorta Bge / 广州中医药大学学报

[Objective] To obtain the tyrosine decarboxylase gene (tyrDC) from Aristolochia contorta Bge.and to assay its cDNA sequence and homologous analysis, thus to remove its nephrotoxieity. [Methods] The primers designed by referring to the conservative amino acid sequences of known plant tyrDC were used to amplify a fragment of cDNA from Aristolochia contorta Bge.by reverse transcription-polymerase chain reaction (RT-PCR). The amplified cDNA sequence was cloned and sequenced to design a pair of specific primers and to amplify a full-length tyrDC cDNA from Aristolochia contorta Bge.by rapid amplification of cDNA ends (RACE). [Results] The length of cloned tyrDC cDNA is 1678 base pairs (bp), which comprises an open reading frame ( ORF) of 1551 bp encoding 516 amino acids and a downstream untranslated region (3'UTR) of 127 bp. The results of sequence comparison indicated that the amino acid sequence deduced from the nucleotide sequence of tyrDC from Aristolochia contorta Bge.shares 76% homology with issued tyrosine decarboxylase of Papaver somniferum L. and 79% homology with tyrosine /DOPA decarboxylase from Thalictrum flavum subsp. glaucum. [Conclusion] The full-length tyrDC cDNA has been amplified from Aristolochia contorta Bge. and the homologous retrieve of tyrosine decarboxylase reveals an extensive sequence similarity among tyrosine decarboxylases of different plants. This will provide evidence for the romoval of nephrotoxicity of Aristolochia contorta Bge. .

Transient expression of recombinant human cytokine genes in transgenic Chinese materia medica cells / 中草药

Object To explore the feasibility of breeding genetic modified (GM) medicine by expressing human cytokine in transgenic Chinese materia medica Methods Human interferon ? gene and RANTES gene available from the amplification in vitro were enzymatically excised, recoveried, and inserted into intermediate vectors The recombinants were identified by double enzyme digestion of EcoRⅠand HindⅢ The plasmids were extracted from Escherichia coli and introduced into A tumefaciens, and the transformants harboring binary vectors were screened by addition of antibiotics of kanamycin (Km) and rifampicin (Rif), and the explants of M charantia and P vulgaris were transformed by co cultivation of leaf disks with A tumefaciens strain Results RT PCR was applied to detect the transient expression of human interferon ? gene and RANTES gene in transformed medicinal herbal calli Conclusion The expression of recombinant human interferon ? gene and RANTES gene in transgenic M charantia and P vulgaris cells was firstly reported, which opens an alternative road to antivirus, especially anti AIDS virus, by using transgenic Chinese materia medica

Gene targeting of squalene synthase in Artemisia annua / 中草药

Objective To increase artemisinin yield in transgenic Artemisia annua plants by regulating metabolic affluxion through metabolic pathway engineering. Methods The gene targeting vector was constructed by squalene synthase (SS) gene of A. annua, green fluorescent protein (GFP) gene, and cytosine deaminase (CodA) gene, and the vector was introduced into Agrobacterium tumefaciens by freeze-thawing procedure. A. annua was transformed through Leaf Disk method and regenerated transgenic plants were screened by the “Step-by-Step Selection”. Results Among the transgenic A. annua plants emitting green fluorescence after expression of GFP gene, the exogenous GFP gene rather than endogenous SS gene was detected in one transgenic plant by PCR as well as hybridization of PCR products. The preliminary data showed that the wild-type SS gene was replaced by mutated SS gene in the transgenic A. annua plant. Conclusion Gene targeting of squalene synthases of A. annua is successful.

Expression of CodA gene from Escherichia coli confering a negative selection phenotype on transgenic Artemisia annua / 中草药

Objective To explore the feasibility of utilizing the cytosine deaminase A (CodA) gene as an effective negative selectable marker in Artemisia annua for gene targeting.Methods The PCR procedure was employed to amplify CodA gene from Escherichia coli.After being cloned and sequenced, the gene was inserted into a plant expression vector, pROKⅡ, and then introduced into Agrobacterium tumefaciens LBA4404 (pAL4404).The leaf disks of A.annua were transformed by the co-cultivation protocol, after which the transformed calli were selected and green shoots of A. annua were regenerated on N6 medium supplemented with 25 ?g/mL Kanamycin (Kan).Then the Kan-resistant transgenic shoots were transplanted onto the MS medium containing 500 ?g/mL 5-fluocytosine (5-FC) plus 25 ?g/mL Kan and continuously cultured for up to two weeks.Results The transgenic shoots have totally died while untransformed shoots still kept normal growth, indicating that A.annua cells introduced into the CodA gene had conferred an expected negative selection phenotype.When detected by RT-PCR, the transgenic shoots displayed a CodA-positive amplified band, but untransformed shoots gave no such CodA-specific amplified pattern.This result suggested that CodA gene had transcribed into corresponding mRNA in A.annua cells with furtherly verifying the result of phenotypic assay.Conclusion The CodA gene can be utilized as an effective negative selectable marker in A.annua for gene targeting.