Clone of Full-length cDNA Sequence of Actin Gene of Eleutherococcus Senticosus and Bioinformatics Analysis / 世界科学技术-中医药现代化

This study was aimed to clone the full-length cDNA sequence of actin gene of Eleutherococcus sentico-sus. And bioinformatics analysis was used. The total RNA was isolated from leaves of E. senticosus , and cDNA was synthesized by reverse transcription of total RNA. Primers were designed according to the conserved se-quence that had been cloned of Actin of E. senticosus . Then, the 3'and 5' cDNA fragments were cloned by nested PCR . The full-length gene was obtained by gene splicing method . Sequencing results were compared and treated with similarity analysis by blast analysis in the GenBank. Protein secondary structure and tertiary struc-ture of Actin of E. senticosus was predicted by online software. The results showed that the full-length cDNA of Actin of E. senticosus is 1507 bp, which named EsActin1, GenBank accession No. KC469585. The conserved sequence, which contained a 1134 bp open reading frame that encoding a 377 amino acid residues, a 5'-UTR of 140 bp and a 3'-UTR of 233 bp. Homologous alignment showed that it shared over 75% nucleotide se-quence similarity and over 94% amino acid sequences similarity with Actins in other plants. It was concluded that this study first isolated and reported the full-length cDNA sequence of actin gene of E. senticosus , and laid a foundation for the molecular biology research of E. senticosus .

Molecular cloning of farnesyl diphosphate synthase from Eleutherococcus senticosus and its bioinformatics and expression analysis / 中国中药杂志

<p><b>OBJECTIVE</b>To clone farnesyl diphosphate synthase (FPS) gene from Eleutherococcus senticosus and analyze the bioinformatics and expression of the gene.</p><p><b>METHOD</b>The FPS full length cDNA was cloned by rapid amplification of cDNA ends (RACE). The data was analyzed by bioinformatics method, the structure and function of FPS was deduced. The expression of FPS in different organ of E. senticosus was detected by RT-PCR.</p><p><b>RESULT</b>The full length of FPS cDNA was 1 499 bp containing a 1 029 bp ORF that encoded 342 amino acids. The deduced protein sequence exhibited two Asp riches conserved motifs (DDXXD). Without transmembrane domain, FPS was located in cytoplasm. RT-PCR result showed that FPS gene expressed in different organs of E. senticosus. The expression amounts of FPS in different organs were different significantly (P < 0.05).</p><p><b>CONCLUSION</b>The FPS gene of E. senticosus was successfully cloned for the first time, and provided a stable foundation for studying on its effect and expression control on E. senticosus saponins biosynthesis.</p>

Effect of endophytic fungi on expression amount of key enzyme genes in saponins biosynthesis and Eleutherococcus senticosus saponins content / 中国中药杂志

<p><b>OBJECTIVE</b>To analyze the effect of endophytic fungi on expression amount of key enzyme genes SS (squalene synthase gene), SE (squalene epoxidase gene) and bAS (beta-amyrin synthase gene) in saponin biosynthesis and saponins content in Eleutherococcus senticosus.</p><p><b>METHOD</b>Wound method was used for back meeting the endophytic fungi to E. senticosus. With GAPDH as internal control gene, the expression of key enzyme genes was detected by real time PCR method. E. senticosus saponins content was measured by spectrophotometry method.</p><p><b>RESULT</b>When wound method back meeting P116-1a and P116-1b after 30 d, the expression content of SS improved significantly (P < 0.05), however the back meeting of P109-4 and P312-1 didnt change the expression of SS. After that SS expression showed reduction-equality-reduction varying trend. Thirty days after back meeting P312-1, the expression content of SE improved significantly (P < 0.05). Ninty days after back meeting P116-1b and P312-1, the expression content of SE improved significantly to 130%,161%, respectively (P < 0.05). After 120 d, back meeting four endophytic fungi, the expression of SE were significantly higher than the control (P < 0.05). Back meeting four endophytic fungi form 60 d to 120 d, the expression of bAS was significantly higher than the control (P < 0.05). The back meeting four endophytic fungi improved E. senticosus saponins content significantly (P < 0.05).</p><p><b>CONCLUSION</b>Endophytic fungi P116-1a, P116-1b, P1094 and P312-1 significantly effected the expression of key enzyme genes SS, SE and bAS and then affected E. senticosus saponins content. Among the genes, bAS was key target gene.</p>

Cloning and sequence analysis on cDNA of squalene epoxidase gene in Eleutherococcus senticosus / 中国中药杂志

<p><b>OBJECTIVE</b>To clone and sequence the cDNA of squalene epoxidase gene in Eleutherococcus senticosus.</p><p><b>METHOD</b>Total RNA of E. senticosus was extracted by the improved isothiocyanate method and reverse transcripted into cDNA. The primers were designed depending on the reported SE cDNA sequences of Panax ginseng. The SE cDNAs in E. senticosus was amplified using RT-PCR strategy.</p><p><b>RESULT</b>Sequencing results showed two different cDNA fragments (SE1, SE2) with 1665, 1629 bp each ORF which encoded 554,542 amino acids, respectively. The identities of nucleotides and amino acids between SE1, SE2 were 91.49%, 92.55%. SE1, SE2 had the highest amino acids similarity to the SE1 of P. notoginseng, 93.45%, 94.87% respectively. SE1, SE2 both had a FAD binding domain. The deduced speculated amino acids of SE1, SE2 each had 2,4 membrane-spanning helices.</p><p><b>CONCLUSION</b>The two SE sequences in E. senticosus were firstly separated and reported, which has made foundation for E. senticosus secondary metabolite engineering researches.</p>

Cloning of Eleutherococcus senticosus calmodulin gene and effect of endophytic fungus on expression amount of gene / 中国中药杂志

<p><b>OBJECTIVE</b>To clone calmodulin (CaM) gene in Eleutherococcus senticosus, and study the effect of endophytic fungi on expression amount of CaM gene.</p><p><b>METHOD</b>The CaM full length cDNA sequence was cloned by rapid amplification of cDNA ends (RACE). The gene was analyzed and corresponding structure and functions were predicted by the bioinformatics methods. The expression amount of CaM gene affected of endophytic fungus P116-1a, P116-1b, P1094 and P312-1 was detected by RT-PCR.</p><p><b>RESULT</b>The full length of CaM cDNA was 856 bp containing an ORF of 450 bp that encoded a protein of 149 amino acids. The homologous of predicted protein was almost 100% with plants like Panax ginseng and Daucus carota. RT-PCR results showed that endophytic fungus improved CaM expression amount significantly (P<0.05). The highest expression amount of CaM occurred 90 d after reinoculated with endophytic fungi P1094, up to 2.96 times of the control.</p><p><b>CONCLUSION</b>The CaM gene of E. senticosus was successfully cloned for the first time. The results demonstrated that endophytic fungus of E. senticosus improved CaM expression amount significantly.</p>

Expression of MMP-3 and extracellular matrix metalloproteinase inducer in benign and malignant breast lesions / 第二军医大学学报

Objective:To explore the expression of MMP-3 and extracellular matrix metalloproteinase inducer(EMMPRIN)in benign and malignant breast lesions and its relevance.Methods:Using immunohistochemical method,we examined the expression of MMP-3 and EMMPRIN proteins in 58 breast cancer specimens,28 premalignant lesions,40 hyperplasia specimens,40 benign tumor specimens and 20 normal breast tissues.The expression of MMP-3 mRNA and EMMPRIN mRNA was examined using in situ hybridization in 20 breast cancer specimens,20 premalignant lesions,20 hyperplasia specimens,20 benign tumor specimens and 20 normal breast tissues.The values of integral optic density(IOD)of the expression was calculated by image analysis system.Results:The expression of MMP-3 and EMMPRIN proteins and mRNA was stronger in breast cancer and premalignant lesions than in the other lesions.The IODs of MMP-3 and EMMPRIN proteins and mRNA in breast cancer and premalignant lesion were significantly higher than those of hyperplasia,benign tumors and normal breast tissues(P

Somatic embryogenesis from petiole in Eleutherococcus senticosus / 中草药

Objective The petiole of Eleutherococcus senticosus was used as somatic embryo,which is widely original plants with the pharmacological active components whose contents could be determined,the somatic embryo in E.senticosus was studied,the aim of this study is to provide the proof of E.senticosus species which have the higher yield of pharmacological active components.Methods Using the petiole of E.senticosus of three years old plants germinated somatic embryos within 15 d to observe the somatic embryogenesis of E.senticosus with the 2,4-D+BA medium.Results After cultured for 28 d with 2,4-D 1.5 mg/L+BA 1.0 mg/L,71.4% of the petiole somatic embryos were directly produced or 8.5 embryos in total were produced via callus.Both of the two methods could be used in the elicitation medium,but the percentage of indirect production was smaller.After transforming into the same or the lower concentration of 2,4-D medium,the somatic embryos gradually matured.At the same time,those of the new somatic embryos were also produced,the percentage of the somatic embryos which were produced by indirect way was increased with it.Conclusion Using the petiole of E.senticosus germination within 15 d could make somatic embryogenesis.It confirms that the somatic embryogenesis and the bodybmeryos inductivity depend on the 2,4-D and BA concentration.

Somatic cell embryogenesis of Acanthopanax senticosus / 中草药

Objective To study somatic cell embryogenesis of Acanthopanax senticosus induced by different concentration of 2,4D and type of explants,which provides theoretic evidence in protection of A.senticosus resources and genetic engineering.Methods Using 3-week seedlings and zygotic embryos(cotyledon, hypocotyls,and roots) of stratificated seeds as explants researches the effect of different hormones on somatic cell embryogenesis of A.senticosus. Results Explants of zygotic embryos of stratificated seeds cultured on MS and 1/2MS media containing 0.5 mg/L 2,4-D generated the highest frequency(57.1%) and the most number(3.3) of somatic cell embryos,which can develop into maturation in the initial medium.But it is more beneficial to generate new somatic cell embryos and to develop primary somatic cell embryos into maturation when transferred into 2,4-D of decreased concentration.And the deve-(lopment) process of somatic cell embryos of A.senticosus is similar to that of zygotic embryos.Conclusion(Somatic) embryogenesis of A.senticosus is realized by culturing explants of zygotic embryos and the inductive rate of somatic cell embryos is related to the concentration of 2,4-D and developmental stage of explants.