Establishment and analysis of in vitro culture system for transgenic Salvia miltiorrhiza hairy roots / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a culture system for transgenic Salvia miltiorrhiza hairy roots.</p><p><b>METHOD</b>Investigated the success rate of different explants, different infection time and different co-culture time to induce hairy roots of S. miltiorrhiza. Co-cultured explants were sterilizated with 400 g x L(-1) Cef water for 5 min, inoculated on MS solid medium supplied with 400 mg x mL(-1) cef and 2.5 g x L(-1) Hyg, and then transfered to the 67-V liquid medium with 2.5 g x L(-1) Hyg after complete sterilization. GFP fluorescence detection was performed to detect positive hairy root lines. PCR method to detect rolC gene which is the specific gene of hairy root. Biomass was determinated in different growth periods of root lines. HPLC was conducted to measure the content of dihydrotanshinone I of transgenic hairy roots.</p><p><b>RESULT</b>Leaf base of S. miltiorrhiza was used as a perfect explant to Induce hairy roots, the success rate can reach 93.3%. Inducing efficiency was up to 63.3% after Agrobacterium infection for 10 min. Co-culture for 2-3 d can reach the best induced effect. It is a high credibiliy to use PCR method combined with detection of GFP fluorescence to identified positive transformants. There is a close contact between biomass increases and secondary metabolite accumulation of transgenic hairy roots.</p><p><b>CONCLUSION</b>Successfully in vitro culture system has been established in transgenic S. miltiorrhiza, and this research can lay foundations for the further genetic engineering applications.</p>

Research of mechanism of secondary metabolites of phenolic acids in Salvia miltiorrhiza hairy root induced by jasmonate / 中国中药杂志

<p><b>OBJECTIVE</b>To study the mechanism of secondary metabolites of some phenolic acids in the hairy roots of Salvia miltiorrhiza induced by methyl jasmonate.</p><p><b>METHOD</b>The hairy roots of S. miltiorrhiza were induced with methyl jasmonate (100 micromol x L(-1)) and collected at 0, 12, 24, 36 h after treatment. Real-time quantitative PCR was used for detecting the mRNA expression level of the key enzyme genes on the secondary metabolites pathway of rosmarinic acid, while a LC-MS method was developed to determine the content of rosmarinic acid, caffeic acid and salvianolic acid B.</p><p><b>RESULT AND CONCLUSION</b>The concentration of phenolic acids grew up and accumulated quickly in the hairy roots with exogenous signal molecule MJ induced, and it was showed that the content of CA and RA reached the maximum after 24 h and the content of LAB reached the maximum in 36 h by MJ induced. The induction mechanism may be activated with different levels of RA synthesis in PAL, 4CL, C4H genes on the key enzyme phenylalanine pathway and TAT, HPPR genes on tyrosine pathway. The time of gene expression was different, among them, 4CL and PAL genes were more important. In a word, the result can provide some basis data about the mechanism of secondary metabolites of phenolic acids for further research.</p>

New analysis of EST-SSR distribution and development of EST-SSR markers in Salvia miltiorrhiza / 中国中药杂志

<p><b>OBJECTIVE</b>To establish the new EST-SSR markers for analyzing the genetic variation of different population of Salvia miltiorrhiza.</p><p><b>METHOD</b>It was dealt with ESTs newly downloaded from Genbank and that of acquired from HMPL lab EGassembler software, and then carried out SSR loci search and SSR type analysis by SSRIT software. After that, it was designed the EST-SSR primer pairs for PCR amplification condition optimization.</p><p><b>RESULT</b>Abundant and high coverage of SSR loci distribution were found in S. miltiorrhiza with having one SSR per 5.8 kb ESTs. Among them, the occurrences of different repeat units were mainly the di- (63.0%) and tri- (35.5%). The CT/AG was the most frequent motif in dinucleotide motif type and the GAA/TCC was the most frequent motif in trinucleotide repeats. Out off 36 primer pairs, 29 primer pairs (80.5%) were successfully amplified in all samples of S. miltiorrhiza while the rest failed to give PCR products at various annealing temperature and Mg2+ concentrations. The selected primer pairs also showed the polymorphism in samples from different S. miltiorrhiza populations.</p><p><b>CONCLUSION</b>The newly establishment of EST-SSR markers showed high SSR loci coverage and genetic polymorphisms in S. miltiorrhiza population. It could be used for genetic variation analysis.</p>

Investigate of DNA extraction of os cervi / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a convenient, practical and high efficient method of DNA extraction of os cervi, and lay the foundation of identification of animal bones.</p><p><b>METHOD</b>The bones of sika deer, red deer, cattle, dog and pig were used to extract DNA under different decalcification time (24,48,72 h) and decalcification temperature (4,25,37,56,70 degrees C), and extract method.</p><p><b>RESULT</b>It proved by experiments that demineralization process promotes the cracking of osteocyte. In a broad of decalcification time and temperature, DNA could be extracted from all bone samples successfully while the quantity varied slightly.</p><p><b>CONCLUSION</b>Samples (about 0.1 g) decalcify with 0. mol x L(-1) EDTA at 4 degrees C for 24 h, then water-bath for 1 h after lysis buffer added, DNA extracted via the method above is of high quality and can be used for PCR.</p>

The establishment of PCR system to identify Bungarus multicinctus rapidly / 药学学报

The purpose of the present study is to establish a rapid and effective PCR method for the identification of B. multicinctus. Based on sequence alignment of B. multicinctus and its adulterants, we found that Cyt b gene is a good molecular genetic marker for the authentication of B. multicinctus. On the basis of the sequence data, a pair of highly specialized primers was designed. The templates were extracted by the DNA purification system. Key factors such as annealing temperature, concentration of Taq enzyme and cycle numbers were analyzed and optimized. The modified PCR program consisted of an initial denaturation step at 95 degrees C for 5 min, followed by 30 cycles of 95 degrees C for 30 s and 55 degrees C for 45 s and a final extension at 72 degrees C for 5 min. Thirteen samples of B. multicinctus were identified accurately from their 20 adulterants in 4 hours. The results indicated it is a highly accurate, rapid and applicable method for the authentication of B. multicinctus.

Molecular cloning and SNP analysis of a acetyl-CoA C-acetyltransferase gene (SmAACT) from Salvia miltiorrhiza / 药学学报

Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the terpene synthesis pathway, catalyzed two units of acetyl-CoA to acetoacetyl-CoA. In order to study the tanshinone biosynthesis in Salvia miltiorrhiza, a novel AACT gene, SmAACT, was cloned using cDNA microarray and RACE strategy. The full length cDNA of SmAACT is 1 623 bp (accession No. EF635969), which contained a 1 200 bp open reading frame (ORF) encoding a 399 amino acid protein. Nine introns were found in the genomic sequence. SmAACT was upregulated by YE and Ag+ elicitors both with cDNA microarray and quantitative RT-PCR analyses along with the accumulation of tanshinones. Sequence homology comparison and phylogenetic analysis all suggested that SmAACT belonged to the class of acetyl-CoA C-acetyltransferase. The transcription level of SmAACT was relatively higher in root than that in stem and leaf tissues. SNP analysis revealed that SmAACT was highly variable in the region of 6 to 9 introns with 33 SNPs in the 600 bp region, there are 5 SNPs in the cDNA region while they are all synonymous cSNPs. Some special genotypes were found in Salvia miltiorrhiza from different areas. SmAACT will be an useful gene for further analyze the mechanism of gene regulation among the tanshinones biosynthesis.

Cloning and polymorphism analysis of SmERF in Salvia miltiorrhiza / 药学学报

The transcription factor of ethylene responsive factor binding protein (ERF) is belonged to AP2/ERF superfamily, which is known to be unique in plants. AP2/ERF proteins have important functions in the transcriptional regulation of a variety of biological processes related to growth and development, as well as various responses to environmental stimuli. An ERF gene from Salvia miltiorrhiza is cloned and divided into ERF gene family group VII of Arabidopsis and Rice. It contains a MCGGAI (I/L) motif referred to as CMVII-1 and a single intron in the 5'-flanking region of the AP2/ERF domain. Sequence analysis reveals that the region of second extron has abundant polymorphism sites. There are 21 single nucleotide polymorphism sites (SNPs) in the 264 bp region, among them, 14 SNPs are synonymous substitutions and 7 SNPs are non-synonymous substitutions. Though analysis of 181 samples from Shandong, Shaanxi and Sichuan Provinces, it reveals that each production area has its own special genotypes, 5 SNPs show significant difference. Cluster based on UPGMA method reveals that different populations from specific province have clustered together. It shows that SmERF gene will be a candidate molecular marker for the identification of Salvia miltiorrhiza from different areas.

Establishment of allele-specific diagnostic PCR method for identification of antlers / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a convenient, quick and accurate molecular method for the identification of crude drugs of antlers due to the difficult discrimination between the genuine antler and its adulterants.</p><p><b>METHOD</b>According to the alignment analysis of full length sequences of Cyth gene from closely relate species of Cervus, one pair of allele-specific diagnostic PCR primers was designed. Factors such as annealing temperature, dosage of polymerase, times of cycles and dosage of template DNA that influence the PCR results were also investigated.</p><p><b>RESULT</b>Based on the study mentioned above, about 323 bp positive band was amplified under the annealing temperature of 65 degrees C in the total volume of 25 microL PCR reaction using the genuine antler DNA as the template. Sequencing results proved that the positive band was the fragment of Cytb gene from both C. elaphus Linnaeus and C. nippon Temminck.</p><p><b>CONCLUSION</b>The established method, with higher specificity and reproducibility, could accurately differentiate genuine antler from its adulterants and would be widely used in Cervus related Chinese crude drugs' identification.</p>

New method of "ingredient difference phonetypical cloning" for functional gene cloning from medicinal plants / 中国中药杂志

This paper introduced a new method of "ingredient difference phonetypical cloning" for functional gene clone of medicinal plants, which might solve the difficulties in isolating genes encoding enzymes for the biosynthesis of secondary metabolites by usual ways. Concepts, mechanisms and methods were systematically introduced and possibility was proved by experiments. The method showed the extra superiority of for the isolation of the genes belonged to unknown metabolic pathway and little information about its sequences. The method provides a new way to isolate functional gene cloning from Chinese herbs and a fundament for the further study on medicinal plant genetic engineering.

Study on genetic relationship of Astragalus membranaceus var mongholicus in different producing area using SRAP / 中国中药杂志

<p><b>OBJECTIVE</b>To study the genetic relationship of Astragalus membranaceus var. mongholicus in different producing area and provide theoretical basis for the evaluation of Astragalus germplasm resources.</p><p><b>METHOD</b>Through quence-related amplified polymorphism (SRAP) analysis, the systematic diagram of genetic relationship was constructed by UPGMA method.</p><p><b>RESULT</b>A total of 141 SRAP markers were scored. By the use of UPGMA cluster analysis with genetic distance, Astragalus could be divided into two provenance plots of Gansu and Shanxi.</p><p><b>CONCLUSION</b>The genetic differentiation among populations of A. membranaceus var. mongholicus is remarkable. SRAP marker could be efficiently used for the study of the genetic relationship of Astragalus.</p>

Functional genomics of Salvia militiorrhiza IV--analysis of ethylene responsive element binding protein gene / 中国中药杂志

<p><b>OBJECTIVE</b>To study the ethylene responsive element binding protein genes of Salvia miltiorrhiza through bioinformatics and characterization of its tissue expression in regenerated plantlets.</p><p><b>METHOD</b>The ethylene responsive element binding protein genes were obtained by cDNA microarray analyze. BLAST was used for alignment, ORF finder software was used to find open reading frame, Prosite database was used to analyze the protein. Semi-quantitative RT- PCR method was used to detect the gene expression level.</p><p><b>RESULT</b>One ethylene responsive element binding protein was obtained, named as SmERF. SmERF had an open reading frame of 699 bp with 5'-URT 87 bp and 3'-URT 166 bp. The putative protein SmERF contains a highly conserved ERF/AP2 domain. Semiquantitative RT- PCR illustrated that SmERF was expressed in all tissues such as root, stem and leaf in regenerated shoots, while the expression level was higher in root than in stem and leaf.</p><p><b>CONCLUSION</b>It was the first time to obtain ERF gene in S. miltiorrhiza and set a good foundation for its further functional study.</p>

Cloning and characterization of geranylgeranyl diphosphate synthase gene of Salvia miltiorrhia / 中国中药杂志

<p><b>OBJECTIVE</b>To obtain geranylgeranyl diphosphate synthase gene of Salvia miltiorrhiza, and conduct bioinformatic and transcript expression analysis of the cloned SmGGPS1 gene.</p><p><b>METHOD</b>The degenerate primers were designed based on the conservative regions of GGPS protein sequences from public databases. The target gene was obtained from root of S. miltiorrhiza by use of homologous cDNA amplification and RACE technologies. The sequence alignment was performed using BLAST. The open reading frame was identified by use of the ORF Finder. The protein domains were defined by use of Prosite software and the signal peptide sequence was predicted by Target P1.1. MEGA4.0 was used to conduct multiple amino acid sequence alignment and construct the phylogenetic tree. Roots and leaves at the seedlings stage and roots, stems, leaves, buds and flowers in the flowering stage were sampled for transcript analysis. Semi-quantitative RT-PCR was used to detect the gene expression level. The complete gene of GGPS was obtained from S. miltiorrhiza genomic DNA by PCR using the cDNA-derived specific primer. The gene structure of GGPS was analyzed by comparison of the genomic DNA and its cDNA.</p><p><b>RESULT</b>The obtained 1 298 bp SmGGPS1 cDNA sequence contains an 1095 bp ORF, encoding 364 amino acids. It is predicted that it has a plastid targeting signal peptide of approximately 52 amino acid at the N-terminal end. It is to believe that this is the polyprenyl synthetase signature, and nucleic acid sequence comparison revealed that SmGGPS1 ORF has more than 60% identity to the reported GGPS. RT-PCR semi-quantitative analysis showed that the gene expresses in the all tested tissues, and with much higher level of expression in the leaves in the flowering stage. SmGGPS1 has a 397 bp intron.</p><p><b>CONCLUSION</b>For the first time the cloning of geranylgeranyl diphosphate synthase gene from S. miltiorrhiza was reported, and it provides a good basis for further functional study of SmGGPS1.</p>