Gene Cloning and Bioinformatics Analysis of Limonene-3-hydroxylase from Schizonepeta tenuifolia / 中国实验方剂学杂志

Objective:This paper aims to clone the cDNA sequence of<italic> limonene</italic>-3-<italic>hydroxylase</italic>（<italic>StL</italic>3<italic>OH</italic>） in <italic>Schizonepeta tenuifolia</italic> and analyze its sequence by bioinformatics. Method:Specific primers were designed based on sequences of<italic> StL</italic>3<italic>OH </italic>gene screened from transcriptome sequencing data of <italic>S. tenuifolia</italic> and the cDNA sequence of <italic>StL</italic>3<italic>OH </italic>gene was cloned by reverse transcription polymerase chain reaction （RT-PCR） and analyzed for its bioinformatics. Result:The <italic>StL3OH</italic> gene cDNA sequence length was 1 598 bp，containing a 1 497 bp long complete open reading frame which encoded 498 amino acids. StL3OH protein had a theoretical relative molecular mass of 56.40 kDa，with a hydrophilic and unstable nature. Bioinformatics analysis showed that StL3OH protein had no signal peptide but had a transmembrane domain which might be located in endoplasmic reticulum. Multiple sequence alignment and cluster analysis showed that the amino acid sequence of MsL3OH protein had a high similarity with StL3OH protein，both of which contained cytochrome P450 heme binding region，belonging to the D subfamily of cytochrome CYP71 family. Codon bias analysis showed that <italic>StL</italic>3<italic>OH</italic> gene preferred guanine/cytosine（G/C） ending codon，with 27 skewed codons， and Nicotiana benthamiana was proven to be the most suitable host for exogenous expression of <italic>StL</italic>3<italic>OH</italic> gene. Conclusion:The cDNA sequence of<italic> StL3OH</italic> gene was cloned from <italic>S. tenuifolia</italic> for the first time，which will provide a basis for further study on the structure and function of StL3OH protein and the regulation mechanism of <italic>StL3OH </italic>gene in the accumulation and biosynthesis of monoterpenes in<italic> S. tenuifolia</italic>.

Study on Sparganium stoloniferum from different regions in China based on appearance characters and ISSR markers / 中国中药杂志

Based on the characteristics and ISSR molecular marker technology, the study is aimed to compare and perform genetic diversity analysis on Sparganium stoloniferum from 7 regions. Molecular identification method was established for S. stoloniferum from Hunan province. Differences among Sparganii Rhizoma samples from seven habitats were analyzed via measuring weight, length, width and thickness of them. Genetic diversity of S. stoloniferum from 7 regions was analyzed by screening out primers amplifying clear band and showing rich polymorphism, then a cultivars dendrogram was built. The target primer was screened out, and the specific band was sequenced. Nine ISSR primers were selected to amplified clear band, rich polymorphism. A total of 73 bands were amplified by nine ISSR primers selected from 27 ISSR primers. On average, each primer produced 8.0 bands. A total of 38 bands were polymorphic, which occupied 52.8% of all bands. The cultivars dendrogram showed the genetic similarity was 0.54-0.94. Genetic similarity coefficient of S. stoloniferum from Jiangsu province, Anhui province and Jiangxi province was big, indicating the differences among them were slight on genetic level. S. stoloniferum from Hunan province is quite different from samples from the other six habitats on appea-rance and genetic level. A specific band(327 bp) in S. stoloniferum from Hunan province was obtained via ISSR-857 primer, and was sequenced. According BLASTn database, there were few sequences similar to the gene fragment and had little correlation with the growth process of plant. ISSR molecular marker technology provides a new idea for the identification of S. stoloniferum. This result confirmed the particularity of S. stoloniferum from ancient Jingzhou.

Expression profiling and functional verification of flavonoid 3'-hydroxylase gene from leaves of Euryale ferox / 中国中药杂志

Leaves of Euryale ferox are rich in anthocyanins. Anthocyanin synthesis is one of the important branches of the flavonoid synthesis pathway, in which flavonoid 3'-hydroxylase(F3'H) can participate in the formation of important intermediate products of anthocyanin synthesis. According to the data of E. ferox transcriptome, F3'H cDNA sequence was cloned in the leaves of E. ferox and named as EfF3'H. The correlation between EfF3'H gene expression and synthesis of flavonoids was analyzed by a series of bioinforma-tics tools and qRT-PCR. Moreover, the biological function of EfF3'H was verified by the heterologous expression in yeast. Our results showed that EfF3'H comprised a 1 566 bp open reading frame which encoded a hydrophilic transmembrane protein composed of 521 amino acid residues. It was predicted to be located in the plasma membrane. Combined with predictive analysis of conserved domains, this protein belongs to the cytochrome P450(CYP450) superfamily. The qRT-PCR results revealed that the expression level of EfF3'H was significantly different among different cultivars and was highly correlated with the content of related flavonoids in the leaves. Eukaryotic expression studies showed that EfF3'H protein had the biological activity of converting kaempferol to quercetin. In this study, EfF3'H cDNA was cloned from the leaves of E. ferox for the first time, and the biological function of the protein was verified. It provi-ded a scientific basis for further utilizing the leaves of E. ferox and laid a foundation for the further analysis of the biosynthesis pathway of flavonoids in medicinal plants.

Analysis on quality and differences of Mentha haplocalyx from different regions / 中国中药杂志

A gas chromatography-mass spectrometry(GC-MS)method was established for the analysis of volatile components in Mentha haplocalyx, and seven principal components were quantified by gas chromatography(GC). Based on these analyses, the differences of volatile components in M. haplocalyx from Jiangsu, Anhui and other regions were compared. The results showed that the volatile oil of M. haplocalyx was divided into four chemical types：menthol-menthone type, pulegone-menthone type, piperitone-menthol type, piperitone epoxide type, and menthol-menthone type was the principal type. Menthol was the highest and pulegone was the lowest. The differences of M. haplocalyx from Anhui and other regions were obvious. The major volatile components and the differences of M. haplocalyx from different regions were confirmed and a quantitative method was established for the determination of volatile components, which provided the basis for improving the quality standard of M. haplocalyx.

Reasearch on evolution and transition of processing method of fuzi in ancient and modern times / 中国中药杂志

Fuzi is a medicine used for rescuing from collapse by restoring yang as well as a famous toxic traditional Chinese medicine. In order to ensure the efficacy and safe medication, Fuzi has mostly been applied after being processed. There have been different Fuzi processing methods recorded by doctors of previous generations. Besides, there have also been differences in Fuzi processing methods recorded in modern pharmacopeia and ancient medical books. In this study, the authors traced back to medical books between the Han Dynasty and the period of Republic of China, and summarized Fuzi processing methods collected in ancient and modern literatures. According to the results, Fuzi processing methods and using methods have changed along with the evolution of dynasties, with differences in ancient and modern processing methods. Before the Tang Dynasty, Fuzi had been mostly processed and soaked. From Tang to Ming Dynasties, Fuzi had been mostly processed, soaked and stir-fried. During the Qing Dynasty, Fuzi had been mostly soaked and boiled. In the modem times, Fuzi is mostly processed by being boiled and soaked. Before the Tang Dynasty, a whole piece of Fuzi herbs or their fragments had been applied in medicines; Whereas their fragments are primarily used in the modern times. Because different processing methods have great impacts on the toxicity of Fuzi, it is suggested to study Fuzi processing methods.