[Prokaryotic expression, functional identification of squalene synthase in Alisma orientale and its immunoassay study].

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.

Application of the ITS2 Region for Barcoding Medicinal Plants of Selaginellaceae in Pteridophyta.

BACKGROUND: Selaginellaceae is a family of nonseed plants with special evolutionary significance. Plants of the family Selaginellaceae are similarly shaped and easily confused, complicating identification via traditional methods. This study explored, for the first time, the use of the DNA barcode ITS2 to identify medicinal plants of the Selaginellaceae family. METHODOLOGY/PRINCIPAL FINDINGS: In our study, 103 samples were collected from the main distribution areas in China; these samples represented 34 species and contained almost all of the medicinal plants of Selaginellaceae. The ITS2 region of the genome was amplified from these samples and sequenced using universal primers and reaction conditions. The success rates of the PCR amplification and sequencing were 100%. There was significant divergence between the interspecific and intraspecific genetic distances of the ITS2 regions, while the presence of a barcoding gap was obvious. Using the BLAST1 and nearest distance methods, our results proved that the ITS2 regions could successfully identify the species of all Selaginellaceae samples examined. In addition, the secondary structures of ITS2 in the helical regions displayed clear differences in stem loop number, size, position, and screw angle among the medicinal plants of Selaginellaceae. Furthermore, cluster analysis using the ITS2 barcode supported the relationship between the species of Selaginellaceae established by traditional morphological methods. CONCLUSION: The ITS2 barcode can effectively identify medicinal plants of Selaginellaceae. The results provide a scientific basis for the precise identification of plants of the family Selaginellaceae and the reasonable development of these resources. This study may broaden the application of DNA barcoding in the medicinal plant field and benefit phylogenetic investigations.

[Pollen morphological characteristics, viability test and storage of endangered medicinal plant Atractylodes lancea].

OBJECTIVE: To study the pollen morphological characteristics, viability test and storage character of the endangered plant Atractylodes lancea. METHOD: Pollen grains morphologies of A. lancea were observed by scanning electron microscope. The optimum culture medium and viability determination methods were screened out by liquid culture and dyeing methods, and then the pollen germination capacities in different storage conditions were detected. RESULT: The pollen grains are quasi-spherical, with tricolpate and spinous sculpture. The optimal culture medium was ME3 + 16% PEG4000 + 10% sucrose, in which the pollen germination capacity reached to 62.1%, while the other three dyeing methods were not able to be applied to detecting the pollen viability of A. lancea. The low storage temperature could significantly prolong the storage time of pollen of A. lancea. At -80 degrees C, pollen viability could be maintained for 60 days. CONCLUSION: Liquid culture method is suitable for the determination of pollen germination of A. lancea, and the rate of pollen germination is closely related to the storage time and temperature. At last, this study provides a foundation for the artificial pollination and cultivating in wildness of A. lancea.

[Molecular cloning of farnesyl pyrophosphate synthase from Alisma orientale (Sam.) Juzep. and its distribution pattern and bioinformatics analysis].

Triterpenes, which have large application potential in the treatment of cancer, are the main active components of genuine medicinal material Alisma orientale (Sam.) Juzep. Farnesyl pyrophosphate synthase (FPPS) is one of the important rate-limiting enzymes in the synthetic pathway of triterpenes. In this study the FPPS full length cDNA of the A. orientale, was cloned via homology-based cloning approach and rapid amplification of cDNA ends (RACE). The full length of the FPPS cDNA was 1 531 bp (accession no. HQ724508), which contained a full 1 032 bp ORF that encoded 343 amino acids. The deduced protein sequence exhibited five conserved motifs, two of which is riched of Asp (DDXXD). The result of real-time quantitative PCR (QRT-PCR) showed that FPPS gene was expressed in different organs of A. orientale. The expression increased from October to the first ten-day period of December, and then decreased. The FPPS gene expression was higher in leaves but lower in leafstalk, tuber and root. HPLC analysis of active components 23-acetyl-alismol B of A. orientale. during different periods indicated that its change trend should be consistent with FPPS gene expression. It can be primarily deduced that FPPS gene should be an important control point in the synthetic pathway of Alisma terpenes. This study may facilitate the quality of medicinal plants through gene engineering in the future.

[Study on rapid propagation of Changium smyrnioides].

OBJECTIVE: For the protection of Changium smyrnioides Wolff germ-plasm resources. METHODS: Rapid propagation was conducted by tissue culture. RESULTS: The best explants were leaves. The optimum culture medium for the induction of callus was MS + 2,4-D 1.0 mg/L + Kt 1.0 mg/L; that of bud was MS + 6-BA 3.0 mg/L + NAA 0.2 mg/L; and that of root was MS + IBA 0.4 mg/L or MS + NAA 0.4 mg/L. CONCLUSION: Rapid propagation of Changium smyrnioides is established with tissue culture, which provides an effective way for the sustainable utilization.

[A research on fast-propagation of Pseudostellaria heterophylla].

The result of fast-propagation of Pseudostellaria heterophylla by tissue culture showed that the best explant was stem tip. It also showed the suitable inducement mediums were MS + 6-BA 2.0 mg/L + NAA 0.2 mg/L or MS + 6-BA 4.0 mg/L + NAA 0.4 mg/L for the clustered bud, MS + 2,4-D 4.0 mg/L + Kt 0.5 mg/L or MS + 2,4-D 3.0 mg/L + Kt 0.5 mg/L for the callus growth and 1/2 MS + NAA 0.3 mg/L for the root growth.