[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.

[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.

[Cloning and distribution pattern of HMGR gene conserved fragment in Alisma orientale].

OBJECTIVE: To clone and study the distribution pattern of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene conserved fragment in Alisma orientale. METHODS: The HMGR conserved fragment in A. orientale was amplified by RT-PCR strategy with the total RNA of young leaves as the template. HMGR mRNA expression in different organs was detected by real-time quantitative PCR (QRT-PCR). RESULTS: The conserved fragment was 458 bp (accession NO. HQ913638). NCBI Blast sequence analysis showed the resulting protein had high homology to HMGR with 86.8% similarity to Artemisia annua, 88.2% to Bupleurum chinense, 88.2% to Eucommia ulmoides, and 85.5% to Salvia miltiorrhiza. The result of QRT-PCR showed that the HMGR gene was expressed in different organs (i. e. leaves, petioles, tubers, and roots) which was higher in leaves relative to other tissues. CONCLUSION: The HMGR gene conserved fragment from A. orientale was cloned and its distribution pattern was detected for the first time. This work provides a foundation for exploring the synthetic pathway and bioengineering of Alisma terpenes.

[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.

[Toxic effects of Hg2+, Cd2+ and Cu2+ on cell membrane system of Cabomba caroliniana A. Gray].

By the observation with electron- and confocal laser scanning microscopy and the determination of physiological and biochemical reactions, the toxic effects of Hg2+, Cd2+ and Cu2+ on the cell membrane system of Cabomba caroliniana A. Gray were investigated. The results showed that under the actions of the three heavy metal ions, the contents of reactive oxygen species (ROS) and malondialdehyde (MDA) in C. caroliniana leaf cells increased, activities of protective enzymes were in disorder, and lipid peroxidation happened. The cell membrane was damaged, membrane permeability increased, and plasmolysis occurred. Meanwhile, the chloroplast swelled or even disintegrated. The excitement of photosynthetic pigments on thylakoids membrane by light was inhibited, and the auto-fluoresent intensity was decreased. The cristae of mitochondria swelled and decreased, mitochondria membrane was damaged, and nuclear membrane was broken. The effects of Hg2+, Cd2+ and Cu2+ on the cell membrane system of C. caroliniana showed a definite dose-effect correlation, and the stability of membrane system played a key role in the resistance of C. caroliniana to the toxic effects of heavy metals. C. caroliniana was sensitive to Hg2+, and the lethal concentration of Hg2+ was ranged from 0.3 to 0.5 mg L(-1). C. caroliniana had relatively higher endurance to Cd2+ and Cu2+, and could be used as the resistant plant for biological control.