Cloning and characterization of the promoters of the key genes CPT, SRPP and REF involved in Periploca sepium rubber biosynthesis / 生物工程学报

Hevea brasiliensis is the main source of natural rubber. Restricted by its tropical climate conditions, the planting area in China is limited, resulted in a low self-sufficiency. Periploca sepium which can produce natural rubber is a potential substitute plant. cis-prenyltransferase (CPT), small rubber particle protein (SRPP) and rubber elongation factor (REF) are key enzymes involved in the biosynthesis of cis-1, 4-polyisoprene, the main component of natural rubber. In this study, we cloned the promoter sequences of CPT, SRPP and REF through chromosome walking strategy. The spatial expression patterns of the three promoters were analyzed using GUS (β-glucuronidase) as a reporter gene driven by the promoters through Agrobacterium-mediated genetic transformation. The results showed that GUS driven by CPT, SRPP or REF promoter was expressed in leaves and stems, especially in the leaf vein and vascular bundle. The GUS activity in stems was higher than that in leaf. This study provided a basis for analyzing the biosynthesis mechanism of natural rubber and breeding new varieties of high yield natural rubber.

Correlation between elements accumulation and active components in velamen and stem bark of Periploca sepium / 中草药

Objective: To study the dynamic variation of Mn, Cu, Zn, Fe, K, Ca, and Mg in velamen and stem bark of Periploca sepium and their correlation with the elements such as periplocin and 4-methoxysalicylic aldehyde in different harvesting periods, and to reveal dominant factors on the accumulation of two active components. Methods: The contents of periplocin and 4-methoxysalicylic aldehyde were detected by FAAS and electric heating wet digestion, and the data were analyzed by SPSS 19.0. Results: The seven elements were varied widely in velamen and stem bark. The variation trends of Ca and periplocin, Fe and 4-methoxysalicylic aldehyde in velamen were similar, equally, those of Fe and 4-methoxysalicylic aldehyde, Ca and Mg in stem bark were similar and like double peaks. Periplocin has highly significant positive correlation with Ca in velamen and significant negative correlation with Zn in stem bark. To 4-methoxysalicylic aldehyde, when in velamen, it has highly significant positive correlation with 4-methoxysalicylic aldehyde and Fe in stem bark, significant negative correlation with Mn in stem bark, and significant positive correlation with Fe in velamen; when in stem bark, it has highly significant positive correlation with Fe in both velamen and stem bark. Conclusion: Ca, Zn, Fe, and Mn are dominant elements of accumulation, the key factors for periplocin are Ca in velamen and Zn in stem bark, and Mg in velamen is indierect acting factors; The key factors for 4-methoxysalicylic aldehyde are Mn in stem bark and Fe, Ca and Mg in stem bark are indirect acting factors.

Inhibitory Effects of Periplocin from Cortex Periplocae on Human Lung Cancer Cell Line QG56 / 天津医药

Objective To investigate the inhibitory effects of periplocin from cortex periplocae (CPP) on human lung cancer cell line QG56 and to discuss its mechanism. Methods QG56 cells were cultured in vitro. The final concentrations of CPP in control group were 1.25, 2.50, 5.00, 10.00 and 20.00μg/L. QG56 cells were treated with ascending concentration of CPP for 24 h, 48 h and 72 h. The cell proliferation was measured using MTT method. The morphological changes of QG56 cells were observed under inverted microscope. Flow cytometry (FCM) was used to detect the effects of CPP on cell cycle and cell apoptosis. The expression of apoptosis associated gene bax mRNA in QG56 cells was detected by RT-PCR. The expres-sion of bax protein before and after treatment of CPP was examined by SP immunocytochemistry. Results The inhibitory ef-fect of CPP on the proliferation of QG56 cells was increased with the increasing concentrations of CPP and the prolonged du-ration of treatment. The morphological changes were displayed in QG56 exposed to CPP. The results of FCM showed that CPP caused cell cycle arrest at G0/G1 phase. The apoptotic rate of QG56 cells was significantly increased after CPP treatment for 48 h (P<0.05). The expression of bax mRNA was increased in QG56 exposed to CPP. The result of immunocytochemis-try indicated that CPP up-regulated the expression of bax protein. Conclusion CPP showed significant inhibitory effect on human lung cancer cell lines QG56 through inducing cell cycle arrest and apoptosis.

Lack of Mutagenicity Potential of Periploca sepium Bge. in Bacterial Reverse Mutation (Ames) Test, Chromosomal Aberration and Micronucleus Test in Mice

OBJECTIVES: The root barks of Periploca sepium Bge. (P. sepium) has been used in traditional Chinese medicine for healing wounds and treating rheumatoid arthritis. However, toxicity in high-doses was often diagnosed by the presence of many glycosides. The potential mutagenicity of P. sepium was investigated both in vitro and in vivo. METHODS: This was examined by the bacterial reverse mutation (Ames) test using Escherichia coli WP2uvrA and Salmonella typhimurium strains, such as TA98, TA100, TA1535, and TA1537. Chromosomal aberrations were investigated using Chinese hamster lung cells, and the micronucleus test using mice. RESULTS: P. sepium did not induce mutagenicity in the bacterial test or chromosomal aberrations in Chinese hamster lung cells, although metabolic activation and micronucleated polychromatic erythrocytes were seen in the mice bone marrow cells. CONCLUSIONS: Considering these results, it is suggested that P. sepium does not have mutagenic potential under the conditions examined in each study.

Improvement of Growth and Periplocin Yield of Periploca sepium Adventitious Root Cultures by Altering Nitrogen Source Supply / 中草药·英文版

Objective To increase the ultimate yield of periplocin in Periploca sepium adventitious root cultures by a two-stage culture based on nitrogen source.Methods Firstly,the effects of nitrogen source(NH-NO-)at different ratios and different total initial nitrogen amounts on the accumulation of biomass and secondary metabolites in adventitious root cultures of P sepium were investigated,and growth and production media for the two-stage culture based on the above results were established.Results The highest biomass and periplocin content were obtained in the culture medium of 15 mmol/L total nitrogen amount with NH-NO(1:2)and 30 mmol/L total nitrogen amount with nitrate as the sole nitrogen source.By adopting a fed-batch cultivation strategy,the dry weight adventitious root,periplocin content and yield were increased by 136%,108%,and 389%,respectively when compared with those of the control,reaching up to 8.13 g/L,157.15 μg/g,and 1277.63 μg/L,respectively.Furthermore,it was found that in the process of two-stage culture,the adventitious roots grew thicker significantly after they were transferred into production medium directly.Conclusion The ultimate yield of periplocin in P.sepium adventitious root cultures could be significantly increased by a two-stage culture based on nitrogen source.