Screening and verification of proteins of Salvia miltiorrhiza polyphenol oxidase interaction / 中国中药杂志

Polyphenol oxidase(PPO) is an important antioxidant enzyme in plants. It has the functions of scavenging active oxygen and synthesizing phenols, lignin, and plant protection factors, and can enhance the plant's resistance to stress and resistance to pests and diseases. Our previous research found that Salvia miltiorrhiza PPO gene can positively regulate salvianolic acid B synthesis. In order to further explore the mechanism, a pGBKT7-PPO bait vector was constructed using the cloned S. miltiorrhiza polyphenol oxidase gene(SmPPO, GenBank accession number: KF712274.1), and verified that it had no self-activation and no toxicity. The titer of S. miltiorrhiza cDNA library constructed by our laboratory was 4.75 × 107 cfu·mL~(-1), which met the requirements for library construction. Through yeast two-hybrid test, 22 proteins that could interact with SmPPO were screened. Only yeast PAL1 and TAT interacted with SmPPO through yeast co-transformation verification. Further verification was performed by bimolecular fluorescence complementary detection(BiFC). Only TAT and SmPPO interacted, so it meant that TAT and SmPPO interacted. TAT and SmPPO were truncated according to the domain, respectively. The first 126 amino acids of SmPPO and tyrosine amino transferase(TAT) were obtained to interact on the cell membrane and chloroplast. SmPPO was obtained by subcellular localization test, which was mainly loca-lized on the nucleus and cell membrane; TAT was localized on the cell membrane. Real-time quantitative PCR results showed that the SmPPO gene was mainly expressed in roots and stems; the TAT gene was expressed in roots, and the expression level in stems and flowers was low. This article lays a solid foundation for the in-depth study of the molecular mechanism of the interaction of S. miltiorrhiza SmPPO and TAT to regulate the synthesis of phenolic substances.

Screening of different AFLP fragments between near-isogenic lines of male sterile and fertile Salvia miltiorrhiza and their comparison analysis / 中国中药杂志

There is distinctive advantage of using male sterile lines to breed new cultivar and produce hybrids, when compared with general breeding method on yield and quality. In our previous work, near-isogenic lines (NILs) of male sterile and fertile Salvia miltiorrhiza have been obtained through continuous hybridization in many years. In this investigation, 378 primer combination were screened by using AFLP and BSA technique, in which 26 markers amplified from seven primers were found to tightly link to male sterile gene. Based on these markers, two linkage genetic maps were constructed. A 2 027,2 028 bp fragment was amplifed from NILs of fertile and sterile S. miltiorrhiza, respectively, using genome walking technique and previous E11/M4-208 marker as template. Four base mutations were found in intron when comparing both fragments. Among all different markers between NILs of male sterile and fertile S. miltiorrhiza, four was found to have 100% identities to chromosome 1, 3 and 5 of Arabidopsis, namely, E01/M09-418, E05/M13-308, E05/M04-750 and E01/M01-204. The E01/M09-418 marker was very close to male sterile gene of S. miltiorrhiza with distance of 2.1 cM, which also had 100% identities to male sterile gene MS2 in Arabidopsis. Both were distributed in chromosome 3 of Arabidopsis. The 2 028 bp fragment also had 100% identities to MS2 gene. Another E05/M04-750 marker that had 100% identities to chromosome 5 of Arabidopsis was found to have high identities to POP085-M05 gene of poplars and low affinity calcium antiporter CAX2 of Arabidopsis with very low E-value. The constructed genetic map and differential fragments with potential functions found in this study provide a solid foundation to lock male sterile genes in S. miltiorrhiza genome and to discover their functions.

Forced running enhances neurogenesis in the hippocampal dentate gyrus of adult rats and improves learning ability / 生理学报

To investigate the effect of forced running in motor-driven wheel on neurogenesis in the hippocampal dentate gyrus (DG) of adult rats, 5-bromo-2-deoxyuridine (BrdU), a thymidine analog was applied to mark cell proliferation. Neuroepthelial stem cell protein (nestin) expression was used to identify neural stem/precursor cells. The BrdU- and nestin-positive cells were examined by immunohistochemical technique. The ability of learning was evaluated by Y-maze test to explore the functional role of the newborn cells in the DG after forced running. It was found that the number of BrdU- and nestin-positive cells in the DG in running groups was significantly increased compared to that in the control group (P<0.05). The effect of forced running on neurogenesis was intensity-dependent. In addition, an improvement of learning ability in Y-maze test was observed after forced running. These findings suggest that forced running in motor-driven wheel could enhance neurogenesis in the hippocampal DG of adult rats and improve learning ability.

Effects of exercise on neurogenesis in the dentate gyrus and ability of learning and memory after hippocampus lesion in adult rats / 神经科学通报·英文版

Objective To explore the effects of exercise on dentate gyrus (DG) neurogenesis and the ability of learning and memory in hippocampus-lesioned adult rats. Methods Hippocampus lesion was produced by intrahippocampal microinjection of kainic acid (KA). Bromodeoxyuridine (BrdU) was used to label dividing cells. Y maze test was used to evaluate the ability of learning and memory. Exercise was conducted in the form of forced running in a motor-driven running wheel. The speed of wheel revolution was regulated at 3 kinds of intensity: lightly running, moderately running, or heavily running. Results Hippocampus lesion could increase the number of BrdU-labeled DG cells, moderately running after lesion could further enhance the number of BrdU-labeled cells and decrease the error number (EN) in Y maze test, while neither lightly running, nor heavily running had such effects. There was a negative correlation between the number of DG BrdU-labeled cells and the EN in the Y maze test after running. Conclusion Moderate exercise could enhance the DG neurogenesis and ameliorate the ability of learning and memory in hippocampus-lesioned rats.