The promoter and the 5'-untranslated region of rice metallothionein OsMT2b gene are capable of directing high-level gene expression in germinated rice embryos.

KEY MESSAGE: Critical regions within the rice metallothionein OsMT2b gene promoter are identified and the 5'-untranslated region (5'-UTR) is found essential for the high-level promoter activity in germinated transgenic rice embryos. Many metallothionein (MT) genes are highly expressed in plant tissues. A rice subfamily p2 (type 2) MT gene, OsMT2b, has been shown previously to exhibit the most abundant gene expression in young rice seedling. In the present study, transient expression assays and a transgenic approach were employed to characterize the expression of the OsMT2b gene in rice. We found that the OsMT2b gene is strongly and differentially expressed in germinated rice embryos during seed germination and seedling development. Histochemical staining analysis of transgenic rice carrying OsMT2b::GUS chimeric gene showed that high-level GUS activity was detected in germinated embryos and at the meristematic part of other tissues during germination. Deletion analysis of the OsMT2b promoter revealed that the 5'-flanking region of the OsMT2b between nucleotides -351 and -121 relative to the transcriptional initiation site is important for promoter activity in rice embryos, and this region contains the consensus sequences of G box and TA box. Our study demonstrates that the 5'-untranslated region (5'-UTR) of OsMT2b gene is not only necessary for the OsMT2b promoter activity, but also sufficient to augment the activity of a minimal promoter in both transformed cell cultures and germinated transgenic embryos in rice. We also found that addition of the maize Ubi intron 1 significantly enhanced the OsMT2b promoter activity in rice embryos. Our studies reveal that OsMT2b351-ubi(In) promoter can be applied in plant transformation and represents potential for driving high-level production of foreign proteins in transgenic rice.

In vitro antioxidant activities, free radical scavenging capacity, and tyrosinase inhibitory of flavonoid compounds and ferulic acid from Spiranthes sinensis (Pers.) Ames.

In this study, ultrasound-assisted extraction (UAE) and other methods of extracting flavonoid compounds and ferulic acid (FA) from S. sinensis were investigated. Five different extraction methods, including water extraction (W), water extraction using UAE (W+U), 75% ethanol extraction (E), 75% ethanol extraction using UAE (E+U), and supercritical CO2 extraction (SFE) were applied in the extraction of bioactive compounds (flavonoids and ferulic acid) in order to compare their efficiency. The highest yield of flavonoids (4.28 mg/g) and ferulic acid (4.13 mg/g) content was detected in the E+U extract. Furthermore, S. sinensis extracts obtained by E+U show high antioxidant activity, and IC50 values of 0.47 mg/mL for DPPH radicals and 0.205 mg/mL for metal chelating activity. The total antioxidant assay shows superoxide radical scavenging capacity and in vitro mushroom tyrosinase inhibition in a dose-dependent manner, suggesting that E+U can be used for extraction of bioactive compounds from S. sinensis.