Identification and characterization of a novel tyrosine aminotransferase gene (SmTAT3-2) promotes the biosynthesis of phenolic acids in Salvia miltiorrhiza Bunge.

Rosmarinic acid (RA) and salvianolic acid B (SAB) are main phenolic acids in Salvia miltiorrhiza Bunge have been widely used in the treatment of cardiovascular and cerebrovascular diseases due to their excellent pharmacological activity. RA is a precursor of SAB, and tyrosine transaminase (TAT, EC 2.6.1.5) is a crucial rate-limiting enzyme in their metabolism pathway. This study identified a novel TAT gene, SmTAT3-2, and found that it is a new transcript derived from unconventional splicing of SmTAT3. We used different substrates for enzymatic reaction with SmTAT1, SmTAT3 and SmTAT3-2. Subcellular localization of SmTAT1 and SmTAT3-2 was completed based on submicroscopic techniques. In addition, they were overexpressed and CRISPR/Cas9 gene edited in hairy roots of S. miltiorrhiza. Revealed SmTAT3-2 and SmTAT1 showed a stronger affinity for L-tyrosine than SmTAT3, localized in the cytoplasm, and promoted the synthesis of phenolic acid. In overexpressed SmTAT3-2 hairy roots, the content of RA and SAB was significantly increased by 2.53 and 3.38 fold, respectively, which was significantly higher than that of overexpressed SmTAT1 strain compared with EV strain. These findings provide a valuable key enzyme gene for the phenolic acids metabolism pathway and offer a theoretical basis for the clinical application.

Transcriptional response to cold and fasting acclimation in Onychostoma macrolepis during the overwintering stage.

In this study, we investigated the transcriptome responses of the liver of Onychostoma macrolepis in by RNA sequencing. The sampling process involved three groups: 1G (0 week, 10 °C), 2G (12 weeks, 0 °C) and 3G (24 weeks, 10 °C). The body weight, viscera index, hepatopancreas index and intraperitoneal fat index of O. macrolepis showed a decreasing trend with the prolonging of overwintering time. The crude fat contents of whole fish, muscle and liver in O. macrolepis after overwintering were significantly lower than those of the fish before overwintering (p < 0.05). In 1G versus 2G group, 2G versus 3G group and 1G versus 3G group, the differently expressed genes (DEGs) were 4630, 3976 and 2311, respectively. These results indicated that different stages of overwintering period had significant effects on gene expression of O. macrolepis, and the influence degree gradually decreased with the extension of overwintering period. The results of Gene ontology (GO) enrichment showed that these DEGs were mainly related to metabolism and immunity, and most of them were down-regulated. In this study, the KEGG pathway classification results showed that signal transduction was the most representative. In addition, KOG enrichment results showed that many DEGs associated with lipid transport and metabolism were down-regulated during the overwintering period. These observations suggested that slowing metabolism and delaying immunity may be the strategies for overwintering adaptation of O. macrolepis.

Comparative transcriptome analysis reveals key insights into male sterility in Salvia miltiorrhiza Bunge.

BACKGROUND: Large-scale heterosis breeding depends upon stable, inherited male sterility lines. We accidentally discovered a male sterility line (SW-S) in the F1progeny of a Salvia miltiorrhiza Bunge from Shandong, China (purple flowers) crossed with a S. miltiorrhiza f. alba from Sichuan, China (white flowers). We sought to provide insights into the pollen development for male sterility in S. miltiorrhiza. METHODS: The phenotypic and cytological features of the SW-S and fertile control SW-F were observed using scanning electron microscopy and paraffin sections to identify the key stage of male sterility. Transcriptome profiles were recorded for anthers at the tetrad stage of SW-S and SW-F using Illumina RNA-Seq. RESULTS: The paraffin sections showed that sterility mainly occurred at the tetrad stage of microspore development, during which the tapetum cells in the anther compartment completely fell off and gradually degraded in the sterile line. There was little-to-no callose deposited around the microspore cells. The tetrad microspore was shriveled and had abnormal morphology. Therefore, anthers at the tetrad stage of SW-S and fertile control SW-F were selected for comparative transcriptome analysis. In total, 266,722,270 clean reads were obtained from SW-S and SW-F, which contained 36,534 genes. There were 2,571 differentially expressed genes (DEGs) in SW-S and SW-F, of which 63.5% were downregulated. Gene Ontology (GO) enrichment analysis indicated that the differentially expressed genes were enriched in 56 functional groups (GO terms); of these, all DEGs involved in microgametogenesis and developmental maturation were downregulated in SW-S. These results were confirmed by quantitative RT-PCR. The two GO terms contained 18 DEGs, among which eight DEGs (namely: GPAT3, RHF1A, phosphatidylinositol, PFAS, MYB96, MYB78, Cals5, and LAT52) were related to gamete development. There were 10 DEGs related to development and maturation, among which three genes were directly related to pollen development (namely: ACT3, RPK2, and DRP1C). Therefore, we believe that these genes are directly or indirectly involved in the pollen abortion of SW-S. Our study provides insight into key genes related to sterility traits in S. miltiorrhiza, and the results can be further exploited in functional and mechanism studies.