Development and Application of Detection Methods for Capture and Transcription Elongation Rate of Bacterial Nascent RNA

Objective Detection and quantification of RNA synthesis in cells is a widely used technique for monitoring cell viability,health,and metabolic rate.After exposure to environmental stimuli,both the internal reference gene and target gene would be degraded.As a result,it is imperative to consider the accurate capture of nascent RNA and the detection of transcriptional levels of RNA following environmental stimulation.This study aims to create a Click Chemistry method that utilizes its property to capture nascent RNA from total RNA that was stimulated by the environment.Methods The new RNA was labeled with 5-ethyluridine(5-EU)instead of uracil,and the azido-biotin medium ligand was connected to the magnetic sphere using a combination of“Click Chemistry”and magnetic bead screening.Then the new RNA was captured and the transcription rate of 16S rRNA was detected by fluorescence molecular beacon(M.B.)and quantitative reverse transcription PCR(qRT-PCR).Results The bacterial nascent RNA captured by“Click Chemistry”screening can be used as a reverse transcription template to form cDNA.Combined with the fluorescent molecular beacon M.B.1,the synthesis rate of rRNA at 37℃is 1.2 times higher than that at 15℃.The 16S rRNA gene and cspI gene can be detected by fluorescent quantitative PCR,it was found that the measured relative gene expression changes were significantly enhanced at 25℃and 16℃when analyzed with nascent RNA rather than total RNA,enabling accurate detection of RNA transcription rates.Conclusion Compared to other article reported experimental methods that utilize screening magnetic columns,the technical scheme employed in this study is more suitable for bacteria,and the operation steps are simple and easy to implement,making it an effective RNA capture method for researchers.

The rice histone methylation regulates hub species of the root microbiota

Plants have a close relationship with their root microbiota,which comprises a complex microbial network.Histone methylation is an important epigenetic modification influencing multiple plant traits;however,little is known about the role of plant histone methylation in the assembly and network structure of the root microbiota.In this study,we established that the rice(Oryza sativa)histone methylation regulates the structure and composition of the root microbiota,especially the hub species in the microbial network.DJjmj703(defective in histone H3K4 demethylation)and ZH11-sdg714(defective in H3K9 methylation)showed significant different root microbiota compared with the corresponding wild types at the phylum and family levels,with a consistent increase in the abundance of Betaproteobacteria and a decrease in the Firmicutes.In the root microbial network,35 of 44 hub species in the top 10 modules in the tested field were regulated by at least one histone methylation-related gene.These observations establish that the rice histone methylation plays a pivotal role in regulating the assembly of the root microbiota,providing insights into the links between plant epigenetic regulation and root microbiota.

The safflower MBW complex regulates HYSA accumulation through degradation by the E3 ligase CtBB1

The regulatory mechanism of the MBW(MYB-bHLH-WD40) complex in safflower(Carthamus tinctorius) remains unclear. In the present study,we show that the separate overexpression of the genes CtbHLH41, CtMYB63, and CtWD40-6 in Arabidopsis thaliana increased anthocyanin and procyanidin contents in the transgenic plants and partially rescued the trichome reduction phenotype of the corresponding bhlh41, myb63,and wd40-6 single mutants. Overexpression of CtbHLH41, CtMYB63, or CtWD40-6 in safflower significantly increased the content of the natural pigment hydroxysafflor yellow A(HYSA)and negatively regulated safflower petal size.Yeast-two-hybrid, functional, and genetic assays demonstrated that the safflower E3 ligase CtBB1(BIG BROTHER 1) can ubiquitinate CtbHLH41,marking it for degradation through the 26S proteasome and negatively regulating flavonoid accumulation. CtMYB63/CtWD40-6 enhanced the transcriptional activity of CtbHLH41 on the CtDFR(dihydroflavonol 4-reductase) promoter.We propose that the MBW-CtBB1 regulatory module may play an important role in coordinating HYSA accumulation with other response mechanisms.

GmCRY1s modulate gibberellin metabolism to regulate soybean shade avoidance in response to reduced blue light

Soybean is an important legume crop that displays the classic shade avoidance syndrome(SAS),including exaggerated stem elongation,which leads to lodging and yield reduction under density farming conditions.Here,we compared the effects of two shade signals,low red light to far-red light ratio(R:FR)and low blue light(LBL),on soybean status and revealed that LBL predominantly induces excessive stem elongation.We used CRISPR-Cas9-engineered Gmcry mutants to investigate the functions of seven cryptochromes(GmCRYs)in soybean and found that the four GmCRY1s overlap in mediating LBL-induced SAS.Lightactivated GmCRY1s increase the abundance of the bZlP transcription factors STF1 and STF2,which directly upregulate the expression of genes encoding GA2 oxidases to deactivate GA1 and repress stem elongation.Notably,GmCRY1b overexpression lines displayed multiple agronomic advantages over the wild-type control under both dense planting and intercropping conditions.Our study demonstrates the integration of GmCRY1-mediated signals with the GA metabolic pathway in the regulation of LBL-induced SAS in soybean.It also provides a promising option for breeding lodging-resistant,high-yield soybean cultivars in the future.