Functional Analysis Of PtrRHH94 Gene In Populus Trichocarpa

The RING family is a super family,its members are involved in diverse biological processes.RING-H2 subfamily,the most abundant RING-type genes,they are involved in a variety of biological processes,including the stress response,hormonal regulation,signal transduction and photomorphogenesis with light signaling and responses.Several members of RING-H2 in poplar were highly or preferentially expressed in xylem,but these genes function has not been identified.We identified the PtrRHH94gene for research and analysis,the main results are as follows:The RING-H2 subfamily has 288 members in Populus trichocarpa,among them,30 genes of highly or preferentially xylem-expressed,which contained multiple xylem development-related cis-elements in the promoters,the PtrRHH94 gene has only two cis-elements.Semi-quantitative RT-PCR results indicate that PtrRHH94 gene was specifically expressed in xylem,but it was almost not expressed or very low in other tissues.We constructed pgwb3-ptrrhh94pro vector with promoter fusion of GUS reporter gene,then the construct is transformed into P.trichocarpa.The GUS staining signal was visible in the xylem of stem and root,which are mainly secondary vascular tissues,and its intensity holds pace with the increase of stem lignification.Based on the Cas9/gRNA gene editing technique,we designed three specific targets on the PtrRHH94 gene and obtained multiple PtrRHH94 knockout mutants in P.trichocarpa,all of them were effectively edited and same phenotype.Compared with the wild type,the young tree height and the number of stems increased after PtrRHH94 gene knockout,but other phenotypes did not change significantly.We analyzed the different stems of the PtrRHH94 gene knockout mutant by paraffin sectioning and histochemical staining.Using toluidine blue staining and scanning electron microscopy to observe the cross section of the stem,we found that the phloem,cambium,xylem,and cell wall thickness did not change significantly.Only the phloroglucinol staining,the lignin staining area of the mutant stem in the 3th and 5th segments was larger than that of the wild type,but the total lignin content did not change.It is suggested that the PtrRHH94 gene may affect stem growth,but it needs to be further verified.The PtrRHH94 gene is a major member of the RING-H2 subfamily of P.trichocarpa that may participate in secondary growth,and plays a negative regulatory role in the growth and development of poplars.PtrRHH94 gene is specifically or highly expressed in xylem,but its mutation does not seem to directly affect the development of xylem,suggesting that it may regulate the development of poplars through ubiquitination-mediated downstream target genes.The study of its function has laid the foundation for further exploring the molecular mechanism of the PtrRHH94 gene.