Functional Study Of NtHD20 Affecting The Density Of Tobacco Glandular Hair

Epidermal trichomes are specialized trichomes-like structures derived from the epidermal cells of the aerial part of plants.They are divided into glandular trichomes and non-glandular trichomes according to whether they have glandular heads,and unicellular epidermal trichomes and multicellular epidermal trichomes according to their morphological structures.Tobacco epidermal trichomes are multicellular secretory glandular trichomes,and their secretions are closely related to tobacco aroma quality.At present,the development mechanism of single-cell epidermal trichomes in Arabidopsis has been comprehensively analyzed,while the development mechanism of glandular trichomes in tobacco is still unclear.The laboratory found that the number of glandular trichomes of tobacco(Nicotiana.tabacum cv.Xanthin)transfected with Ch IFN-γ(chicken interferon γ)increased significantly.The differentially expressed genes were screened by gene chip,and the gene Nt HD20(LOC107765920),which may be related to tobacco glandular trichomes density,was obtained.Nt HD20 belongs to the HD-ZIP I protein family and contains a homeodomain and a leucine zipper domain.The HD-ZIP protein family includes four subfamilies,among which HD-ZIP IV is related to the development of plant epidermal trichomes,but the effect of HD-ZIP I on tobacco glandular trichomes density is unknown.In this study,Nt HD20 was used as the object.By overexpressing and silencing Nt HD20 in tobacco(Nicotiana.tabacum cv.Xanthin),the changes in glandular trichomes density were calculated,and the expression levels of genes related to glandular trichomes development and the changes in endogenous hormones were detected in the overexpressed plants.To explore the effect of Nt HD20 on tobacco glandular trichomes density.The main findings are as follows:1.Nt HD20 tissue expression analysisThe expression of Nt HD20 in different tobacco tissues was detected by q RT-PCR.The results showed that Nt HD20 was expressed in different tissues of tobacco,and the expression level was the lowest in roots,while the expression levels in flowers,stems and leaves were significantly higher than those in roots,which were 116 times,17 times and 10.4 times higher than those in roots,respectively.times.2.Nt HD20 overexpression and silencingTobacco was genetically transformed by Agrobacterium-mediated leaf disc transformation.After Kan resistance screening,16 Nt HD20-overexpressing tobacco lines were identified by GUS chemical tissue staining,genomic PCR and q RT-PCR.At the same time,the p TRV2-Nt HD20 vector was constructed,and the expression level of Nt HD20 was verified by q RT-PCR after VIGS-induced silencing.The results showed that the expression level of Nt HD20 was significantly reduced in the silenced plants.3.The effect of Nt HD20 on the density of tobacco glandular trichomesTobacco leaves were stained with 0.2% rhodamine B,observed under a superdepth-of-field microscope,and the density of glandular trichomes was counted.The results showed that the density of glandular trichomes in the overexpression plants was significantly lower than that of the control,which was 34.66% lower than that of the control,while the density of glandular trichomes in the silenced plants was significantly higher than that of the control,which was 42.42% higher than that of the control,indicating that Nt HD20 is the initiation of the development of tobacco glandular hairs.negative regulatory transcription factor.4.Nt HD20 affects the expression of genes related to glandular trichomes developmentReal-time quantitative PCR was used to detect the expression levels of glandular trichomes development-related genes in tobacco overexpressing Nt HD20.The results showed that the expression level of Nt Cyc B2(Nitab4.5-0014697g0010.1)in Nt HD20 transgenic tobacco was 1.77 times that of the control;Nt MYB123-like(LOC107773379),Nt Wo-like(LOC107766155)and Ntab SPL6-1(LOC107789814)were 0.14,0.48 and 0.49 times that of the control group,respectively.This indicates that overexpression of Nt HD20 gene causes up-regulation of Nt Cyc B2 expression and down-regulation of Nt MYB123-like,Nt Wo-like,Ntab SPL6-1 expression.It is speculated that Nt HD20 may directly or indirectly regulate the expression of Nt Cyc B2,Nt MYB123-like,Nt Wo-like and Ntab SPL6-1 genes through unknown pathways.,thereby affecting the development of tobacco glandular trichomes.5.Nt HD20 affects the content of tobacco endogenous hormones ABA and JAThe changes of endogenous hormone content in tobacco overexpressing Nt HD20 were detected by high performance liquid chromatography tandem mass spectrometry.The results showed that the average contents of endogenous ABA and JA in the overexpressed plants were significantly reduced,which were 43.67% and 83.84%lower than those of the control,while the contents of GA3 and SA had no significant difference compared with the control.It indicated that overexpression of Nt HD20 gene inhibited the accumulation of endogenous ABA and JA in plants.It was speculated that Nt HD20 may regulate the accumulation of ABA and JA through an unknown pathway,thereby affecting the development of tobacco glandular trichomes.In conclusion: Nt HD20 is a negative regulator of tobacco gland trichomes density.It may inhibit the accumulation of ABA and JA,down-regulate important genes for glandular trichomes development(ab SPL6-1,MYB123,Wo),and up-regulate the cyclin gene Cyc B2,thereby Decreases tobacco glandular trichomes density.