Identification And Functional Analysis Of PnMYB Transcription Factor Genes In Panax Notoginseng

Plants are affected by various environmental factors including abiotic stress and biotic stress during their growth and development.These stress environments are not conducive to the growth and development of plants,and even cause economic losses.Panax notoginseng（Burk.）F.H.Chen is a unique traditional Chinese medicinal herb which belongs to ginseng genus of Acanthaceae.It has many pharmacological effects.And the market demand for P.notoginseng is increasing year by year.However,due to P.notoginseng preference for a shady and humid environment and its long growth cycle,it is extremely prone to breeding diseases and pests during its growth process,among which root rot is the most serious harm,which is a major obstacle restricting the sustainable development of P.notoginseng.The pathogens of root rot include bacteria and fungi,among which Fusarium oxysporum is one of the main pathogens.MYB transcription factors play an important role in plant defense responses.Therefore,in this study,MYB transcription factor PnMYB4、PnMYB6、PnMYB7 genes were cloned from P.notoginseng,and analyzed their expression characteristics and functions in disease resistance.The research work and main results obtained in this paper are as follows:1.Bioinformatics analysis of MYB transcription factors in P.notoginseng.Cloning and obtaining three P.notoginseng MYB transcription factor genes,namely PnMYB4,PnMYB6,and PnMYB7.The base numbers of the three PnMYB genes are1056 bp,1043 bp,and 894 bp,the amino acid numbers of the proteins are 351,347,and 297,and the relative molecular weight of 38788.56 Da,37286.50 Da,32404.64Da,and isoelectric points of 8.66,5.09,and 8.66.In terms of protein stability,the instability coefficient of MYB gene of P.notoginseng are 63.65,71.73,and 74.28,and the total average hydrophilicity are-0.64,-0.518,and-0.477,respectively.The molecular formulas of these three proteins were C₁₆₇₃H₂₆₅₉N₄₉₉O₅₃₀S_17、C₁₆₂₆H₂₅₄₇N₄₆₃O₅₂₄S₁₀、C₁₄₁₀H₂₂₅₆N₄₁₂O₄₄₀S₁₂;The secondary and tertiary structures of PnMYB4,PnMYB6 and PnMYB7 proteins were predicted.Conserved domain analysis showed thatthe proteins encoded by PnMYB4,PnMYB6 and PnMYB7 genes had R2R3-MYB domain.2.One-year-old P.notoginseng was infected by F.oxysporum and treated with exogenous SA.Treated with sterile water was used as the control group,the expression patterns of three genes（PnMYB4,PnMYB6,PnMYB7）in P.notoginseng after infection with F.oxysporum and treatment with exogenous salicylic acid were analyzed by quantative Real-time PCR.The expression level of PnMYB4 increased gradually with the increase of time of FO treatment.The expression level of PnMYB7reached the peak at 48 h after FO treatment,while the expression level of PnMYB6was the highest at 4 h treatment and then decreased;In exogenous SA treatment,the expression of PnMYB4 was first down-regulated and then increased from 48 h to 72h.The expression level of PnMYB6 was increased,but the increase was small at 24h,while that the expression level of PnMYB7 was first down-regulated and then up- regulated,the lowest at 24 h and the highest at 48 h;Within co-treatment with FO and SA,the expression of PnMYB4 showed an increasing trend,with the highest expression at 72 h;The expression level of PnMYB7 showed increase trend and reached its peak at 72 h;while the expression level of PnMYB6 reached the peak at 4h and then decreased gradually.However,the expression level began to rise after 48 h.3.Clarify the transcriptional activation role of PnMYB transcription factor and determine the transcriptional activation domain.Using yeast two hybrid system,the experimental results showed that the transcription factors PnMYB4,PnMYB6,and PnMYB7 have transcriptional activation activity,and the transcriptional activation domain is located at the C-terminal.4.The subcellular localization of PnMYB was clarified.The results showed that p RI101-GFP-PnMYB4 and p RI101-GFP-PnMYB7 were expressed in the nucleus of tobacco leaf cells,indicating that PnMYB4 and PnMYB7 were located in the nucleus.Fluorescence of p RI101-GFP-PnMYB6 was not observed in tobacco leaves.5.The function of PnMYB in transgenic tobacco was clarified.In order to study the role of the role of PnMYB4,PnMYB6,PnMYB7 in plant disease resistance,tobacco slices with transient expression of PnMYB4,PnMYB6,PnMYB7 gene was inoculated with F.oxysporum to detect the change of disease spots.The results showed that after 5 days of F.oxysporum inoculation,the experimental group of tobacco leaves expressed PnMYB4,PnMYB6,and PnMYB7,showed slight decay and browning after inoculation with FO,while the control group of tobacco leaves with empty vector showed more severe browning and decay,and the leaf lesion area was much higher than the experimental group.These results suggested that the transient transformation of PnMYB4,PnMYB6 and PnMYB7 genes enhanced the resistance to F.oxysporum.The fusion expression vector p RI101-GFP-PnMYB7 was transferred into tobacco to construct transgenic tobacco lines.q RT-PCR analysis showed that PnMYB7 was successfully expressed in transgenic tobacco,aand the transcription levels of defense genes PAL4,WIPK,Definin,and PR1c in the PnMYB7 transgenic tobacco lines were significantly increased.The above results showed that PnMYB4,PnMYB6 and PnMYB7,as new P.notoginseng MYB genes,could be induced by infection of F.oxysporum and exogenous SA treatment.Transient transformation and expression of PnMYB4, PnMYB6 and PnMYB7 in tobacco can enhance the resistance of tobacco leaves to F.oxysporum infection.The overexpression of PnMYB7 increased the expression of four defense genes PAL4,WIPK,Definsin and PR1c in transgenic tobacco leaves.Therefore,PnMYB7,as a candidate functional gene for disease resistance,can be used in genetic engineering to improve plant disease resistance.The research in this paper will help us to understand the interaction between P.notoginseng and F. oxysporum at molecular level and reveal the molecular mechanism of P.notoginseng MYB transcription factor in resistance to F.oxysporum.