| Transcription factor is a type of protein molecule that can be combined with a specific sequence of upstream of a gene to ensure that the target gene is expressed at a specific time and space with a specific intensity.Some transcription factors are closely related to plant growth and development,while others are involved in plants responding to various environmental stresses.The study results indicated that AtMYC1,AtTGA1a and AtTRF7 play a regulatory role in plant defense responses,of which AtMYC1 mainly plays an important role in JA signaling pathway,while AtTGA1a and AtTRF7 mainly play a regulatory role in SA signaling pathway,but usually SA And JA defense signals are antagonistic to each other.The results of Bimolecular fluorescence complementation indicated that there is a strong interaction signal between AtMYC1 and AtTGA1a and AtTRF7.Verticillium wilt is a soil-borne vascular disease caused by hemi-pathogens,both SA and JA signal pathway play important roles in the plant against Verticillium dahliae.The effects of overexpression of these transcription factors that regulate mutually antagonistic signaling pathways on the resistance to Verticillium wilt in plants and its mechanism need further understanding and exploration.In this paper,transgenic Arabidopsis and tobacco were used as materials,using co-transformation and hybridization methods to obtain double-gene transgenic plants,and disease resistance identification of detached leaves and whole plants was carried out to clarify the effect of AtMYC1,AtTGA1a and AtTRF7 interaction on resistance to Verticillium wilt,and to further detect the changes in the expression level of defense signaling pathway marker genes after the increase of transcription factor levels.In order to explore the possible mechanism of resistance of transcription factors that regulate mutually antagonistic signal pathways to Verticillium wilt.The main results are as follows:1.Constitutive expression of AtMYC1/AtTRF7 and AtMYC1/AtTGA1a increases the resistance to Verticillium wilt in Arabidopsis and tobacco(1)Simultaneous constitutive expression AtMYC1/AtTRF7 genes increase the resistance to Verticillium wilt in Arabidopsis and tobaccoCo-transformation yielded transgenic Arabidopsis and tobacco that constitutively expressed AtMYC1 and AtTRF7 genes,and AtMYC1 and AtTRF7 single genes.Transgenic Arabidopsis thaliana plants were inoculated with Verticillium dahliae for11 days by using root irrigation method.The disease index of wild-type Arabidopsis was 62.92,and the disease indexes of AtMYC1-AtTRF7-6,AtMYC1-AtTRF7-7 and AtMYC1-AtTRF7-10 lines were 36.68,33.60 and 44.38,respectively,while the disease indexes of AtMYC1-13,AtMYC1-26,AtTRF7-1 and AtTRF7-11 lines were58.65,50.56,46.41 and 44.31,respectively.The results showed that the disease index of AtMYC1/AtTRF7 double-gene transgenic Arabidopsis was lower than that of single-gene transgenic Arabidopsis,and the disease index of all transgenic Arabidopsis was significantly lower than the wild type control.The same results were obtained by using in vitro leaf inoculation for disease resistance identification.Similarly,the root irrigation method was used to detect the resistance of AtMYC1and AtTRF7 transgenic tobacco plants and their hybrid F1generation to Verticillium dahliae.The results showed that the disease indexes of WT,AtMYC1-7,AtMYC1-9,AtTRF7-4,AtTRF7-4×AtMYC1-7 and AtTRF7-4×AtMYC1-9 were 71.34,58.77,56.22,61.35,26.04 and 43.75,respectively.The results showed that the disease index of AtMYC1/AtTRF7 double-gene transgenic tobacco was significantly lower than that of single-gene transgenic tobacco,and the disease index of all transgenic tobacco was significantly lower than that of wild-type control.The results showed that constitutive expression of AtMYC1/AtTRF7 can further increase the resistance to Verticillium wilt in Arabidopsis and tobacco.(2)Simultaneous constitutive expression AtMYC1/AtTGA1a genes increase the resistance to Verticillium wilt in Arabidopsis and tobaccoCo-transformation yielded transgenic Arabidopsis and tobacco that constitutively expressed AtMYC1 and AtTGA1a genes,and AtMYC1 and AtTGA1a single genes.Transgenic Arabidopsis thaliana plants were inoculated with Verticillium dahliae for11 days by using root irrigation method.The disease index of wild-type Arabidopsis was 62.92,and the disease indexes of AtMYC1-AtTGA1a-2,AtMYC1-AtTGA1a-15and AtMYC1-AtTGA1a-23 lines were 28.75,23.16 and 48.81,respectively,while the disease indexes of AtMYC1-13,AtMYC1-26,AtTGA1a-7 and AtTGA1a-10 lines were58.65,50.56,54.59 and 48.09,respectively.The results showed that the disease index of AtMYC1/AtTGA1a double-gene transgenic Arabidopsis was lower than that of single-gene transgenic Arabidopsis,and the disease index of all transgenic Arabidopsis was significantly lower than the wild type control..The same results were obtained by using in vitro leaf inoculation for disease resistance identification.Similarly,the root irrigation method was used to detect the resistance of AtMYC1and AtTRF7 transgenic tobacco plants and their hybrid F1generation to Verticillium dahliae.The results showed that the disease indexes of WT,AtMYC1-7,AtMYC1-9,AtTGA1a-6,AtTGA1a-6×AtMYC1-7 and AtTGA1a-6×AtMYC1-9 were 71.34,58.77,56.22,38.75,37.50 and 20.83,respectively.The results showed that the disease index of AtMYC1/AtTGA1a double-gene transgenic tobacco was lower than that of single-gene transgenic tobacco,and the disease index of all transgenic tobacco was significantly lower than that of wild-type control.The results showed that constitutive expression of AtMYC1/AtTGA1a can further increase the resistance to Verticillium wilt in Arabidopsis and tobacco.2.Transient expressions of AtMYC1/AtTRF7 or AtMYC1/AtTGA1a improve resistance to Verticillium wilt in cottonThe agroinfiltration method was used to obtain cotton cotyledons with transient expression of AtMYC1/AtTRF7 or AtMYC1/AtTGA1a double genes,and AtMYC1,AtTRF7 and AtTGA1a single genes.At the same time,agroinfiltration method was used to inoculate Verticillium dahliae for 5 days.The disease index of non-transgenic CK was 66.1,the disease index of cotton cotyledons transformed by AtMYC1/AtTGA1a and AtMYC1/AtTRF7 were 32.18 and 35.61,and AtMYC1,AtTGA1a and AtTRF7 were 49.23,52.86 and 44.41 respectively.The results showed that the disease index of the double-gene transgenic cotyledons was significantly lower than that of the single-gene transgenic cotyledons and CK,and the single gene was also lower than that of CK.It shows that AtMYC1/AtTRF7 and AtMYC1/AtTGA1a can better improve the resistance of cotton to Verticillium wilt.3.AtMYC1/AtTGA1a activates SA,JA and NO defense signaling pathwaysReal-time PCR method was used to detect the expression levels of SA,JA,and NO defense response marker genes in transgenic Arabidopsis and tobacco.The results showed that among AtTGA1a transgenic Arabidopsis and tobacco lines,only the expression level of ICS1 or PR1 was higher than that of the wild-type control.Among AtMYC1 transgenic Arabidopsis or tobacco lines,only the relative expression of LOX2 or NOS1 was higher than that of the wild-type control.In AtMYC1/AtTGA1a transgenic Arabidopsis and tobacco lines,the relative expression levels of PR1,ICS1,LOX 2,NOS1 or HIN1 were higher than those of the wild-type control.It shows that the transcription factor AtTGA1a can activate the SA signaling pathway;AtMYC1can affect the SA and JA signaling pathways;while the presence of AtMYC1 and AtTGA1 can not only activate the SA signaling pathway,but also activate the JA,NO signaling pathways and Hypersensitive Reaction,which can more effectively improve the resistance to Verticillium dahliae in Arabidopsis and Tobacco.This study proves that AtMYC1 and AtTGA1a,AtMYC1 and AtTRF7 can further improve the resistance to Verticillium wilt in tobacco and Arabidopsis,and explores the mechanism of resistance improvement,providing a basis for the research on the defense of hemi-biotroph pathogens. |
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