Preliminary Study On Molecular Mechanism Of GbDREB And GbABR1 Resistance To Verticillium Wilt In Cotton

Gossypium hirsutum is one of the most important economic crops in the world.Verticillium wilt in cotton resulting in gerat economic losses by severely affected cotton yield and fiber quality.The prevention of traditional chemical methods does not solve this problem very well.Therefore,the cultivation of disease-resistant varieties of cotton has become an effective and environmental friendly to solve this problem.This makes it important to deeply dig out and utilize the important function of disease-resistant genes in cotton and reveal the molecular mechanism of cotton resistance to Verticillium wilt,which will accelerate the breeding process of cotton disease-resistant molecules and promote the development of cotton industry.Based on the previous study of our group,the GbDREB silencing plants more susceptible symptoms,as evaluted by the ratio of diseased leaves.To elucidate the mechanism of GbDREB participation in the resistance response of Verticillium dahliae in cotton,the GbDREB gene was studied using molecular biology techniques such as VIGS(virus-induced gene silencing).A specific segment of the VIGS vector was constructed.The results showed that the incidence and disease index of GbDREB silencing cotton increased after inoculation with Verticillium dahliae.The experimental results on the cut-off plants showed that the vascular bundle browning degree of GbDREB silencing plants was greater than that the control plants(TRV:00 plant material).The trypan blue staining results showed that the staining of TR V-GbDREB plants was deeper than the control plants.The above results indicated that the downregulation of GbDREB increased the susceptibility of cotton plants to V.dahliae infection.Overexpression of GbDREB vector was used to transform A rabidopsis thaliana plants and screened for homozygote.The infection of Verticillium dahliae to Arabidopsis thaliana plants by injuring the roots.The results showed that GbDREB overexpression plants and wild-type plants both have wilting symptoms.The statistical disease indexes were 50%(D6),60.42%(D11),58.12%(D1),and 60%(WT),respectively.Statistical phenotypic results showed no significant differences.Further qRT-PCR detected the transcription levels of pathogenic response genes AtPR3 and AtPR4,showed that there was no significant difference in the transcription of AtPR3 and AtPR4 compared to the wild type for three days after inoculation.Collectively,our results suggest that GbDREB overexpression in Arabidopsis does not significantly increase its resistance.The expression of GbDREB was enhanced by the exogenous application of JA,SA and ETH,while the exogenous application of ABA and IAA had no significant difference.Therefore,the involvement of GbDREB in the regulation of resistance to cotton may be participate in hormone(JA,SA,ET)signals.To further explore how GbDREB is involved in the regulation of cotton disease resistance,yeast two-hybrid screening libraries were used to select upstream and downstream proteins that interact with GbDREB.The GbDREB protein non-toxic and self-activating activity.The truncated fragment verification results showed that the transcriptional activation region of the GbDREB protein is located at the C-terminus 122-176 amino acids.At the same time,the GbABR1 gene of the ERF(Ethylene responsive gene)subfamily also proved its role in the regulation of Verticillium wilt in cotton through the previous VIGS experiment results.After inoculation by Verticillium dahliae,GbABR1 began to up-regulate after 24 hours,and reached the highest level on the 3rd day,the expression level was still up-regulated after 5 days.Subsequently,there was no significant difference between infected plants and the control plants.The GbABRl overexpression plants infected Verticillium dahliae,the statistical disease index was 67.96%(A9),65.35%(A11)and 60.96%(WT),respectively.The results showed the index of diseased in GbABRl overexpressing Arabidopsis lines and WT plants were no obviously difference after inoculation with V.dahliae.After hormone treatment,GbABR1 quickly responded to the treatment of JA and ETH,indicating that the gene is related to the cross-regulation pathways of the two hormones JA and ETH.Self-activation and toxicity tests were showed that GbABR1 has obvious self-activation.The results of the truncation test showed that the transcriptional activation region was located at the C-terminus 293-370 amino acids.