| Verticillium wilt is a soilborne vascular fungal disease mainly caused by V.dahliae.Once it occurs,it is difficult to control it.Moreover,the frequent variation and long survival time of V.dahlaie in the soil caused that the control of Verticillium wilt is more difficult.It is an effective stratigy to control plant diseases by genetic engineering.For Verticillium wilt,it is necessary to use a broad-spectrum and high-efficiency resistant genes.It is of great significance in genetic engineering of Verticillium wilt resistance to search for new gene and enlarge the resource of resistance gene in plant.An anti-fungal protein was isolated from the latex of Broussonetia papyrifera in our previous work.In order to further study the disease resistance of the anti-fungal protein gene,the full-length coding sequence of the anti-fungal protein was obtained by RACE and YADE methods in this paper;The anti-fungal protein was expressed by yeast expression system and its anti-fungal activity was further verified;The resistance of transgenic Arabidopsis and tobacco to Verticillium wilt by overexpressing the anti-fungal protein gene were evaluated;And the resistance of cotton to Verticillium wilt by transient expression of the gene in the cotyledon were also evaluated.The main results are as follows:1.Cloning of BpChiI full-length coding sequenceAccording to the partial coding sequence of anti-fungal peptide,specific primers were designed and the 5’and 3’terminal sequences of the target gene were amplified by RACE and YADE methods.A 1245bp whole sequence containing 975 bp ORF frame was finally obtained.Bioinformatics analysis found that the antimicrobial protein contains a chitin binding domain and a chitinase catalytic domain,which belong to class I chitinase of 19 family.Therefore,the anti-fungal protein was named BpChiI.The molecular weight of BpChiI is 31k Da.This protein is a hydrophilic protein and it contains a signal peptide of 38 amino acids.2.BpChiI inhibited the germination of A.brassicae sporeBpChiI was expressed by Pichia pastoris system.And the influence of BpChiI to the germination of A.brassicae spores was further evaluated by the concentrated fermentation broth.The results showed that the germination rate of A.brassicae spores incubated with 8.5 mg/m L and 17.0 mg/m L total protein was 77.47%and 44.37%at 6 h after incubation,respectively,however,that of the control incubated with p PIC9K control was 94.62%.This indicated that BpChiI could inhibit the germination of A.brassicae spores.Results also showed that the information of hyphe,which treated with the lower concentration of total protein,was later than that of the control.These results indicated that BpChiI could inhibit the spore germination and mycelial growth.3.Overexpression of BpChiI could enhance the resistance to Verticillium wilt in Arabidopsis,tobacco and cotton(1)Overexpression of BpChiI can enhance the resistance of Arabidopsis to Verticillium wiltThe resistance of BpChiI transgenic Arabidopsis to Verticillium wilt was identified by root-dipping method with 10~8 spores/m L of V991 strain.At 13 days after inoculation,the disease index of wild type control was 47.97,and the disease indexes of transgenic strains BpChiI-2,BpChiI-5,BpChiI-6 and BpChiI-7 were 31.10,23.80,22.73,and 20.64,respectively.The results showed that the disease index of transgenic Arabidopsis was reduced by about 50%.The phenotype of plant showed that almost all leaves of WT showed the symptom of chlorosis at 13d after inoculation with V991 strain.However,only part leaves showed disease symptoms in BpChiI transgenic plants.The results,which the q RT-PCR analysis of the relative number of V.dahliae and the pathogen isolation by PDA in the petioles of Arabidopsis thaliana,showed that the number of V.dahliae in transgenic Arabidopsis was significantly lower than that in WT plants.This result demonstrated that BpChiI could enhance the resistance of Arabidopsis by decreasing the colonization of V.dahliae in Arabidopsis.(2)Overexpression of BpChiI enhanced the resistance of tobacco to Verticillium wiltTobacco seedlings with 2-3 leaves were inoculated with 10~7 spores/m L V991spore suspension by root-dipping method.At 54d after inoculation with V991,the disease index of WT tobacco was 50.00,while the disease index of transgenic tobacco lines BpChiI-7,BpChiI-19,BpChiI-21,BpChiI-22,BpChiI-25,BpChiI-44,BpChiI-51and BpChiI-69 were 27.50,30.00,31.39,28.76,34.17,25.00,28.89 and 21.40,respectively.The phenotype of inoculated plants showed that the number of wild-type tobacco with severe symptom was much more than that of BpChiI transgenic tobacco.And most leaves of WT tobacco showed the severe symptom of chlorosis or necrosis.However,only part leaves of BpChiI transgenic plant showed symptom of chlorosis or necrosis.This indicated that BpChiI could reduce the symptoms of tobacco and improve its resistance to Verticillium wilt.(3)Transient expression of BpChiI increased the resistance of cotton to Verticillium wiltTransient expression of the anti-fungal protein BpChiI in cotyledons of cotton accompanying with inoculated with the strain V991(1×10~7 spores/m L).5 days after Agroinfiltration,the disease index of the control injected with GV3101 was 73.17,however,that of the cotyledons with transient expression of BpChiI was 40.86,which decreased by 44.16%.Result indicated that BpChiI could improve the resistance of cotton to Verticillium Wilt.4.Influence of BpChiI on defense signaling pathways such as SA and JA in Arabidopsis during V.dahlaie infectionThe expression pattern of marker genes of SA,JA and NO signaling pathways were analyzed by q RT-PCR after inoculation with V.dahliae.Results showed that the expression pattern of SA,JA and NO marker genes PR1,PDF1.2 and NOS1 in transgenic Arabidopsis was similiar as that of WT control after inoculation with V991.This indicated that BpChiI did not affect the plant defense signals during V.dahliae infection.This study proved that the anti-fungal protein BpChiI could inhibit the germination of A.brassicae spores and effectively improve plant resistance to Verticillium wilt.And we provided a new gene for genetic engineering of plant disease resistance. |
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