Functional Analysis Of Clathrin Light Protein TaCLC1 Involved In The Regulation Of The Resistance For Wheat To Fusarium Head Blight

Fusarium head blight,as a worldwide wheat disease,is a wheat fungal disease caused by Fusarium graminearum,this brings a large loss to wheat production worldwide each year.In addition to yield loss,which affects the quality and commercial value of wheat.In severe cases,it can also poison people and livestock and even takes serious harm to humans and live stock.Clathrin-mediated endocytosis is the main mode of intracellular and extracellular signal transduction and material transport,and plays an important role in the growth and development of organisms.In recent years,the research on plant clathrin has gradually increased,and it has been found that plant clathrin can regulate membrane transport,salt stress,nodule formation,auxin signal,and hypocotyl formation,but plant clathrin is resistant to fungal diseases.There is less research on the aspect and the specific function is not clear.In this study,the susceptible material Jimai 22(JM22)and the disease-resistant material Jimai 22-Fhb7(JM22-Fhb7)were inoculated with Fusarium graminearwm(F.g)and analyzed by transcriptome sequencing and found that the transcriptional level of TaCLC1 in JM22-Fhb7 was significantly higher than that of JM22.Thus the further research was conducted on the function of TaCLC1 in regulating wheat resistance to FHB.The main results and conclusions are as follows:(1)We isolated a clathrin light chain gene from wheat,named TaCLC1(GenBank ID: KT345966).The TaCLC1 contains a 936 bp open reading frame(ORF),and encodes a 311 amino acids polypeptide of molecular weight 32.9 kDa.TaCLC1 is a single copy gene,located in 7BL chromosome.And TaCLC1 was high homology with the light chain proteins of barley,corn and sorghum.The TaCLC1::GFP fusion protein was transiently expressed in tobacco epidermal cells,and the fusion protein was localized in the cytoplasm using a laser confocal microscope.(2)The results of qRT-PCR showed that TaCLC1 was expressed in roots,stems,leaves and panicles,and the expression level was highest in leaves.At the same time,the transcription of this gene was induced by various hormones such as SA,MeJA and F.g.(3)The results of gene silencing induced by Barely stripe mosic virus showed that the area of spots in BSMV::TaCLC1 plants was significantly higher than that of in BSMV::00.The relative number of F.g in BSMV::TaCLC1 plants is also more than that in BSMV::00.That TaCLC1 can inhibit the growth of F.g in wheat.(4)TaCLC1 overexpression lines for tobacco and arabidopsis were obtained using transgenic technology.F.g was inoculated into wild type Arabidopsis thaliana and Arabidopsis thaliana of overexpressing TaCLC1 gene.After 5 days,the incidence of each strain was counted.It was found that the leaves of wild type Arabidopsis thaliana leaf lesion area less than 25%.The number accounted for 18% of the total,75% of the leaves with lesions less than 25% on the leaves of transgenic Arabidopsis,and 25% more than 75% of the leaves in the transgenic lines;also found in wild-type Arabidopsis thaliana.The relative number of F.g is about 2 times that of the transgenic strain.F.g was inoculated on leaves of wild-type tobacco and transgenic tobacco,and it was found that there were significant lesions on the leaves of wild-type tobacco,while there were only slight lesions on the leaves of transgenic tobacco,and the pathogen was in the leaves of transgenic tobacco.The amount of mycelial growth was significantly less than that of wild type tobacco.TaCLC1 plays an important role in regulating wheat resistance to FHB.(5)The expression levels of TaPAL and TaOPR,the key genes for the synthesis of SA and JA in wheat plants with the TaCLC1 gene down-regulated,were detected.The level of expression of these genes was less than that of the wild type after infected with F.g.The transcription levels of disease-associated protein genes in transgenic tobacco and Arabidopsis were detected.It was found that the transcription levels of PR1,PR2,PR3,and PR5 in TaCLC1 overexpressing plants were significantly higher than those in WT.The above results indicate that TaCLC1 may resist pathogen infection by promoting the expression of disease-associated protein genes.