| Cotton is an important cash crop and fiber crop as well as a potential grain,feed and oil crop.Cotton Verticillium wilt is a soil-borne vascular bundle disease caused by Verticillium dahlium,which is one of the most important diseases to harm cotton and is known as " cotton cancer ".At present,there are few researches on the pathogenic mechanism of cotton verticillium wilt caused by Verticillium dahlium.Therefore,it is of great significance to explore the pathogenic mechanism of mi RNAs in Verticillium dahlium for breeding new varieties resistant to Verticillium dahlium.In this paper,we studied the pathogenic mechanism of Verticillium wilt in cotton through the transboundary regulation of mi RNAs from Verticillium dahlium.The main research results are as follows:1、 By using RNA sequencing technology,the mi RNAs of 24 Verticillium dahlium bacteria were identified by sequencing and bioinformatics analysis.The expression of mi R24 was detected in cotton infected with Verticillium dahlium by q PCR technique,and the precursor of mi R24 was cloned from the genome of Verticillium dahlium.2、 The target genes of mi R24 were not predicted in the genome of Verticillium dahliae,46 predicted target genes in cotton.Through the GO enrichment,it was found that the function of the target genes are mainly concentrated in cell parts and cell in the cell component,and molecular function.Biological information analysis and preliminary experimental results show that mi R24 candidate target genes is a related to disease resistance of coding serine/threonine protein kinase gene Gh Stk10;The expression patterns of mi R24 and Gh Stk10 in cotton were opposite at 0,6,12,24 and 48 h after Verticillium dahlium colonization by q PCR.3、 In order to verify the targeting relationship between mi R24 and the gene Gh Stk10,q PCR and GUS staining were combined.GUS staining and relative expression levels of mi R24 and Gh Stk10 genes were detected in leaves of tobacco plants with transient transformation of GUS,mi R24 precursors and Gh Stk10 gene overexpression vectors.Histochemical staining of tobacco leaves transformed with the GUS and Gh Stk10 gene overexpression vectors respectively showed blue,while the histochemical staining of tobacco leaves transformed with the mi R24 precursor overexpression vector had no color.The blue color of tobacco leaves was lighter after co-transformation of mi R24 precursor and Gh Stk10 gene overexpression vector.The expression levels of mi R24 and Gh Stk10 transcripts were significantly increased in tobacco leaves transformed with mi R24 precursor and Gh Stk10 overexpression vector respectively.However,the expression levels of mi R24 were significantly increased in tobacco leaves cotransformed with mi R24 precursor and Gh Stk10 overexpression vector,while the expression levels of Gh Stk10 were significantly decreased.The results proved the targeting relationship between mi R24 and Gh Stk10 gene.4、 Transient transformation technology was used to transform GUS,mi R24 precursor and Gh Stk10 gene overexpression vector in cotton cotyledon.The q PCR results showed that the expression of mi R24 and Gh Stk10 gene was significantly increased,and the expression of Gh Stk10 gene was decreased in cotton cotyledon transformed with mi R24 precursor.The cotyledon of transiently expressing Gh Stk10 inoculated with trypan blue lighter color,higher CAT and SOD content,lower fungi biomass,and transiently overexpressed Shannong SF06 plants were more resistant to the disease.However,the cotyledon with transient expression of mi R24 precursor inoculated,trypan blue was deeper,the contents of CAT and SOD were lower,and the biomass of fungi was higher,and the transient overexpression variety Shannong SF06 was less resistant to disease.The interaction between mi R24 and target gene Gh Stk10 in the interaction between cotton and Verticillium wilt was further verified.5、 Using VIGS technique to reduce the expression of Gh Stk10 gene in the upland cotton variety Shannong SF06,and it was found that the phenotypes of the control and the silent plants were significantly different at 15 and 20 days of infection.Compared with the TRV:00 plants,the silenced Gh Stk10 plants showed obvious wilting,yellow,necrosis,higher disease index,darker stem vertical section color and more colonized fungi;After inoculated leaves,trypan blue has a darker color,the contents of CAT and SOD were lower,and the biomass of fungi was higher,indicating that the effective silencing variety Shannong SF06 plants was more susceptible to the disease. |
