| Verrticillium dahliae,is a kind of soil-borne fungus,has wide host range.Cotton Verticillium wilt caused by V.dahliae is one of the destructive vascular disease in cotton,and also is one main barrier of producing high quality cotton in China,causing great damage on the economy;Cotton Verticillium wilt is extremely difficult to control,so known as cotton ’cancer’.At present,pathogenic mechanism of V.dahliae is yet unclear.And ideal methods of disease control have not been developed.A new way and thought to prevent and control cotton Verticillium wilt effectively is that screening and identifying the possible pathogenic genes in V.dahliae genome from molecular level to well understand the pathogenic mechanism.Endoglucanase belongs to cell wall-degrading enzymes in plants,which is important for the cellulose degradation.Plant pathogenic fungi,including V.dahliae,produce a range of cellulases to facilitate host infection and colonization.And,the higher endoglucanase activity in V.dahliae was related with aggressiveness.Thus,the predicted endoglucanase genes in V.dahliae plays an important role in plant penetration or in early stages of colonization,may be pathogenic factors of cotton diseases.The study analyzed function of endoglucanase genes in pathogenicity of V.dahliae by RNAi technology.We made 9 endoglucanase genes from GHFs 5,12 silenced simultaneously and then gained the mutants of V.dahliae by RNAi technology.After that,through RT-PCR,colonial morphology,growth rate,the spore production capacity and infection analysis on wild type and mutant strains to study function of endoglucanase genes in pathogenicity of V.dahliae,provided a reference for verifying if endoglucanase gene is essential for virulence of V.dahliae,and made for understanding the pathogenic mechanism of V.dahliae well,then provided a new way and basis for preventing and controlling cotton Verticillium wilt.Main results for this study listed as follows:1.Using bio informatics methods,9 cDN A sequences of glycoside hydrolase family 5 and 12 endoglucanase genes were searched from V.dahliae genome database.And sequence alignment analysis was done to contribute to the selection of non-homologous fragments of 40 bp,and 9 sequences of 40 bp formed the target gene VdF5012 of 360 bp that was synthesized.2.An efficient gene silencing system suit for V dahliae was constructed as follows.(1)Getting fusion fragment containing eGFP gene with overlap PCR,and then the binary vector rh-pPK2 containing eGFP fragment was constructed;(2)Hairpin structure(intron-hairpin RNA,ihpRNA)silencing vector and reverse dual promoter silencing vector containing target gene were constructed;(3)Knock-down transformants of V.dahliae were obtained by using ATMT method.(4)The screening system for gene knock-down transformants of V.dahliae was been optimized by PCR testing of eGFP gene and microscopic observation ofgreen fluorescence signal intensity.3.The characteristics of wildtype,mutant strains and parent strain expressing eGFP protein were analyzed and compared.(1)Semi-quantitative RT-PCR of eGFP and 9 target genes were analyzed,in which 18S in fungi was the internal control.Result was that the expression level of 9 target genes in parent strain was lower than wildtype,silencing efficiency of target genes in mutant strains KD A5 and KD B3 was higher than KD_B7,in which target genes were hardly silenced;(2)Conidiospore suspension was adjusted to 107 conidia mL-1,and then inoculated 5 μL on the center of PDA plate.Colony diameter change recorded everyday showed that the gene knockdown strains grew slower than wildtype,parent strain grew similarly to wildtype;(3)Inoculating 200 μL conidiospore suspension with concentration of 107 conidia mL-1 into 25 mL PDA,and cultivated(25℃3 200 rpm)for 2 week,the spores amount was recorded weekly.The final concentration of spores indicated that spore production capacity of knockdown mutants relatively reduced compared with wild type and parent V.dahliae;(4)Cotton seedlings were cultivated with water culture,and then,the roots suspended in the conidia with concentration of 107 conidia mL-1 for 40 min,observed the pathogenic condition.It turned out that the disease of cotton seedlings inoculated with mutants was weaken than that inoculated with wildtype or parent strain,and that disease index of cotton seedlings inoculated with KD_A5 and KD_B3 was lower,Accordingly,silencing of endoglucanase genes affected the pathogenicity of V.dahliae on cotton host. |
