Screening Of The Tn5 Transformants Of Xanthomonas Axonopodis Pv.Manihotis And Cloning Asl_Xam Gene

Cassava bacterial blight is a worldwide disease caused by Xanthomonas axonopodis pv.Manihotis.This disease has become the most serious disease in domestic in planting area of cassava which also has been one of the factors that limiting the sustainable and healthy development of cassava industry in our country.At present,there are few research reports on the pathogenic mechanism of cassava bacterial blight.This research use GR4 which is a susceptible cassava germplasm to be the plant materials for transformants screening.After the first-round and the second-round screening,61 pathogenicity-decreased tansformants and 54 pathogenicity-loss transformants were screened out from 10510 Tn5 transformants by leaf-clipping.By further analysis,all of the pathogenicity-decreased and pathogenicity-loss transformants are Tn5 transformants,and most of them are single copy insertion.According to the physiological and biochemical characteristics analysis,extracellular protease,cellulase and amylase activity of three transformants(Xtn102-01,Xtn 199-5 7 and Xtn142-67)are decreased and the decreasing trends are the same.According to the T-DNA insertion mode analysis of 29 pathogenicity-decreased or pathogenicity-loss transformants,Tn5 transposon preferred to insert in AT-rich region in Xam-GX11 9bp direct repeat sequences would be formed after the insertion,and the probability of guanine at the head base of 9bp sequences and cytosine at the tail base are high;Tn5 transposon preferred to insert in the coding sequence of gene and there were no preferences for direction.10 genes from pathogenicity-decreased or pathogenicity-loss transformants which were inserted by T-DNA were cloned.The non-polar knock-out mutants of alaTXam,aslXam,clsXam,kdpAXam,traFXam,xam7341 and xam5926 genes were constructed.In these mutants,the growth-rate of the knock-out mutants of Xam7341 and Xam5926 genes slowed down significantly;the pathogenicity of aslXam knock-out mutant was loss completely only.The Aslxam complement strain(GX11?Asl+Asl)of knock-out mutant(GX11?Asl)was constructed,and the pathogenicity of GX11AAsl+Asl restored.But compared with wild type strain GX11,the pathogenicity of GX11?Asl+Asl was a little weaker.According to the biological characteristics analysis,the extracellular enzyme activity,growth rate and motility of GX11,GX11AAsl and GX11AAs1+AAsl has no significant difference.The extracellular polysaccharide secretion of GX11 ? Asl decreased significantly.