Transformants Library Construction And Analysis Of Xanthomonas Axonopodin Pv.Manihotif Of Cassava

Cassava, yams (Dioscorea spp.) and sweet potatoes (Ipomoea batatas) are important sources of food in the tropics. In China Cassava is a important tropical economic crops. Cassava bacterial blight caused by Xanthomonas axonopodis pv. manihotis (short for Xam) is one of the most important diseases distributed at cassava-growing areas worldwide, cassava bacterial blight can cause up to12-100%crop loss. The important cassava-growing areas in our country have been surveyed by our reseach group.The results showed that cassava bacterial blight was the most destructive disease in cassava plantations, and it has become a potential threat to the development of cassava industry. However, molecular pathogenesis of the pathogen is little known, which deterred the research and development of the prevention and control technology. Based on our previous study, a strong virulent strain Xam-GX11was screened, its genome was sequenced. Besides the transformants library of Xam-GX1I has been generated, with hopes these can benefit for screening and cloning pathogenic genes.The extracted Xam-GX11genomic DNA was high quality with the concentration of1863.4ng/μL, and the OD280/260ratio was1.84. It’s draft genomes was sequenced by Illumina Solexa platform and assembled via de novo. The sizes of genome was5.4Mb, containing115scaffold,4764genes. The average length of these genes was917nt.A transformation method for Xam-GX11was initially formed using a Tn5-based transposome and electroporation.The thalli were collected for preparing competent cells when it’s OD600was o.4, then aliquots of competent cells weere mixed with0.5μL (lOng) transposome.Under the electroporation conditions of1.8KV voltage,25μF and200Ω, the best effect of transposition was observed. The efficiency was5.66±0.38clones/107cfu, in our experiment,20382transposon insertion mutants were obtained now.45randomly selected Xam-GX11transformants were transferred to fresh YPG plates without kanamycin and incubated at28℃. The bacterial cultures were re-inoculated20times onto fresh YPG plates every2-3days. Finally, after20successive subcultures, all transformants retained the kanamycin resistance, suggesting that the transposon inserted in the transformants was stable.15Xam-GX11transformants were randomly selected to extract genomic DNA. The transformants were analyzed by polymerase chain reaction (PCR) using primers TMK1and TMK2complementary to the sequence of the transposon. Every transformant obtained 0.5Kb of amplification products, but Xam-GX11did not. The polymorphic fragments of Xtn21-05, Xtn21-68and Xtn34-4were selected to clone and sequence, suggesting that they were completely consistent with kanamycin resistant gene coding sequence.10randomly selected transformants of Xam-GX11were analyzed by Southern Blot hybridization. Genomic DNAs were digested with EcoR I and hybridized with the probe derived from the transposon. EcoR I has no target site within the probe. The result showed that single copy insertion mutants was50%.The leaf clipping methods was choosen for large scale screening mutants defected in pathogenicity of Xam-GXll according our experimental results by comparingthe leaf piercing and spraying inoculation method. The leaf clipping methods was the most applicable method for large scale of pathogenic screening experiment. The healthy and free from disease, fully expanded living plant leaves of cassava cultivars SC9were used as inoculation material for mutant screening experiment. By the first and the second-round screening,94disease-related transformants were screened out from9872transformants.15transformants were randomly selected, and analyzed by PCR, about0.5kb bant was amplified from the DNA of all selected transformant, indicating that Tn5has been inserted into the the genome of wild bacteria(Xam-GX11). By southern blot analysis, we found that11mutants were single copy insertion,4mutants were double or more copies insertion.15genomic sequence flanking tag were obtained by Tail-PCR. The PCR products were sequenced and searched agains the genomic sequence of Xam-GXll by Basic Local Blast Alignment Search tool, then analyzed by Blast search in GenBank. Result showed that10mutants losed pathogenicity because of exogenous gene fragment insertion. Among them the exogenous gene fragment inserted into the gene encoding aspC in mutant (Xtn57-56), which is a disease-causing gene. Two fragments were inserted in between two predicted gene, the other three have not matched with any genomic sequence of f Xam-GX11.