Screening And T-DNA Flanking Sequence Analysis Of Arabidopsis Thaliana Mutants Against Phytophthora Infestans

Arabidopsis thaliana is a plant of Crucifereae. The Arabidopsis Genome is 125Mb,having five chromosomes, which had been sequenced in 2000.Several Phytophthora infestans-susceptible mutants had been obtained based onthe different reactions of Arabidopsis activated tagging mutants after being inoculatedby the fungus, and flanking sequence of Arabidopsis T-DNA insertion had also beenobtained by TAIL-PCR technology and genomic DNA from disease-susceptible mutantsas template. Some disease resistant-related genes had been found using NCBI sequencehomology analysis.Based on the 258 Arabidopsis activated tagging mutants being inoculated by thefive Phytophthora infestans different isolates we found that AtTM31, AtTM34, AtTM40,AtTM113, AtTM246 were higher susceptibility to isolate 17-B-1 and AtTM114 weremediate susceptibility to isolate 17-B-1 and AtTM32, AtTM144, AtTM243, AtTM244were lower susceptibility to isolate 17-B-1; AtTM32, AtTM34, AtTM40, AtTM113were higher susceptibility to isolate W040604 and AtTM31, AtTM244 were mediatesusceptibility to isolate W040604 and AtTM114, AtTM144, AtTM243, AtTM246 werelower susceptibility to isolate W040604; AtTM31 were higher susceptibility to isolateW041002 and AtTM 32, AtTM34, AtTM40, AtTM113, AtTM243, AtTM246 weremediate susceptibility to isolate W041002 and AtTM114, AtTM144, AtTM244 werelower susceptibility to isolate W041002; AtTM32, AtTM40 were higher susceptibilityto isolate W041904 and AtTM34, AtTM144, AtTM243, AtTM246 were mediatesusceptibility to isolate W041904 and AtTM31, AtTM113, AtTM114, AtTM243werelower susceptibility to isolate W041904; AtTM31, AtTM32, AtTM40, AtTM246 werehigher susceptibility to isolate W041406 and AtTM34, AtTM114, AtTM243, AtTM244were mediate susceptibility to isolate W041406 and AtTM113, AtTM144 were lowersusceptibility to isolate W041406.Thermal asymmetric interlaced PCR (TAIL-PCR) was efficiently used to amplifythe T-DNA flanking regions. Arbitrary primers AD1, AD2, AD4 and specific primers(Lex2, Lex4 and Lex5),(Lex3, Lex4 and Lex5) were the most suitable combination.Among 10 specific tertiary TAIL-PCR products sequenced, 2 mutants were found with T-DNA insertion within gene regions and 2 with T-DNA insertion in the region betweentwo genes and 5 with vector sequences.In AtTM 34, T-DNA was inserted at 133bp in chromosomeâ…¢and was in the exonof gene AT3G13620.1 which was Amino acid permease family protein. In AtTM 246,T-DNA was inserted at 2672bp in chromosome V and was in the fifth exon of geneAT5G24830.1. The function was pentatricopeptide repeat containing protein.Southern blotting analysis showed that AtTM 34 have one copy of T-DNAinsertion and AtTM 246 have two copies of T-DNA insertions.