Isolation And Functional Analysis Of The Flanking Gene Of T-DNA Insertional Ustilaginoidea Virens Mutants A880-1and B1015

In this study, mutant library of Ustilaginoidea virens was constructed using Agrobacterium tumefaciens-mediated transformation (AtMT) and promoter trapping technology, and more than9800T-DNA insertional mutants were obtained. More than5600mutants were in A mutant library using wild-type70-22and more than4200mutants in B mutant library using wild-type P1.Ten mutants, with T-DNA inserted in the downstream region of the promoter of U. virens, were screened through fluorescence microscopy. Six mutants, named as A880, A2000, A2331, A4829, A5248and A5297were form A mutant library and four mutants, named as B111, B367, B913and B4865were from B mutant library. In analysis of growth rate for these ten mutants, A880-1, one purified mutant of A880-1, grew slower in potato sucrose agar (PSA), TB3and minimal mediums (MM) contrasting to wild-type70-22. In MM, malformed hyphae were found and numerous conidia were produced at the terminal of these hyphae, so mutant A880-1was used for further research. The hypha and spores of A880-1could emit green fluorescence under fluorescent microscopes. The insertion of the T-DNA in genome of A880-1was validated by polymerase chain reaction (PCR). The single copy number of T-DNA in A880-1was defined by Southern blot. A2038base pair (bp) long promoter sequence of A880-1and upstream of gfp in T-DNA was cloned using High-efficiency thermal asymmetric interlaced PCR (hi-TAIL PCR) and this promoter was named as puvS80. None homologous sequence was found after puv880being transformed into NCBI. Sequence analysis revealed that puv880contained TATA-box, CAAT-box and some other potential eukaryotic regulators elements. Three TATA-box-like structures and one reverse complementary sequence of TATA-box were also found in puv880. Basing on5’deletion analysis, four fragments of puv880, which were444bp,650bp,864bp and1045bp, were amplified together with T-DNA in genome of A880-1. Then these four fragments were transformed into genome of Magnaporthe oryzae wild-type Guy11respectively through PEG-mediated transformation and all fragments exhibited promoter activity. This result validated the promoter activity of puv880and the core promoter sequence of puv880was no longer than444bp. Combination with the results of growth rate, puv880might be relevant to grow and conidiation of U. virens under unfavorable conditions, and would a trail for explain the molecule mechanism on conidiation.A mutant of U. virens B1015lost pathogenicity was screened through pathogenicity analysis. B1015grew slower obviously in PSA, TB3and MM than wild-type P1. In potato sucrose liquid medium (PS), B1015almost didn’t grow, and none spore was found either. These results show that the expression of gene related to grow and sporulation was affected by the insertion of T-DNA.The insertion of the T-DNA in B1015was validated by PCR and the single copy number was defined by Southern blot. Two flanking sequence of T-DNA, which were247bp long (U.vRB) and309bp long (U.vLB), were amplification by hi-TAIL PCR. The sequence of gfp and most of its terminator of T-DNA were lost after amplifying using genome DNA of B1015as template DNA and primers, F1and R1, which were designed basing on U.vRB and U.vLB. This result suggested that some sequence of T-DNA was lost with the insertion of the T-DNA.