ISOLATION AND CHARACTERIZATION OF A PROMOTER FROM RICE FALSE SMUT FUNGUS USTILAGINOIDEA VIRENS

J.K. Hu, Z.Y. Chen, CH.H. Gu, M.N. Yu, J.J. Yu, Y.F. Nie, L. Huang, J.Q. Qiao, X. Huang, J. He, Y.F. Liu

Abstract


A promoter was cloned from Ustilaginoidea virens, a rice false smut fungus. A U. virens mutant library was constructed using a promoter-trapping vector harboring the enhanced green fluorescent protein (egfp) as reporter. Six transformants emitting green fluorescence were screened from 6,500 transformants. One transformant, A880-1, which grew slower and generated more conidia, was chosen for further studies. The insertion of T-DNA in A880-1 was validated by PCR, and the copy number was defined by Southern blot analysis. High-efficiency thermal asymmetric interlaced PCR (hi-TAIL PCR) yielded a 2,038 bp long sequence cloned from genome DNA of A880-1, this sequence being upstream of egfp in T-DNA. The 1,045 bp long upstream region of egfp was used for analysis. This sequence consisted of a CAAT-box, a TATA-box, three TATA-box-like structures, and four other potential eukaryotic regulator elements. This promoter, designated as puv880 (GenBank accession No. KC355189), was the first promoter isolated from U. virens. Four different constructs carrying different 5’ deletions of this promoter, along with the downstream egfp sequence, were obtained and transformed into Magnaporthe oryzae strain Guy11 through PEG-mediated transformation to identify the smallest functional promoter region of puv880. All constructs exhibited promoter activity, and the smallest deletion was no more than 444 bp in size. Compared with the wild-type strain 70-22, the growth of A880-1 was slower and produced more conidia at the terminal of hyphae on minimal medium. Studying puv880 can facilitate the understanding of the conidiation and mycelium growth of U. virens.

Keywords


Ustilaginoidea virens;enhanced green fluorescent protein promoter;5’ deletion function analysis;cloning;PCR

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DOI: http://dx.doi.org/10.4454/JPP.V95I3.039

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