Functional Analysis Of Conidiation Associated Gene Cm1107in Coniothyrium Minitans

Coniothyrium minitans is an important biocontrol agent of rapeseed stem rot caused by Sclerotinia sclerotiorum, which is distributed worldwide. A T-DNA insertional library with30500mutants, of which ZS-1T2029is a mutant deficient in conidiation, was constructed in our lab. Some ESTs whose expressions were up regulated was ditected through suppression subtractive hybridization (SSH). In this research, Cm1107, related with conidiation of C. minitans, was preliminarily studied through reversed genetic methods on the basis of prior research. The results were as followed:By analyzing the genome sequence of C. minitans with bioinformatic methods, it revealed Cm1107gene, a1395bp fragment with two exons and one intron (64bp), had a single copy in the genome. Sequence analysis through blastx showed Cm1107had high identity with basic Helix-loop-helix (bHLH) transcription factor, what’s more,3’end of Cm1107contained Helix-loop-helix (HLH) domain (cd00083). The bHLH transcription factor superfamily contains a series of proteins which are involved in processes related with growth and has a mutual motif, the basic region and HLH domain. Some researches had proved several bHLH transcription factors were correlated with fungal sexual and asexual sporulation growth.The expression of Cm1107was detected with RT-PCR, and the result showed Cm1107gene expresses during all the development stages of C. minitans, and the expression at spore germination period reaches its peak.Gene knock-out vector was constructed according to the homologous recombination principle, and was transformed into spores of C. minitans with Agrobactirium tumfaciens mediated transformation (ATMT). Three positive transformants (KO1107) were obtained. When cultivated in potato dextrose agar (PDA), the growth rate and mycelial morphology of KO1107did not change remarkably compared with that of wild type ZS-1, while spores of KO1107were bigger than those of wild type. Percent of abnormal spores of KO1107were over16%, but in wild type abnormal ones were less than2%. Statistic results revealed spores washed from the surface of the colony of KO1107declined remarkably compared with ZS-1, however, the number of spores of internal mycelial colony of both KO1107and ZS-1did not have remarkable differences. When KO1107and ZS-1were dual cultured with S. sclerotiorum1980, black pycnidia of ZS-1were generated over the contact region of ZS-1and1980and on the surface of1980colony, but pycnidia of KO1107were even not generated at the contact region of KO1107and1980. In the experiment of C. minitans parasitizing sclerotia of S. sclerotiorum Ep-1PNA367, it revealed lots of pycnidia of ZS-1were generated on the surface of sclerotia of Ep-1PNA367, while little conidia and high mounts of white mycelia of KO1107were generated on the surface of Ep-1PNA367, moreover, the rot rate of parasitical ability on sclerotia of KO1107declined remarkably compared with ZS-1. Over-expression vecotor was constructed through cloning Cm1107gene, and was transformed into spores of C. minitans with ATMT. Five over-expression transformants (OE1107) were obtained, and further researches were continued.Research results above preliminarily revealed Cm1107gene functioned on the spore growth of C. minitans. A new gene related with conidiation of C. minitans was proposed in this paper, furthermore, new discoveries about signaling passways of conidiation of C. minitans might be found.