| Coniothyrium minitans, an important sclerotial mycoparasite of Sclerotinia sclerotiorum with worldwide distribution, has great potential for biological control of plant diseases caused by S. sclerotiorum and other species in the genus of Sclerotinia. In this paper, the conditions for transformation of C. minitans with Agrobacterium tumefaciens have been primarily studied, and the quality of the insertial transformation library was evalauted.The vector (pTFCM) used for transformation of C. minitans was builded up mainly based on pCAMBIA1301 that carries CaMV 35S promoter and hygromycin-resistance gene. To make the pTFCM vector, the CaMV35S promoter was replaced with trpC promoter derived from Aspergillus nidulans by double digestion with restiction enzymes. The modified vector pTFCM was used to do transformation work of C. minitans. It proved that the pTFCM works well on tansformation of C. minitans.After co-incubated with the conidia of C. minitans and cells of A. tumefaciens on the cellophane membrane touched on the Co-IM plate for 2 days, the membrane was removed into new sterilized Petri dish and covered with PDA containing 60 ug/mL of hygromycin and 750 |ag/mL of cephalosporin, and incubated at 20癈 for 3-5days. Colonies emerging from the selective medium were transferred into fresh PDA plate containing 60 |j.g/mL hygromycin only and looked as candidates of transformant. To identify the transformation, genomic DNA from twenty randomly selected candidates were used to conduct PCR with primers designed from T-DNA of vector pTFCM, all tested candidates cantianed the expected DNA fragment (1165bp). Based on the proof, about 10000 transformants have been obtained so far.The conditions for transformation were studied mainly on conidia sources, conidia amount used. The amount of conidia used might affect the transformation efficiencysignificantly, when conidia concentration was 108spores/mL, transformants obtained was much higher than those of 109 spore/mL and 107spore/mL. More transformants were obtained when conidia from 17-day cultured PDA slant used,.At this condition, 50 transformants per dish was achieved.To examine the stablility of transformants, ten transformants were randomly selected to check the hygromycin-resistance stability for 6 asexual generations on PDA containing 60ug/mL hygromycin B and the result showed that all tested transformants could grow on hygromycin B containing PDA plate and produce conidia.To evaluate the copy number of T-DNA in transformants, 58 transformants randomly selected were used to do genomic Southern blot analysis,.48 transformants were proved to be single-copy insertion, 4 transformants were double-copied, the percent of single-copy insertion transformants is about 82.3%.Twenty transformants were randomly selected from the 10000 transformants to check some visual phenotypes including colony morphology, growth rate, sporulation and pathogenicity on S. sclerotiorum. Results showed that no significant changes of the twenty transformants compared to that of original strian ZS-1, but the growth rate is slower than that of ZS-1. However, some insertial mutants, such as mutants lost the ability to produce pycnidium, mutants changed the black pigment, and growth stunt mutant, could be seen from the transformation library. None mutant that lost the pathogenicity to the sclerotium of S. sclerotiorum have been found out.TAIL-PCR was successfully used to amplify the T-DNA flank genomic DNA segments. The subsequent works (sequence analysis) done by colleagues showed that many of the flank sequences have been obtained and the associated genes been determined. |
