Screening Of Alkali-tolerance Deficient Mutants From A T-DNA Insertional Library Of Coniothyrium Minitans And Cloning Of Alkali-associated Genes

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 Sclerotinia spp. C.minitans ZS-1 could growth under pH2 to pH 12, which indicates that ZS-1 has alkali-tolerance associated genes. Our work is to screen alkali-tolerance deficient mutants from a T-DNA insertional library of C. minitans and clone alkali-associated genes and accumulate gene material for alkali-tolerance crop. Four alkali-tolerance deficient mutants were obtained from the insertional library of strain ZS-1 constructed ago and their biological characteristics were studied. The DNA fragments flanked the T-DNA in these 4 mutants were amplified by Tail-PCR, and one fragment was cloned and identifided.In this study, 4 mutants, ZS-1T2613,ZS-1T2979,ZS-1T3446 and ZS-1T20266, were screened from 4300 insertional mutants, and the frequency of insertional mutation is 9.3×10-4; ZS-1T3446 could not grow at a pH value of 8.0, the other three could grow much slower at pH 8.0, but stop growing at pH 9.0. Comparing with ZS-1, four mutants show significant difference in the growth rate and production of antibacterial substance. ZS-1T2979 failed to sporulate on both normal and alkalic PDA medium and it's colony is white, while the other three did not show any significant difference from the original strain ZS-1. Our results showed that T-DNA inserted in a phenotypic control gene. 4 mutants were amplified by Tail-PCR. The results showed that aim fragment was amplified only from ZS-1T2979 when LB and AD were used together. The difference between the second and the third amplifed fragment is 150bp (The between L3 and L2) which showed that the amplified fragment maybe the aim fragment.