Establishing REMI Transformation System And Primary Analysis Of Molecular Karyotype Of Exserohilum Turcicum

Firstly, wild-type isolate strain 01-23 of Exserohilum turcicum as material, a series of process of preparing the protoplasts were optimized. Based on that, we established a system of restrictive enzyme mediated integration (REMI) transformation and a system of analysis of molecular karyotype.The optimized conditions preparing protoplast of E. turcicum included that: (1) PDA medium containing 1%glucose is used to produce conidiophore; (2)Before enzyme-digested, the material is disposed by vortex; (3) The enzyme solution contains 1.25%Lyallzyme, 1.25% Drislase and 1.25% Snailase, and the material is digested for 4 hours. In this way, the output of protoplasts increased impactfully which reach. At the aspect of purification, centrifuging 10min at the speed of 3000g could decrease the loss of protoplasts mostly. And the best way to filtrate protoplasts is that: the protoplasts was filtrated with one layer of lens wiping paper, followed by 50μM nylon membrane. For regeneration of protoplasts, it is the best approach to make them incubate in RPD for a night long and transfer to RPDA the next day. In addition, the method utilizing suspended liquor of mycelia to prepare protoplasts. was advanced. Using this method, about 2×10⁶～5×10⁶ protoplasts were got per mL.It is the first time for us to succeed ransformanting E. turcicum and establishing REMI transformating system, which include:(1) Collect and purify the protoplasts and put 10⁶ protoplastes into 100μL STC buffer; (2)Add in 2μg linear plasmid PAN7-1 and 20U Hindâ…¢per 10⁶ protoplastes; (3)Before and after adding in PTC(PEG6000), the STC buffer is placed on ice for 20 minutes equally; (4) Transformed protoplasts are firstly incubated in RPD overnight and then transferred to RPDA medium. After 3 days, the RPDA is covered with 10mL PDA containing hygromycin B; (5)The inhibited concentration of hygromycin B is 60μg/mL. According to the scheme mentioned above, 225 transformers were got in all. The biology characteristic of these transformants were compared with that of wild-type isolate 01-23. The compared contents were colonial morphology, hypha growth weight, conidial modality, sporiferous ability and pathogenicity with wild-type. In result, it was found that (1) The colonial morphology of M-55, M-8 and M-11 were distinctly different from the wild-type isolate; (2)The hypha growth of M-14 was increased obviously and M-11 was decreased clearly; (3) M-25 did not produce conidiophore and the pathogenicity was lost completely; (4) Compared with wild-type, the sporiferous ability of M-36 and M-42 was depressed, while M-59 and M-68 increased.We compared the efficiency of three different ways to prepare intact chromosome. They are agarose-embedding protoplasts, grose-embedding conidiophores, liquid nitrogen freeze-gringd. As a result, we found that the agarose-embedding protoplasts was the best one, and got there chromosomes from mating-type-A F₁42 and two chromosomes from mating-type-a F₁42 in the way. And that indicated that the two mating types chromosomes differ.