【作者】 马继芳；

【导师】 董金皋；

【作者基本信息】 河北农业大学 ， 植物病理学， 2003， 硕士

【摘要】 本文以01-12、01-15为亲本菌株进行有性杂交，共分离得到79个单子囊孢子后代。将79个后代菌株分别与亲本菌株01-12和01-15回交进行交配型测定，与亲本菌株01-12同属一个交配型的后代菌株有36个，与亲本菌株01-15同属一个交配型的后代菌株有20个，分离比例明显偏离1:1，表明交配型性状可能由多对基因控制。此外有3个后代菌株为两性菌株，20个为中性菌株。 试验发现，新分离菌株的交配能力明显高于经继代培养3年以上的菌株，复壮后再分离的继代菌株其交配能力有所增强。此外，将基本Sach培养基中的FeCl₃定量为每1000mL水中加入0.02mg后，可促进子囊孢子的成熟，使子囊壳的产生趋于稳定。本试验针对玉米大斑病菌子囊孢子的情况建立了一种简单易行的单孢分离手段。 以36个子囊孢子后代为研究对象，进行了玉米大斑病菌子囊孢子后代与其亲本基于RAPD的遗传多态性分析。结果发现，子囊孢子后代的遗传图谱较亲本出现明显变异。21个随机引物共扩增出144个RAPD标记，其中126个为特异性标记，占87.5％。菌株间的相似系数在0.6450～0.7914之间。此外，后代与亲本的相似性并无一定规则，甚至部分出现双亲的相似性较亲代与子代相似性高的现象，可见玉米大斑病菌遗传机制的复杂性。在双亲遗传物质重组的过程中，可能亲本菌株01-12的遗传物质更占优势，而01-15的遗传力相对较弱。 本试验对玉米大斑病菌的RAPD扩增程序做了进一步摸索，建立了适宜的、较为稳定的反应体系和扩增程序，即：在25μL体系中，加入Taq酶1U，dNTP200μM，10×PCR buffer 2.5μL，随机引物0.8μM，模板DNA 50ng，用灭菌超纯水补足至25μL。扩增程序为：95℃预变性3min；之后94℃变性1min，45℃退火50sec，72℃延伸2min，进行40个循环；接下来72℃延伸5min；最后在4℃下保存。 对亲本及子囊孢子后代的酯酶同工酶分析表明，亲本与后代菌株在同工酶谱上存在显著差异，其中Rf=0.042的酶带为区分两亲本的特异性酶带。个别后代菌株出现较大变异。 本文还对玉米大斑病菌原生质体的制备条件进行了初步摸索，研究了酶系统、渗透压稳定剂、酶解时间、酶解温度对原生质体制备的影响。结果表明，玉米大斑病菌原生质体制备的适宜条件为:取PD液体培养基振荡培养4d后的玉米大斑病菌菌丝，用试管形玻璃组织研磨器将其磨碎，以0.8:nol几甘露醇做渗透压稳定剂，加入10 mg/mL纤维素酶+l 0 mg/mL蜗牛酶的混合酶，28℃卜静置酶解72h。更多还原

【Abstract】 Two isolates belonging to opposite mating type, 01-12 and 01-15, were selected as parent isolates to induce ascospore production of Setosphaeria turcica, and 79 single ascospores were isolated. 79 crossing progenies belonging to which mating type were identified by sexual cross with parental isolates. 36 isolates belonged to ’a’ mating type, and were the same as parent isolate 01-12, and 20 isolates belonged to ’A’ mating type and were the same as parent isolate 01-15. But the ratio of two mating type deviated 1:1 apparently, as deduced that the mating character was decided by polygene site. Otherwise, 3 isolates were bisexual and 20 isolates were neuter.The experimental results had found that new isolates collected had greater mating ability than those isolates which subcultured for 3 years. In the favorite conditions that the quantity of FeCl3 was 0.02 mg per 1000 mL medium, the number of mature ascospores and the stability of asci occurrence were greatly increased. A simplified method for isolating monosacospores was also set up in this test.Progenies of 36 ascospores selected were used to study the genetic diversity of parent isolates and sexual crossing progenies of S. turcicum based on RAPD analysis. The results indicated that there were evident variation on genetic map between parent isolates and progenies isolates. There were total of 144 RAPD markers with 21 arbitrary primers, and 126 markers presented the diversity and the percentage was 87.5%. The similarity coefficient of diferrent strains was 0.6450~0.7914. Moreover, there were no definite rules as to whether markers in progenies were more similar to parents by similarity analysis, even though that within parents were more similar than between parent and progeny. It showed that the genetics of Setosphaeria turcica were very complex. Parent isolate 01-12 maybe had stonger genetic ability than parent isolate 01-15 in the course of sexual cross.The stabilized RAPD reaction system was established in this paper. In 25 Lreactions, there were 1 units of Taq DNA polymerase, 200 M of mixed dNTP, 2.5 L of 10 PCR buffer, 0.8 M of arbitrary primer and 50ng of genomic DNA, the rest volume filled with aseptic ddH2O. Amplification was performed by the following program: initial denaturation at 95 for 3 min followed by 40 cycles of denaturation at 94 for 1 min, annealing at 45 for 50 sec, extension at 72 for 2 min, and final extension at 72 for 5 min, kept at 4 at last.There were great diversity in the esterase isozyme map among parent isolates and progenies. The specific band in Rf=0.042 location was a distinctive marker for parents. A progeny occurred apparent mutation.Mycelia of S. turcica could be used to prepare the protoplast. The yield of protoplasts was significantly correlated with enzymes, osmotic stabilizer, temperature and treating time. The proper condition shown in this paper was that the ground mycelium of S. turcica was treated by mixed enzymes (10 mg/mL Cellulase and 10 mg/mL Snailase) for 72 hours at 28癈 after shocking cultrued in PD medium for 4 days, and the mixed enzymes had a better effect when they were dissolved with 0.8 mol / L mannitol osmotic stabilizer.更多还原