【作者】 赵培宝；

【导师】 李多川；

【作者基本信息】 山东农业大学 ， 植物病理学， 2008， 博士

【摘要】 轮枝镰孢菌(Fusarium verticillioide)是一种重要的植物病原真菌,可引起玉米苗枯病、穗腐病等,纤细齿梗孢(Olpitrichum tenellum)是轮枝镰孢菌上的一种菌寄生真菌,为了研究纤细齿梗孢-轮枝镰孢菌-玉米之间的识别、互作、寄生和侵染致病机制,笔者以与信号传导有关的蛋白激酶基因为切入点并结合随机插入突变对此进行了探讨。摸索掌握了两种真菌原生质体制备技术;建立并优化了轮枝镰孢菌的REMI和ATMT遗传转化体系,获得了数十个突变体;克隆得到两种真菌的4个丝氨酸-苏氨酸蛋白激酶基因;并通过同源重组成功敲除了轮枝镰孢菌的2个蛋白激酶基因,明确了它们的生物学功能。1.掌握了两种真菌的原生质体制备技术,摸索了影响两种真菌原生质体制备的条件,包括菌龄、酶的种类和浓度、酶解时间和温度以及稳渗剂等,找到了两种真菌原生质体制备的最佳条件,其中轮枝镰孢菌采用PDB培养14h的菌丝,以0.7mol/LNaCl为稳渗剂,在30℃、100rpm下用20mg/mL溶壁酶酶解4h,原生质体制备量最大;而纤细齿梗孢采用PDB培养36~48h的菌丝,在30℃、100rpm下用30mg/mL溶壁酶酶解5h原生质体制备量最大。2.以含有潮霉素磷酸转移酶基因的质粒pUC ATPH转化轮枝镰孢菌FT1菌株和纤细齿梗孢OL1菌株的原生质体,前者取得了成功,共获得转化子300多个,转接5代能稳定遗传,PCR验证潮霉素基因已插入转化子基因组DNA中,通过表型观察和致病性测定,发现许多生长异常突变体,2株致病性减弱突变体;而纤细齿梗孢没有得到转化子。3.在植物转化载体pROKII基础上导入带有真菌启动子的潮霉素B抗性基因表达盒PtrpC-hph-TtrpC,构建了一个适合真菌转化的载体pROKII-PtrpC-hph-TtrpC,冻溶法转入农杆菌LBA4404。利用这一转化载体,成功地建立起了轮枝镰孢菌的ATMT遗传转化体系,获得了T-DNA插入突变体;而转化纤细齿梗孢却没有取得成功。摸索优化了转化条件,在镰孢菌孢子浓度106个/mL、农杆菌OD600=0.15~0.20、乙酰丁香酮浓度为200μmol/mL的条件下共培养36h转化效率最高,可达60~120个/106个孢子。共获得转化子1000多个,连续转接5代能够稳定遗传,PCR验证潮霉素B抗性基因已整合进转化子基因组DNA中,部分转化子表现为生长和形态异常。4.利用兼并PCR,TAIL PCR和RACE技术相结合克隆得到两种真菌的四个丝氨酸-苏氨酸蛋白激酶基因,分别命名为fpk1、fmk1、opk1和omk1 (GenBank登录号分别为:EF405958、EU417814、EU417815和EU479712)。其中fpk1由ATG到TAA的DNA全长1854bp,包含3个内含子,长度分别为66bp、54bp、54bp;cDNA全长1680bp,编码559个氨基酸的多肽,序列比对和分析显示其属于cAMP依赖的蛋白激酶PKA。opk1基因自ATG到TAA的DNA全长2223bp,包含2个内含子,分别长108bp、84bp,cDNA全长2031bp,编码676个氨基酸的多肽,序列比对和cAMP依赖的蛋白激酶同源性较低,可能属于另外一种Ser/Thr蛋白激酶。fmk1和omk1属于Fus3/Kss1类MAPK基因,cDNA全长均为1068bp,编码355个氨基酸的多肽,前者DNA全长1242bp,包含3个内含子,长度分别为58bp、56bp和60bp;后者DNA全长1198bp,包含2个内含子,长度分别为57bp和73bp,内含子边界均符合GT-AG规则。5.依据同源重组原理,以pUCATPH和pBS为基础构建了基因fpk1和fmk1的敲除突变载体pBS-K-hph和pUCATPH-KS-KX,以REMI介导的转化手段转化轮枝镰孢菌原生质体,成功筛选到了fpk1和fmk1基因敲除突变体。通过对突变体的表型特征进行研究分析,明确了两基因的生物学功能,证实两基因与菌丝生长、胞壁分化、孢子产生和萌发以及致病性等密切相关。更多还原

【Abstract】 Fusarium verticillioide is an important plant pathogenic fungus all over the world, and can cause maize seedling blight , maize ear rot,etc. Olpitrichum tenellum is an important biotrophic mycoparasite of Fusarium verticillioide,In order to probing into the mechanism of biological recognition, interaction and parasitization between Fusarium verticillioide with Olpitrichum tenellum and maize ,This research try to clone the protein kinase genes from the two fungi ,and study their function in the process of interaction,and look for the factors by establishing muntants throμgh DNA insertion. As a resμLt ,4 protein kinase genes were cloned , two protein kinase’s function were understood by gene disrupting . Also the genetic transformation system of Fusarium verticillioide were established by using the system of REMI and ATMT, and abtained many mutation .1.Generation of fungal protoplast is an essential tool for genetic transformation system. To establish protoplast-mediated genetic transformation systems of F. verticillioide and Olpitrichum tenellum, conditions for the protoplasts isolation and regeneration of the mycelia of F. verticillioide and Olpitrichum tenellum were examined, including the effects of the hyphal age, enzymes and their concentration, time and temperature of digestion, osmotic stabilizers on the protoplast preparation and the effect of osmotic stabilizers on the protoplast regeneration. The resμLts indicated that optimum conditions for preparing protoplasts of F. verticillioid were: the condia cμLtured in liquid PDB medium for 14 hours, 0.7mol/L NaCl used as the osmotic stabilizer, 20mg/mL Lywallzyme used to digest the hyphae for 4 hours at 30℃. And 0.7mol/L sucrose was the osmotic stabilizer for the regeneration of protoplasts. Optimum conditions for preparing protoplasts of Olpitrichum tenellum were: the condia cμLtured in liquid PDB medium for 36~48 hours, 0.7mol/L NaCl used as the osmotic stabilizer, 30mg/mL Lywallzyme used to digest the hyphae for 5 hours at 30℃.2.To study the pathogenicity mechanism, the restriction enzyme-mediated integration approach were used to transform the protoplasts of F. verticillioide with vector pUCATPH which has hygromycin B-resistant gene, over 300 transformants were obtained.It indicated that the hygromycin B-resistant gene have been integrated into their genome by PCR amplification. Most of the transformants were fμLly stable after five rounds successive cμLture,many phenotypic mutants and 2 weak pathogenicity mutants were gained by REMI. But no transformant was got after transforming Olpitrichum tenellum.3.The plant expression vector pROKII was ligated to hygromycin B resistance gene cassette (PtrpC-hph-TtrpC) with the fungus promoter from plasmid pUCATPH. Thus, the vector pROKII-PtrpC-hph-TtrpC was constructed, and then transferred into Agrobacterium LBA4404. By using the system of Agrobacterium tumefacines-mediated transformation, we successfμLly transformed F. verticillioide and obtained T-DNA insertion mutants. But no transformant was got after transforming Olpitrichum tenellum.Under the optimal conditions (F. verticillioide spores:106 /mL;A.tumefaciens OD600:0.15~0.20; acetosyringone:200μg/ mL ; co-cμLtivation:36h), transformation efficiency reached 60~120 transformants per 106spores. More than 1000 transformants were obtained. Most of them were quite stable after five rounds of successive cμLtures. PCR amplification showed that the T-DNA was integrated into the genome. The transformation system is the basis for studying on pathogenicity mechanism and functional gene of the fungi.4.By using degenerate PCR、TAIL-PCR and RACE technique,four protein kinase genes were cloned in the two fungi.They were named of fpk1、fmk1、opk1 and omk1 .All their DNA and cDNA fμLl-length sequences were acquired ,and their GenBank accession were EF405958,EU417814,EU417815 and EU479712.fpk1Included 1854bp DNA sequence from ATG to TAA,with 1680bp coding region , three intron (their length :66bp,54bp and 54bp),the predicated protein of fpk1 gene had 559aa.Sequence analyse indicated that fpk1 belong to PKA.opk1Included 2223bp DNA sequence from ATG to TAA,with 2031bp coding regin , two introns (their length :108bp,84bp),the predicated protein of opk1 gene had 676aa.fmk1Included 1242bp DNA sequence from ATG to TAA,with 1068bp coding regin , three introns (their length :58bp,56bp,60bp),the predicated protein of fmk1 gene had 355aa.omk1Included 1198bp DNA sequence from ATG to TAA,with 1680bp coding regin , two introns (their length :57bp,73bp),the predicated protein of omk1 gene had 355aa.5.The fpk1 and fmk1 gene-disruption vetor were constructed based on the gene homologous combination theory. The gene-disruption vetor were PBS-kinase-hph and pUCATPH-KS-KX, and hygromycin B resistance gene as the screening label. By transforming protoplasts with the gene-disruption vector, a fpk1 gene-disruption isolate and fmk1 gene-disruption isolate were successfμLly screened from more than 200 transformants. homologous combination was testified by PCR and southern blotting in the mutation .It was clear that the two gene’s function were related with hyphal development, conidiophore producing,virμLence and cell wall biosynthesis.更多还原