【作者】 赵巍；

【导师】 韩建民； 董金皋；

【作者基本信息】 河北农业大学 ， 植物学， 2011， 硕士

【摘要】 玉米大斑病是由玉米大斑病菌（Setosphaeria turcica）引起的一种真菌病害。病菌的致病过程受到cAMP、MAPK和Ca²⁺信号途径的调控。蛋白激酶C（protein kinase C,PKC）是一种依赖磷脂和钙的蛋白激酶,是Ca²⁺信号途径中的成员。本实验室成功克隆了玉米大斑病菌PKC基因,利用PKC特异性抑制剂研究发现,抑制剂（浓度10μmol/L）处理的分生孢子在玉米叶片表面虽也能正常萌发并形成附着胞,但不能直接穿透叶片表皮细胞,叶片上未出现病害症状。在此基础上,本研究构建了PKC基因的反义表达载体,利用PEG介导转化玉米大斑病菌原生质体,获得4个PKC基因反义表达突变体,对其培养性状、分生孢子萌发、附着胞形成、致病力等进行了研究,以明确蛋白激酶C在玉米大斑病菌致病过程中的作用。为全面深入了解玉米大斑病菌信号转导途径在调控病菌致病过程中的作用奠定基础。1.不同培养条件下PKC基因表达规律研究表明,培养基以蔗糖为碳源或以NaNO₃为氮源时利于玉米大斑病菌PKC基因表达。Mg²⁺利于病菌PKC基因表达,但Mn²⁺、Cu²⁺、Zn²⁺抑制病菌PKC基因表达。山梨醇造成的渗透胁迫抑制病菌PKC基因的表达,且抑制程度与培养基中山梨醇浓度呈正相关。NaCl造成渗透胁迫时,低浓度NaC（l0.3⁰.5 mol/L）抑制了病菌PKC基因的表达,高浓度NaC（l0.7⁰.9 mol/L）对病菌PKC基因表达没有影响。2.构建了PKC基因反义表达载体,利用PEG介导转化玉米大斑病菌原生质体,通过潮霉素B抗性筛选及PCR、半定量RT-PCR及Northern杂交鉴定获得了4个PKC基因反义表达突变体。研究发现,正常培养条件下,4个突变体的菌落生长速度与野生型无差异;当培养基中含有反义表达诱导物奎宁酸时,4个突变体的菌落生长速度明显小于野生型。经15 mmol/L奎宁酸溶液处理后,4个突变体的孢子萌发率仅为3%⁵%,野生型孢子萌发率达90%;将萌发后的孢子用奎宁酸溶液处理后,野生型菌株可以形成附着胞,并穿透玻璃纸,突变体附着胞形成率显著下降（形成率3%⁵%）,且其附着胞不能穿透玻璃纸。4个突变体产生的HT-粗毒素对感病玉米叶片的致病能力没有明显改变。突变体接种经针刺伤表皮的感病寄主叶片,发现病斑面积明显比野生型小;直接接种感病寄主叶片则不致病。玉米大斑病菌PKC基因的表达规律及反义表达突变体的获得明确了基因的功能,丰富了植物病原真菌的生物信息学资源,为深入了解植物病原真菌信号转导途径奠定了基础。更多还原

【Abstract】 Northern Corn Leaf Blight, caused by Setosphaeria turcica, is one of the important fungous diseases. The pathogenicity of S. turcica to maize is involved in cAMP、MAPK and Ca²⁺signal pathway. Protein kinase C （PKC）, a serine/threonine kinases, is depended on phospholip and Ca²⁺. The protein kinase C gene of S. turcica, named as PKC, was cloned and analyzed in our Lab. After inoculating maize seedlings with the spore suspension containing PKC-specific inhibitor （chelerythrine）, the spore could germinate and form normal appressorium on the leaf surface but invade maize seedlings from the stoma instead of penetrating directly leaf epidermis. There was no lesion appeared on the leaves three weeks after inoculation. The anti-sense vector was transformed into protoplasts of S. turcica by PEG-mediated transformation method and four PKC gene anti-sense mutants were obtained. Cultural characteristic, conidial germination, appressorium formation and pathogenicity of the mutants were studied in this paper to analyze function of protein kinase C. The results will be beneficial to understand how pathogenicity of S. turcica to maize is involved in cAMP、MAPK and Ca²⁺ signal pathway.1. The expression of PKC in S. turcica was increased on PDA media containing saccharose or NaNO₃ or Mg²⁺ ,the expression of PKC was decreased on PDA media containing Mn²⁺or Cu²⁺or Zn²⁺.The expression level of PKC was lower on PDA media containing sorbitol and the more concentration sorbitol, the less expression level. The expression of PKC was increased on PDA media containing NaCl（0.3⁰.5 mol/L）, the expression of PKC was not changed on PDA media containing NaCl（0.7⁰.9 mol/L）.2. We obtained four anti-sense mutants of S.turcica PKC gene which had been identified by hygromycin B resistance, PCR, semi-quantitative RT-PCR and Northern blot analysis. The growth rate of the mutants cultured on PDA media was same with wide type （WT）, but mutants were much lower than WT on PDA media containing quinic acid （15 mmol/L）. When conidia of WT and mutants were inoculated in 15 mmol/L quinic acid solution, the conidial germination ratio of WT was 90%, while that of the mutants were 3%⁵%. In contrast, culturing the germinated conidial in 15 mmol/L quinic acid solution, appressorium formed in WT but not in mutant strains. Penetration experiments showed that appressorium coming from WT could penetrate the cellophane but could not from mutants. Virulence of HT-toxin of mutants on maize leavies were the same with that of WT. While the inoculation of mutant strains did not cause visible lesions on the intact maize leaves, the inoculation of mutant strains caused visible lesions on the scratched maize leaves, but the lesion area was significantly reduced compared with WT.更多还原