玉米大斑病菌PKC基因的功能研究
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摘要
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。为了明确蛋白激酶C与分生孢子萌发、附着胞形成及致病性之间的关系,本文利用PKC特异性抑制剂—白屈菜赤碱(chelerythrine)研究了玉米大斑病菌分生孢子萌发、附着胞形成以及对玉米叶片表皮细胞的侵入等主要致病过程;利用Genome Walking技术克隆了PKC基因5’端上游约1.5 kb的DNA序列,采用生物信息学技术分析了该区域含有的主要元件,预测了该基因的功能;进一步优化了玉米大斑病菌原生质体的制备条件,构建了PKC基因同源重组载体和反义诱导表达载体,利用PEG介导系统转化玉米大斑病菌原生质体,获得转化菌株。
利用PKC特异性抑制剂—白屈菜赤碱(chelerythrine)的研究发现,在含有10μmol/L抑制剂的PDA培养基上,玉米大斑病菌菌丝的生长受到了明显抑制,菌落的颜色没有变化,仍为灰黑色;抑制剂处理的分生孢子能正常萌发,形成的附着胞颜色与形状都没有变化,但是附着胞不能直接穿透玉米叶片的表皮细胞,而是趋向于从气孔侵入叶片内部;抑制剂处理后的孢悬液接种玉米叶片3周后没有呈现出病害症状。从含有10μmol/L抑制剂的PDA培养基上培养的玉米大斑病菌菌丝中提取的HT-毒素在玉米叶片上仍显示出较强的致病活性,表明白屈菜赤碱对玉米大斑病菌产生HT-毒素没有明显影响。
基于pBS和pSO1质粒分别构建了玉米大斑病菌PKC基因敲除载体和反义表达载体。利用PEG介导的转化系统,转化玉米大斑病菌原生质体获得27个基因敲除阳性转化子。对继代培养后的阳性转化子进行PCR验证,获得12个潮酶素磷酸转移酶基因插入的转化菌株。转化菌株的菌落平坦、菌丝纤细,产孢能力降低。
获得质量高、数量多的原生质体是遗传转化的关键技术之一。在稳渗剂较适宜的条件下,菌龄、酶种类与浓度配比、酶解温度及酶解时间是影响原生质体形成的最重要的5个因素,所以本文研究了这5个因素对玉米大斑病菌原生质体制备的影响。结果表明,制备玉米大斑病菌原生质体最佳条件为菌龄24 h,酶组合与酶浓度为1%溶壁酶+1%蜗牛酶+1%崩溃酶,酶解温度30℃,酶解时间4 h。在此条件下,可以产生10个左右原生质体(显微镜×400),约6×105个/mL。
利用Genome Walking技术获得PKC基因5’端上游约1.5 kb的DNA序列。生物信息学分析没有发现类似TATA盒的元件,存在热激转录因子(HSF)结合元件和SP1、AP1等结合元件,推测PKC基因在该菌耐热性、交配型基因表达和寄主信号识别等方面发挥作用。
以上结果初步明确了玉米大斑病菌PKC基因与病菌分生孢子萌发、附着胞形成、侵入及菌丝生长之间的关系。为进一步研究玉米大斑病菌的致病机理以及植物与病原物分子互作和开发新的植物病害防治方法奠定了基础。
Protein kinase C (PKC) located in the middle of Ca2+ signaling pathways. It is a phospholipid and calcium-dependent protein kinase that might play an important role in the processes of conidium germination, appressorium formation and pathogenicity. The functional analysis of PKC gene in Setosphaeria turcica were done by gene-specific inhibitor analysis, constructing PKC gene knock-out mutants based on the gene homologous combination theory and PEG gene transformation system, 5’upstream analysis of PKC gene and the function prediction.
It was found by PKC gene-specific inhibitor (chelerythrine) analysis that the color of the colony growing on the PDA containing 10μmol/L inhibitor was still normal, but the mycelial growth was inhibited. There also wasn’t any change in the color and shape of appressorium while treated with inhibitor. After inoculating maize seedlings with the spore suspension containing specific inhibitor, the appressorium formed from germinating spore on the leaf could not penetrate directly through the skin of epidermal cells and only to invade leaf tissue from the stoma. It was found that 10μmol/L inhibitor didn’t significantly affect the activity of HT-toxin and no lesions appeared on the leaf after three weeks inoculation.
High quality and large quantity of protoplasts was one of the key technologies of genetic transformation. The yield of protoplasts was significantly correlated with the age of hyphae, enzyme system, concentration, temperature and treatment time. The optimum condition for preparing protoplasts was that the conidia cultured for 24 h was digested in enzyme mixture of 1% Lywallzyme, 1% Snailase and 1% Drislase for 4 h in 30℃, and about 10 protoplasts could be produced under above conditions (microscope×400).
The PKC gene knock-out vector was constructed based on the gene homologous combination theory, then transformed into S. turcica protoplasts and 12 mutants were screened by hygromycin B resistance from 27 transformants. In contrast to the wild strain 01-23, these mutants had smooth colony, slender hyphae and sporulation ability.
A 1.5kb gene flank region was obtained by Genome Walking. Bioinformatic analysis showed that it had a lot of cis-acting elements corresponding SP1、AP1 and HSF, but no typical TATA box. It was calculated that PKC gene perhaps played an important role in the regulation of mating type gene expression, host signal identification and hot tolerance.
These results indicated that PKC gene played an important role in the processes of conidium germination, appressorium formation and pathogenicity of S. turcica, Which laid a foundation for investigating pathogenic mechanism of S. turcica, molecular interaction between plant and pathogen, and new methods of disease prevention.
利用PKC特异性抑制剂—白屈菜赤碱(chelerythrine)的研究发现,在含有10μmol/L抑制剂的PDA培养基上,玉米大斑病菌菌丝的生长受到了明显抑制,菌落的颜色没有变化,仍为灰黑色;抑制剂处理的分生孢子能正常萌发,形成的附着胞颜色与形状都没有变化,但是附着胞不能直接穿透玉米叶片的表皮细胞,而是趋向于从气孔侵入叶片内部;抑制剂处理后的孢悬液接种玉米叶片3周后没有呈现出病害症状。从含有10μmol/L抑制剂的PDA培养基上培养的玉米大斑病菌菌丝中提取的HT-毒素在玉米叶片上仍显示出较强的致病活性,表明白屈菜赤碱对玉米大斑病菌产生HT-毒素没有明显影响。
基于pBS和pSO1质粒分别构建了玉米大斑病菌PKC基因敲除载体和反义表达载体。利用PEG介导的转化系统,转化玉米大斑病菌原生质体获得27个基因敲除阳性转化子。对继代培养后的阳性转化子进行PCR验证,获得12个潮酶素磷酸转移酶基因插入的转化菌株。转化菌株的菌落平坦、菌丝纤细,产孢能力降低。
获得质量高、数量多的原生质体是遗传转化的关键技术之一。在稳渗剂较适宜的条件下,菌龄、酶种类与浓度配比、酶解温度及酶解时间是影响原生质体形成的最重要的5个因素,所以本文研究了这5个因素对玉米大斑病菌原生质体制备的影响。结果表明,制备玉米大斑病菌原生质体最佳条件为菌龄24 h,酶组合与酶浓度为1%溶壁酶+1%蜗牛酶+1%崩溃酶,酶解温度30℃,酶解时间4 h。在此条件下,可以产生10个左右原生质体(显微镜×400),约6×105个/mL。
利用Genome Walking技术获得PKC基因5’端上游约1.5 kb的DNA序列。生物信息学分析没有发现类似TATA盒的元件,存在热激转录因子(HSF)结合元件和SP1、AP1等结合元件,推测PKC基因在该菌耐热性、交配型基因表达和寄主信号识别等方面发挥作用。
以上结果初步明确了玉米大斑病菌PKC基因与病菌分生孢子萌发、附着胞形成、侵入及菌丝生长之间的关系。为进一步研究玉米大斑病菌的致病机理以及植物与病原物分子互作和开发新的植物病害防治方法奠定了基础。
Protein kinase C (PKC) located in the middle of Ca2+ signaling pathways. It is a phospholipid and calcium-dependent protein kinase that might play an important role in the processes of conidium germination, appressorium formation and pathogenicity. The functional analysis of PKC gene in Setosphaeria turcica were done by gene-specific inhibitor analysis, constructing PKC gene knock-out mutants based on the gene homologous combination theory and PEG gene transformation system, 5’upstream analysis of PKC gene and the function prediction.
It was found by PKC gene-specific inhibitor (chelerythrine) analysis that the color of the colony growing on the PDA containing 10μmol/L inhibitor was still normal, but the mycelial growth was inhibited. There also wasn’t any change in the color and shape of appressorium while treated with inhibitor. After inoculating maize seedlings with the spore suspension containing specific inhibitor, the appressorium formed from germinating spore on the leaf could not penetrate directly through the skin of epidermal cells and only to invade leaf tissue from the stoma. It was found that 10μmol/L inhibitor didn’t significantly affect the activity of HT-toxin and no lesions appeared on the leaf after three weeks inoculation.
High quality and large quantity of protoplasts was one of the key technologies of genetic transformation. The yield of protoplasts was significantly correlated with the age of hyphae, enzyme system, concentration, temperature and treatment time. The optimum condition for preparing protoplasts was that the conidia cultured for 24 h was digested in enzyme mixture of 1% Lywallzyme, 1% Snailase and 1% Drislase for 4 h in 30℃, and about 10 protoplasts could be produced under above conditions (microscope×400).
The PKC gene knock-out vector was constructed based on the gene homologous combination theory, then transformed into S. turcica protoplasts and 12 mutants were screened by hygromycin B resistance from 27 transformants. In contrast to the wild strain 01-23, these mutants had smooth colony, slender hyphae and sporulation ability.
A 1.5kb gene flank region was obtained by Genome Walking. Bioinformatic analysis showed that it had a lot of cis-acting elements corresponding SP1、AP1 and HSF, but no typical TATA box. It was calculated that PKC gene perhaps played an important role in the regulation of mating type gene expression, host signal identification and hot tolerance.
These results indicated that PKC gene played an important role in the processes of conidium germination, appressorium formation and pathogenicity of S. turcica, Which laid a foundation for investigating pathogenic mechanism of S. turcica, molecular interaction between plant and pathogen, and new methods of disease prevention.
引文
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