【作者】 赵鹏；

【导师】 杨谦；

【作者基本信息】 哈尔滨工业大学 ， 生物学， 2014， 硕士

【摘要】 本论文所研究的task1基因为MAPKs家族的成员，在真核细胞信号传递网中功能显著，在前期的研究中发现task1对生物防治真菌的生防功能具有一定的调控作用，因此对task1深入研究有利于生物防治真菌在分子方面的内在调节机制的剖析意义重大。棘孢木霉本身的生物防治能力突出，并含有task1，因此研究野生型棘孢木霉的task1基因表达状况，对于棘孢木霉调节生防能力意义重大。获得的纯化的目的基因task1，成功的整合入表达载体中，构建重组的表达载体，通过电转化和真菌共培养等方法，在筛选平板上获得诸多的阳性的转化子。对于得到的转化子进行分子生物学分析，在分子鉴定后，选取其中最优良的菌株，进行形态学、酶学及生防特性的测定。在对于形态学的比较中发现，当目的基因task1在原始菌株中表达上调后，转化子的菌丝状态、生长和产孢状况优越于原始的野生菌株，并且菌丝缠绕密集，产孢数优于原始菌株，为其2.25倍。在对两种菌的酶活性测定中，得出转化子的C1、滤纸酶活、CX、β-葡萄糖苷酶等酶活性影响较大，并均有很大的提升。因此，进行了转化子的纤维素酶的实时定量PCR，发现转化子的纤维素的基因的表达量也有很大的提高，进一步证明酶活性提高原因在task1的上调作用。在生防对峙实验中，对选取的两种病原菌的杀伤能力突出。阳性转化子对立枯丝核菌、西红柿镰刀菌、的抑制率分别为74.8%、76.5%，而原始菌株的抑制率则为57.2%、69.15%。可以看出阳性转化子对于立枯丝核菌的抑制率远高于原始菌株，而对于红柿镰刀菌的抑制率差异不大。更多还原

【Abstract】 Task1, a member of MAPK superfamily, plays a important role of eukaryotic cellsignaling network, and is significant for the biocontrol fungus at the molecular spects ofthe internal adjustment mechanism analysis. Trichoderma asperellum, which showssome strong ability of biocontrol, and contains task1, so the research of the expressionof task1gene of wild type Trichoderma asperellum, is significant for adjustingbiocontrol.Purification of the target gene task1is obtained, and successfully integrated into anexpression vector to construct the recombinant expression vector, transformed byelectroporation and fungi co-cultured and other methods, we got a lot of positivetransformants on screening plates. After molecular biology analysis, select thebesteststrains measured morphology, enzymology and biocontrol.Throμgh the comparative of morphology, found that when the gene task1upregulated in the original strain, mycelium state, growth and sporulation conditions ofthe transformants are superior to the original wild-type strain, and mycelium isentangled densely, the number of spore is more2.25-fold than the original wild-typestrain.The enzyme activity of transformants derived C1, filter paper activity, CX,β-glucosidase greater effect.Therefore,the real-time quantitative PCR of cellulase expression of transformantsfound cellulose genes have greatly improved,further proof,activity improved isdue totask1upregulation. In experimentof biocontrol confrontation, selected threepathogen-killing ability is prominent.The inhibition rates of positive transformants toRhizoctonia solani and Fusarium oxysporum are74.8%,76.5%, while inhibition rate ofthe original strain are57.2%,69.15%. It is obvious that inhibition rate of positivetransformants for R. solani is much higher than the original strain, and the inhibitionrate for F. oxysporum is a little difference.更多还原