【作者】 杨力明；

【导师】 杨谦；

【作者基本信息】 哈尔滨工业大学 ， 环境科学与工程， 2008， 博士

【摘要】 在当今世界大力倡导可持续发展的大环境下,可持续发展思想及环保意识提高了生物防治技术的重要性,受到了国际社会的极大关注。构建出高效、广谱及价格低廉的生物农药来替代化学农药,最低限度的减少环境污染,达到绿色生产、绿色环保的目的。哈茨木霉（Trichoderma harzianum）是一种优良的生物防治真菌,在植物病害,尤其是土壤传染病害的生物防治中具有重要的应用价值。哈茨木霉主要通过重寄生作用、溶菌作用、抗生素作用、蛋白酶作用以及对空间和营养的竞争机制达到防治植物病原菌的目的。在各种恶劣的生存条件下哈茨木霉的抑菌功能受到了极大限制和破坏,限制了其生防潜力的进一步发挥。因而,提高哈茨木霉的防治效果,及保证其在逆境条件下生防功能顺利实现具有重要的意义。为增强哈茨木霉防治效果和对外界抵抗能力,利用真菌表达载体pBI121,pCAMBIA1301和克隆载体pUC18,成功构建了用于根癌农杆菌介导的丝状真菌遗传转化表达载体pCA-GChiⅤ和pCA-GSOD。以潮霉素抗性基因为选择标记,目的基因的启动子为CaMV35S,两种载体分别含有几丁质酶（ChiⅤ）基因、超氧化物岐化酶（SOD）基因。对影响哈茨木霉转化的因素进行优化,获得了该系统转化哈茨木霉的最佳条件。经过潮霉素抗性选择培养,Southern杂交,RT-PCR及传代分析得到稳定遗传的转化子。采用与三种植物病原菌室内拮抗实验,检测重组ChiⅤ和SOD后的哈茨木霉转化子的抑菌活性。结果表明通过根癌农杆菌介导的转化系统所获得转ChiⅤ哈茨木霉转化子具有良好的抑菌活性,对植物病原菌都表现出很强的拮抗作用。通过根癌农杆菌介导的转化系统所获得SOD哈茨木霉转化子保持了哈茨木霉菌的抑菌活性,对哈茨木霉原菌和SOD哈茨木霉转化子进行高温和盐胁迫实验进行了比较,结果证实,哈茨木霉原菌在逆境下不能正常存活;而SOD木霉转化子转化后的哈茨木霉菌,在逆境下仍能正常存活,且对植物病原菌仍具备良好的拮抗作用。ChiⅤ基因在哈茨木霉中的表达,明显提高了哈茨木霉的抑菌能力。SOD基因在哈茨木霉中的表达,提高了哈茨木霉对高温和高盐的抗逆性,保证了哈茨木霉能够在高温和高盐的条件下发挥其生防功能。从而,为哈茨木霉的抑菌功能在更广泛的领域中应用奠定了基础。在获得的哈茨木霉（T. harzianum）EST序列基础上,为提高哈茨木霉菌抗外界干扰能力,克隆了与抗碱性盐和干旱相关的sod和hsp24基因的DNA序列,克隆获得的这些序列已在GenBank上注册。将克隆得到的sod和hsp24基因构建原核表达载体pET-sod、pET-hsp24,通过双酶切、测序检测证明重组载体构建成功。通过IPTG诱导使sod、hsp24基因在大肠杆菌（Escherichia coli）BL21中表达,SDS-PAGE检测分别获得15.7kD、24.0kD的蛋白带,蛋白大小与预期相符,说明它们已经在大肠杆菌（E. coli）中表达,并检测了sod蛋白粗酶活和hsp24的耐热功能,证明了哈茨木霉（T. harzianum）基因能够在原核生物中良好表达。为提高哈茨木霉菌（T. harzianum）的sod和hsp24基因表达效率,我们将sod和hsp24基因构建到带有高效启动子的酿酒酵母（ Saccharomyces cerevisiae）表达载体pYES2上,并转入酿酒酵母（S. cerevisiae）INVSc1细胞,得到了携带sod、hsp24外源基因的酵母转化子。逆境胁迫实验表明转sod、hsp24基因酵母对Na₂CO₃和干旱的耐受能力明显提高,表明sod、hsp24基因与哈茨木霉（T. harzianum）的抗逆境胁迫相关,这对研究哈茨木霉（T. harzianum）在自然界常见逆境下的生物防治能力具有重要意义。以上基因的克隆和功能研究为哈茨木霉（T. harzianum）其它功能基因的克隆和研究建立了完善的技术平台,对哈茨木霉（T. harzianum）重要生物防治基因的进一步发掘和利用具有重要意义。同时,将几丁质酶基因和超氧化物歧化酶基因分别转化到哈茨木霉（T. harzianum）中并表达,以及转化后的哈茨木霉菌的功能认定,使我们获得了良好生物防治效果的基因工程菌。为今后应用提供了科学依据。本项研究克隆了涉及生物防治和胁迫响应方面的基因,这些基因的获得将在揭示生物防治机理、研究哈茨木霉（T. harzianum）逆境下的生物防治能力变化以及其体内高价值酶基因的发掘等方面也具有重要意义,是系统地、多方位地对哈茨木霉（T. harzianum）的生物防治分子机理研究的开始。更多还原

【Abstract】 In the sustainable development circumstance of the world,awareness of sustainable development and environmental protection improved the important bio-technology, which got the focus of the international society. Using the high-effect, broad and cheap biology pesticide instead of chemical pesticide could decrease the environment pollution for green produce and green environmental protection.Trichoderma Harzianum, an important plant biocontrol fungus with wide application, had important applied value in the biocontrol of plant diseases, especially diseases infected through soil. T. harzianum can produce many kinds of antibiotics and ergosterols against plant fungal pathogens based on different mechanisms, such as the production of antifungal metabolites, competition for space and nutrients and mycoparasitism. It was widely used in biocontrol of plant disease. But because of various adverse suvivial conditions, the antimicrobial efficacy of T. harzianum was restricted and badly damaged, so it was necessary to enhance the antimicrobial efficacy and the adversity resistance of T. harzianum in order to realize its function of biocontrol.We used the vector of pBI121,pCAMBIA1301 and clone vector of pUC18 to successfully construct express vector pCA-GChiⅤand pCA-GSod for filamentous fungi transformation mediated by Agrobacterium tumefaciens. Including the ChiⅤand Sod gene, hygromycin resistance gene as selected marker and CaMV35S promoter with section of T-DNA the vector pCA-GChiⅤand pCA-GSod could be used for genetic transformation of mycelial fungus. The construction of vector pCA-GChiⅤand pCA-GSod, suggests a stratergy for the enhancement of ChiⅤand Sod expression in T. harzianum and improvement of T. harzianum’environmental resistance. The best conditions of this system to transformate T. harzianum was got by analysis every transformation factors.Positive transformant was harvested by the selected method, southern blot analysis, RT-PCR and passage culturing.Antifungal activities of the ChiⅤand Sod transformants of T. harzianum was tested by means of dual culture with three kinds of plant pathogens. The ChiⅤtransformants of T. harzianum results showed that it improve the inhibition activity to these pathogens compared with original strain of T. harzianum. The SOD transformants of T. harzianum results showed that there was no obvious difference in inhibition activity to these pathogens between original strain of T. harzianum and its transformants obtained by A. tumefaciens. These results indicated that our transgenic operation didn’t influence its antifungal ability. Then stress experiment of wild T. harzianum and its transformant were tested in high temperature and salt conditions. The result indicated that transformant still suvived. Moreover, dual cultured with plant pathogens, the SOD transformant also keep the inhibition activity to these pathogens.however, wild T. harzianum could not live in high temperature and salt conditions.The ChiⅤgene expression in the T. harzianum obviously improved the ability to the inhibit the pathogens. The SOD gene in the T. harzianum improved the ability to the high temperature and salt.This established the foundation of the application for more fields.Based on the EST sequences of T. harzianum obtained by our laboratory, The cloned sod and hsp24 were inserted into prokaryotic expression vector, respectively, and the recombinant expression vectors were named pET-sod and pET-hsp24. Double enzymes digestion and sequencing were performed for confirming whether the expression vector constructed successfully. Genes of sod and hsp24 were expressed respectively in E. coli BL21 induced by IPTG, the protein bands of 15.7kD and 24kD were observed respectively by SDS-PAGE.The enzyme activity of SOD and survival to high temperature of hsp24 were tested. The result conformed to expectation, indicating that these genes have been expressed in E. coli, also suggesting that genes from T. harzianum could express well in E.coli. All these genes had registered in GenBank.Genes of sod and hsp24 from T. harzianum were respectively inserted into pYES2 vector, an expression vector of Saccharomyces cerevisiae with high efficiency promoter, and transformed them into the cells of S. cerevisiae INVSc1, generating transformants of sod and hsp24. Stress testing showed that S. cerevisiae cell transformed with sod and hsp24 both enhanced their tolerance to Na₂CO₃ and drought, indicating the two genes are involved in stress tolerance in T. harzianum.The cloning and functional studies of these genes has built a solid technical platform for cloning and study other genes from T. harzianum, and have a significant effect in further exploiting and application of important biocntrol genes in T. harzianum. In this study, genes involved in the functions of biocontrol and stress response have been cloned. The cloning of these genes will make an important role in investigating the biocontrol of T. harzianum under stress and exploiting its important enzymes genes, and is the beginning of studying the biocontrol mechanism of T. harzianum systemically and broadly. The chiⅤand sod gene was transformed into T. harzianum and well expressed in T. harzianum, which means that we have obtained a genetic engineering strain efficiently. This study enhanced the resistance of T. harzianum, ensured its excellent biocontriol function in various adverse conditions so as to widen the application area of T. harzianum.更多还原