【作者】 王娜；

【导师】 王玉成；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2014， 硕士

【摘要】 植物病害是阻碍农业和林业增产的巨大难题,其中,由真菌引起的疾病占植物病害的80%,传统的防治方式主要依赖于化学农药,但这对环境和人类健康都有重大影响,相比之下,生物防治会更安全些,因而被认为是取代化学制剂的主要后备军。据报道,一些木霉菌株具有高效的防治植物疾病的能力,而且水解酶被认做是抵御过程中的最主要力量。深绿木霉是众所周知生防能手,它能产生枯草杆菌蛋白酶(SS),这种蛋白酶在生物防治发展中有重要的作用。本研究是从生防菌株深绿木霉ACCC30153中克隆出了枯草杆菌丝氨酸蛋白酶基因SS42。SS42基因中包含一个开放读码框(ORF),编码区伞长1328bp,有三个内含子编码434个氨基酸组成的蛋白质,蛋白质分子质量42.55kDa,等电点5.53,为疏水蛋白。BlastP结果显示,SS42序列是Peptidases_S8_S53_superfamily家族成员,且与里氏木霉QM6a的蛋白酶相似性最高,达到98%。SignalP显示SS42氨基酸在第21和22位有个长达21个氨基酸的信号肽。Swissmodel软件预测到SS42蛋白酶椭圆型的三维结构。用荧光定量RT-qPCR的方法分析九种不同的培养基培养下的深绿木霉SS42基因的表达。九种培养基分别是,MM,C饥饿,N饥饿,1%山新杨茎粉,1%山新杨叶粉,1%叶枯痫原菌细胞壁,5%叶枯病原菌发酵液,1%杨树烂皮病病原菌细胞壁,5%杨树烂皮病病原菌发酵液培养基。定量结果显示在不同条件处理下,SS42基因表达均为上调状态,而且在1%叶枯病菌细胞壁培养下4h时表达倍数最高,是未处理时的177.29倍。综合比较实验数据说明,深绿木霉ACCC30153菌株既能与植物也能与植物病原菌互作,且在互作过程中SS42基因可以高效表达。将SS42基因与pGEX-4T-2质粒连接,转化入大肠杆菌BL21感受态,用IPTG诱导异源表达。SDS-PAGE电泳检测出一条清晰的条带,大小在68kDa,这表明重组质粒pGEX-SS42成功导入了大肠杆菌,并产生重组蛋白。用不同浓度的IPTG、不同温度、不同时间以及不同起始OD值等条件下培养重组菌株,得到的表达产物均为包涵体蛋白,再用电纯化法对蛋白进行纯化,得到清晰、单、浓度较高的rSS42重组蛋白。用福林酚法对重组蛋白酶rSS42进行活性研究,重组蛋白酶在30-60℃,pH4-8.5条件下仍有稳定活性。重组菌株培养4h时,分泌的重组蛋白表达量最高,活性最大,40℃,pH=7环境下蛋白酶活性最高为20U/mL用深绿木霉ACCC30153与五种植物病原菌(尖镰孢菌、核盘菌、叶枯病菌、杨树烂皮病菌、立枯丝核菌)对峙培养,整个培养过程中,木霉在生存空间、营养摄取上始终占据绝对优势,能不同程度的抑制病原菌的生长。用重组蛋白酶rSS42对五种病原菌进行抑菌实验表明,rSS42对病原菌抑制作用效果明显,直观证明了其高效的生防功能。总之,深绿木霉ACCC30153菌株中的枯草杆菌丝氨酸蛋白酶SS42基因的成功获取为蛋白质功能和性质的研究奠定了基础；成功表达得到重组蛋白rSS42为并证明其高效的生防效能,为蛋白酶制剂类生物农药的研制奠定了基础。更多还原

【Abstract】 The plant disease is a great influence on agriculture and forestry production. Fungal disease is accounting for80%of plant disease. Chemical pesticides play an important role in the prevention and treatment in traditional which is a huge damage to the environmental and human beings health. Biological control of plant fungal pathogen is much safer so that it is the prime candidate in the search for reducing dependency on chemical pesticides. Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of hydro lytic enzymes is considered as the main mechanism involved in the antagonistic process. The fungus Trichoderma atroviride is a well known biocontrol agent. It produces subtilisin-like serine protease （SS）, which is important in the development of biological control.In this study, a subtilisin-like serine protease gene SS42has been cloned from biocontrol T.atroviride ACCC30153. The SS42gene contains an open reading frame （ORF）,1328bp in length, interrupted by three short introns, and encodes a protein of434amino acids with a molecular weight of42.55kDa and a pI of5.53. BlastP search indicated that SS42protease is a close homologue of Peptidases_S8_S53superfamily, and sharing the highest similarity of98%with T. reesei （XP₀06966748）. Furthermore, signalP prediction showed that the SS42amino acid sequence was cleaved by signal peptidase between positions21and22（AAA|||MP）. We also predicted a oval three-dimensional structure of the proteins with Swissmodel.The expression of SS42gene was analyzed by RT-qPCR after T. atroviride ACCC30153cultured in nine different medias:MM, C starvation, N starvation,1%stem powder,1%leaf powder.1%Cytospora chrysosperma or1%Alternaria alternata cell wall,5%C. chrysosperma or5%A. alternata fermentation liquid culture condition. The results indicated that the expression of the SS42gene was all up-regulated regulated by treatments of different culture condition, and showed the highest expression at4h,177.29times higher than pretreatment with1%A. alternata cell wall. The results of RT-qPCR indicated that both plants and phytopathogen could interact with Trichoderma ACCC30153, and SS42gene highly expressed in the process.The cDNA of SS42was inserted into the pGEX-4T-2vector and transformed into Escherichia coli BL21for heterologous expression induced by IPTG. A clearly visible band with molecular mass about68kDa in the SDS-PAGE gel indicated that the transformant harboring the gene SS42had been successfully translated in E.coli and produced a recombinant protein. The bacterial was cultured under different concentrations of IPTG, different temperature, different OD and so on, of which the product was also inclusion body protein. Finally, the protein was purified by power showing a single clear, high concentration gel on SDS-PAGE.We carried out a study of enzyme activity on serine proteinase rSS42by Folin method. Recombinant serine proteinase remains relatively stable enzyme activity at30-60℃and pH4-8.5. When transformant BL21-SS42was induced with1mM IPTG for4h, the serine protease exhibited its maximal activity of almost20U/mL at40℃and pH9.0.Dual culture with Trichoderma atroviride and five plant pathogens （Fusarium, Sclerotinia, blight fungus, Cytospora, Rhizoctonia solani）,the research showing that Trichoderma always occupy an absolute advantage of living space and nutrition resulting to inhibit the growth of pathogens. It was carried out of fungi-repressing test with recombinant serine proteinase rSS42, the result found that rSS42inhibited the growth of five plant pathogens,which indicated the recombinant serine proteinase had the capability of biocontrol.In conclusion, the cloning of Trichoderma atroviride ACCC30153SS42gene had laid the foundation for the research on function and protein property. The proteinase was secreted into the culture medium by Escherichia coli BL21in a functionally activity form, and the expression serine proteinase could inhibit the growth of some fungal pathogen.It provided a material basis for developing a biological pesticide of recombinant protein SS42.更多还原