【作者】 范海娟；

【导师】 王志英； 刘志华；

【作者基本信息】 东北林业大学 ， 森林保护学， 2013， 博士

【摘要】 木霉菌对多种植物真菌病害具有优良的生物防治效果,为了研究木霉的生物防治机理,木研究首先构建了哈茨木霉ACCC30371菌株的转录组,获得高质量的Unigene基因,之后对转录组获得的与生物防治相关的基因进行生物防治功能分类;进一步筛选到两条生物防治功能基因刺激植物响应蛋白基因epl1和蛋白酶基因ss,并对之进行了功能研究。研究结果如下:以1/2PD、MM(含葡萄糖10g/L)、N饥饿、C饥饿、以及1%植物病原真菌核盘菌(Sclerotinia sclerotiorum)、链格孢菌(Alternaria alternata)、立枯丝核菌(Rhizoctonia solani)、金黄壳囊孢菌(Cytospora chrysosperma)细胞壁研磨粉分别诱导培养哈茨木霉ACCC30371,构建转录组,获得4Gbp数据,其中96.68%的数据测序错误率不高于1%,以此标准进行数据统计,共得到25013条Unigene,平均长度为1135nt,长度大于3000nt的为2571条;其功能注释到Nr蛋白库的16360条,SwissProt蛋白库9875条,KEGG蛋白库10026条,注释到COG蛋白库的7164条,GO蛋白库的1508条;构建获得共计16723条功能基因。通过对哈茨木霉转录组Unigene基因分析,共获得与真菌病害生防相关的基因四大类,包括竞争作用机制类基因14条,重寄生机制相关基因311条,抗生机制相关基因76条,刺激植物响应蛋白基因1条,共计402条基因。经分析认为哈茨木霉的生物防治机制是以重寄生为主的综合防治机制,抗生作用和诱导系统抗性也在其中占有重要地位。这对于进一步研究木霉生防机制、开发利用生防功能基因和研制生物杀菌剂具重要意义。克隆获得了刺激植物响应蛋白基因epl1全长cDNA序列、启动子序列。cDNA序列已提交至GenBank,序列号为KC876056。分析发现该蛋白属于Cerato-platanin家族(Pfam07249),将Epl1蛋白对深绿木霉、绿色木霉、棘孢木霉、哈茨木霉基因组进行相似性搜索,共发现相似序列15条,蛋白保守区长度在113-120之间,哈茨木霉CBS 226.95染色体骨架6上的Epl1-Tha与Epl1蛋白相似性为100%。用RT-qPCR技术研究了在8种营养条件下epl1基因转录水平上的表达:MM培养基中epl1基因表达量随时间呈周期性的增加;接触植物成分(山新杨根、茎、叶研磨粉)后表达量持续升高,最高达处理前的32倍以上;C饥饿时表达量短时间内迅速升高,后期维持稳定状态;N饥饿时上调表达量较小;杨叶枯病菌细胞壁及发酵液存在时,表达量上升,但变化程度低于MM同一时间点;总体上8种诱导方式均能使epl1上调表达。成功构建了epl1的重组载体pGEX-epl1及重组菌株BL21-epl1,SDS-PAGE蛋白电泳发现符合预期分子量40.5kDa的蛋白质大量表达,为蛋白的生产、分离与功能研究奠定了基础。克隆获得了枯草杆菌丝氨酸蛋白酶基因ss全长cDNA序列,cDNA序列已提交至GenBank,序列号为 KC876057。其蛋白属于 Peptidases_S8_PCSK9_ProteinaseK_like(Pfam00082)家族。将SS蛋白对上述4个近源种基因组搜索,获得21条相似序列,哈茨木霉CBS 226.95染色体骨架6上的SS1-Tha与目标蛋白相似性为100%。对四个基因组上丝氨酸蛋白酶编码基因启动子区调控元件结合基序进行分析,发现丝氨酸蛋白酶与木霉生防作用密切相关。用RT-qPCR技术研究上述8种营养条件下ss基因转录水平上的表达,发现在MM、C饥饿和植物根成分存在时各时间点表达量是对照的0.7-3.8倍之间,总体上8种诱导处理方式对哈茨木霉ACCC30371菌株ss基因的表达量影响不大。成功构建了蛋白酶基因ss的重组载体pGEX-ss及重组菌株BL21-ss,SDS-PAGE蛋白电泳发现符合预期分子量68.7kDa的蛋白质大量表达,本研究为哈茨木霉生物农药的研制提供理论支持,对蛋白酶类生物农药的研制具有一定的参考价值。更多还原

【Abstract】 Excellent biocontrol effect of Trichoderma on various plant fungal diseases was identified,in order to study the biocontrol mechanism of Trichoderma,transcriptomic database of Trichoderma harzianum ACCC30371 strain was constructed firstly and quality Unigenes were got,after that biocontrol genes from transcriptome were classified according to function of biocontrol;two biocontrol functional genes were selected from database and studied further,eliciting plant response protein gene epll and protease gene ss.The results are as follows:Transcriptome of T.harzianum ACCC30371 strain was constructed after it grown in 8 culture media as follows:1/2PD,minimal medium MM(containing dextrose lOg/L),C starvation medium(derived from MM without dextrose),N starvation medium(derived from MM without ammonium sulphate),and C starvation medium harboring 1%(w/v)powder of phytopathogenic fungi Sclerotinia sclerotiorum,Alternaria alternate,Rhizoctonia solani,Cytospora chysosperma respectively.The transcriptome database has a size of 4Gbp and 96.68%of it has a sequencing error rate not higher than 1%.Data statistics was carried out according to this criterium and 25013 Unigene sequences were obtained.Mean length of them is 1135 nt and 2571 of them are over 3000 nt.16360 Unigenes were annotated to Nr protein database,9875 were annotated to SwissProt database,10266 were annotated to KEGG database,7164 were annotated to COG database,1508 were annotated to GO database along with their protein functional annotations.16723 functional genes were found.402 bio-control genes were classified into four major categories by analyzing transcriptome data of T.harzianum,including 14 genes related to competition,311 genes related to mycoparasitism,76 related to antibiosis and 1 related to eliciting plant response.It reveals that T.harzianum ACCC30371 has integrated biocontrol mechanisms and mycoparasitism is the most important in proportion,antibiosis and induced systemic resistance(ISR)play also important roles.The results possess extremely important significance in further researching bio-control mechanism of Trichoderma and developing and utilizing biocontrol functional genes and biological fungicides.The full-length cDNA and promoter were cloned of gene epll.The cDNA sequence of it has been submitted to GenBank and number is KC876056.Protein of it belongs to Cerato-platanin protein family(Pfam07249).15 similarity proteins of Epll were found on four Trichoderma genomes,which came from T.atroviride,T.virens,T.harzianum,T.asperellum.The conserved domains of them had a length range of 113-120.Epll-Tha which was located on chromosome scaffold 6 of T.harzianum CBS 226.95 had a similarity of 100%with Epl1.The expression of gene epll on transcriptional level under 8 nutrient conditions was studied using RT-qPCR technique and revealed that expression level of epll increased periodically over time when T.harzianum grown in the MM medium.And the amount of expression of epll increased continuously when T.harzianum grown in medium which powder of roots,stems and leaves of Shanxin-poplar was added respectively and even over 32 times control team amount.The amount of expression of epll increased rapidly from T.harzianum grown in C starvation medium and tended to be constant after having reached a certain level and small amount of up-regulated expression of epll was found when T.harzianum grown in N starvation medium.The amount of expression of epll rose when mycelial powder or zymotic supernatant of A.alternata was added in medium,but the degree of change was lower than that of MM at the same time point.Overall,regulated expression of epll appeared when T.harzianum grown in the eight inducing media.The ecombinant vector pGEX-epll and the recombined strain BL21-epll were successfully constructed.SDS-PAGE analysis determined an over-expression protein band,which had a molecular weight of 40.5-kDa similar to the predicted.It laid the foundation for protein production,separation and functional studies.The full-length cDNA of gene ss was cloned successfully.The cDNA sequence of it has been submitted to GenBank and number is KC876057.Protein of it belongs to Peptidases_S8_PCSK9_ProteinaseK_like protein family(Pfam00082).21 similarity proteins of SS were found on four Trichoderma genomes,which came from T.atroviride,T.virens,T.harzianum,T.asperellum.SS1-Tha which was located on chromosome scaffold 6 of T.harzianum CBS 226.95 had a similarity of 100%with SS.Analysis of regulatory-elements-binding-motifs in promoters of these serine protease coding genes on four Trichoderma genomes indicated that serine protease is closely associated with control of Trichoderma spp.The expression of ss on transcriptional level under 8 nutrient conditions was studied using RT-qPCR technique and revealed that the expression level of gene ss changed between 0.7and 3.8 times of control team amount.Overall,expression level of ss changed little when T.harzianum grown in the eight inducing media.The recombinant vector pGEX-ss and the recombined strain BL21-ss were successfully constructed.SDS-PAGE analysis determined an over-expression protein band,which had a molecular weight of 68.7-kDa similar to the predicted.Results of this study provide a theoretical support for the development of biological pesticides from T.harzianum and a practical reference for exploiting proteases in biological pesticides.更多还原