【作者】 胡仕凤；

【导师】 高必达； 陈捷；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2007， 博士

【摘要】 木霉菌是传统的植物病害生物防治真菌,然而随着生态农业的快速发展,木霉菌的单一功能很难满足农业清洁生产多方面的需求,因此筛选出具有更多生态功能的木霉菌具有重要意义。除了防病功能外,本研究针对目前生态农业中对农作物秸秆处理、土壤磷化合物的降解、杂草的抑制等需求,通过限制性内切酶介导的基因整合技术（restriction enzyme mediated DNA integration,REMI）对野生型木霉菌进行分子改良,以求获得多功能木霉菌资源,为进一步创制新型木霉菌剂奠定基础。两株木霉菌T.atroviride T23和T.koningii CICC 40144经过REMI转化,共得到201株突变株,并继代培养、PCR检测和Southern杂交,证明外源质粒pV₂已经成功整合到出发菌基因组中,且单拷贝率达64.3%,形成了优化的木霉菌分子改良技术体系,为今后克隆功能基因奠定了重要基础。对不同条件下REMI突变株菌丝生长及产孢的情况进行了研究,发现这些突变株在pH2-10范围内均可生长,pH在4-6时,菌丝生长最快;30℃时菌丝的产量最高;液体培养基中以蔗糖和果糖为碳源,以酵母膏、蛋白胨为氮源时突变株的菌丝生长最好,菌丝干重最大。总体上看,经过转化之后获得的突变株在利用碳源、氮源方面比出发菌T23有了明显的提高。对不同条件下突变株孢子萌发的情况进行了研究,发现在15℃-40℃时突变株孢子均可萌发,但最适温度为25℃-35℃;培养28h各突变株孢子萌发率均达到100%;孢子萌发pH值范围为2-8,在pH10时几乎不萌发;光照可以促使孢子萌发,湿度越大,孢子萌发率越高。当湿度为100%时,突变株TE7的孢子萌发率最高,几乎达到100%。用聚丙烯酰胺凝胶电泳技术对木霉菌T23及其4株突变株的可溶性蛋白和酯酶进行了比较。研究发现各突变株之间,突变株与出发菌之间可溶性蛋白和酯酶的谱带均有差异。4株突变株在不同程度上均存在着蛋白条带的丢失现象,其中突变株TE7丢失了5条蛋白带;酯酶同工酶之间的差异更为明显,且谱带数量也明显少于可溶性蛋白谱带。这些结果说明外源质粒DNA插入木霉菌染色体之后导致基因组结构发生改变,从而引起了由基因组控制的蛋白的改变,此研究为今后进一步定位和克隆基因奠定理论基础。为了对木霉菌株更多功能的挖掘,通过筛选获得了纤维素酶活明显高于出发菌的的突变株11株（TK-2R-3、TK-2R-9、TK-2R-11、TK-2R-14、T30-B3、T30-C7、T23-A2H等）,且出发菌纤维素酶活越高,其突变株纤维素酶活提高幅度越小。最终获得了一株高产纤维素酶木霉突变株TK-2R-14,其CMC酶活达37.6IU/mL,比出发菌提高8.05%,FPA酶活达16.9 IU/mL,比出发菌提高19.01%。通过正交试验确定了突变株TK-2R-14固体发酵时最佳培养基组分为稻草粉:麦麸为9:1,料水比1:1,硫酸铵1%,Tween-20 0.1%;通过单因素优化试验,确定其最佳培养条件为:接种量5%-7.5%,培养温度30℃,培养时间96h,pH值自然,该突变株的FPA酶活和CMC酶活分别达6.097 IU/g、8.123 IU/g。从康氏木霉T30的REMI突变株中筛选到四株解磷能力较强的突变株TK-2、TK-38、TK-46、TK-47,以TK-46最显著,其解磷能力比出发菌提高41%,达363.79μg/mL。对突变株TK-46的解磷条件进行了研究,发现其在以麦芽糖为碳源、氯化铵或硫酸铵为氮源、pH7.0的条件下,解磷作用最好,且对CaHPO₄的溶解能力远大于AlPO₄,但不能溶解FePO₄。同时发现pH值下降并不一定引起解磷,但解磷可导致pH值下降。从木霉菌REMI突变株中,筛选出了8株有抑草活性突变株TA2,TB3,TB7,TE2,TE5,TH8,TJ6和TJ9,并对水稻生长安全。在对发酵液分离后进一步检测发现,无论是孢子液、孢子超声波破碎液还是去除孢子的发酵液对稗草根的抑制作用均优于苗;且对苗的抑制作用主要表现为完整的孢子,破碎的孢子对苗基本没有抑制作用,去除孢子的发酵液对苗的抑制作用也不明显。另外还发现,突变株TE2的抑草活性是由存在于发酵液中的某些次生代谢物产生的,与分生孢子无关;但突变株TH8的抑草活性刚好相反,其抑草活性由分生孢子产生,而与其代谢产物无关。最后筛选获得了一株抑草活性较强且功能较全面的目标菌株TB3。对筛选出来的抑草活性较高的木霉REMI突变株TB3的抑草素活性组分进行了分析,通过萃取及蛋白沉淀等方法分离出突变株发酵液的不同组分,然后进行抑草活性检测,结果发现该突变株TB3所产生的这种抑草活性物质即不是胶霉毒素,也不是二氢绿胶霉素,可能是一种蛋白质,该蛋白质用丙酮沉淀会失去其活性,但用硫酸铵沉淀活性保存完好,且适应于用高饱和度硫酸铵沉淀。该蛋白质究竟是一种什么样的蛋白,需进一步研究。为了阐明木霉突变株表型改变的本质,采用功能蛋白质组学的差异蛋白分析方法,比较了突变株TK-2R-1与出发菌T.koningii CICC 40144之间的蛋白质差异,在2-D电泳图谱中获得了8个蛋白表达量存在差异的蛋白点,经质谱分析,鉴定出了4个蛋白点,这些蛋白点均与木霉的生长代谢有关。利用质粒拯救技术研究了突变株TB3和TK-2R-1、TK-2R-14、TK-2R-15插入位点分子特性,得到了经REMI突变后限制性内切酶插入位点侧翼的序列,但是在Blast数据库中基本没有找到与之相对应的功能基因序列,故猜测其可能是一些未知的氨基酸序列,这些序列经ORF Finder查找,没有找到足以大到编码一种蛋白质的ORF,这些序列的作用还有待进一步研究。更多还原

【Abstract】 Trichoderma spp.was the traditional bio-control fungi to plant diseases.The single-functional Trichoderma spp.cannot meet the needs of clean agriculture production.Therefore,it was very important that multifunctional Trichoderma strains were screened.For obtaining multifunctional Trichoderma strains and producing new agent of Trichoderma spp.,the wild strain of Trichoderma spp.was improved through restriction enzyme mediated DNA integration.We studied the effects of Trichoderma mutants on degradation of crop debris,degradation of soil phosphate-compound and inhibition of weeds besides the function of preventing plant disease.For building Trichoderma spp.resource and screening,REMI was used to construct mutants from T.atroviride strain T23 and T.koningii strain CICC 40144. Totally,201 mutants were obtained.Successive culture,PCR and Southern blot analysis were applied to determine that plasmid pV2 was integrated into Trichoderma spp.gene.Frequency of single copy transformation reached 64.3%,which favored further study such as plasmid rescue.The conditions of mycelium growth and spore germination of mutants of Trichoderma strain T23 has been studied.The mutants could grow in a wide range of pH value（from 2 to 10）,but the optimal pH value for mycelium growth was from 4 to 6.The optimal temperature for mycelium growth was 30℃.Sucrose and fructose were the best carbon sources and yeast extracts and peptone were the best nitrogen sources.As a whole,mutants were better than Trichoderma strain T23 in the aspect of using carbon and nitrogen.The spores of mutants could germinate in a wide range of temperature（from 15℃to 40℃）,but the optimal temperature for spore germination was from 25℃to 35℃.In incubating for 28 hours,the rate of spore germination reached 100%.The spore could germinate in a wide range of pH value（from 2 to 8）,but didn’t germinate when pH value was 10. Illumination accelerated spore germination.The spore germination need direct proportion to the RH（relative humidity）.As in the air,conidia of TE7 could germinate complete when RH reached 100%.The soluble proteins and esterase isozyme of Trichoderma strain T23 and four mutants were analysed with polyacrylamide gel electrophoresis.The results showed there were differences among mutants and between wide strain and mutants.Some of the bands of protein was lost in four mutants.TE7 has lost five bands of protein.The difference between esterase isozyme was more obvious.The strips of esterase isozyme were less than protein.It confirmed the protein were changed when a plasmid insertion,which established the base of theory for allocation and the clone gene further.For excavating more functions of Trichoderma spp.,11 mutants were screened for improved cellulase activity as compared with their parent strain.Higher cellulase activity of parent strain had,less improvement of mutants.Moreover,one mutant, TK-2R-14,was found with the highest CMC（37.6 IU.mL-1） and FPA（16.9 IU.mL-1） activities among all screened mutants,which were increased by 8.05%and 19.07%, respectively.The optimal condition for solid fermentation of TK-2R-14 was carried out for higher cellulase production.The results based on orthogonal experiment showed that the optimal solid materials components were determined as follows: ratio of rice straw to bran 9:1,the ratio of solid materials to water 1:3 in conjunction with（NH₄）₂SO₄ 1%and Tween-20 0.1%.Moreover the single fermentation factors were also crucial to the mutant for improving cellulase enzyme activity,inoculum volume 5%-7.5%,culture temperature 30℃.culture time 96 h and neutral pH value were best for fermentation.The FPA and carboxyl methyl cellulase activity of TK-2R-14 were 6.097 IU/g and 8.123 IU/g respectively under these optimal conditions.Four phosphate-degradading mutants were screened from T.koningii REMI mutants,named TK-2,TK-38,TK-46 and TK-47.TK-46 of them was the best because it’s phosphate- degradading capacity was 363.79μg / mL,increased 41% compared with their parent isolates.Studied the optimal condition of TK-46 for phosphate-degradading,the results showed that the optimal carbon source was maltose,the optimal nitrogen source was ammonium chloride or ammonium sulfate, the optimal pH value was 7.0.The phosphate-degradading capacity to CaHPO₄ was far greater than to AIPO₄.But it didn’t dissolve FePO₄.It was discovered that the decreasing pH value of culture mediums was not necessarily to the degradation of phosphate,but the degradation of phosphate could lead to pH value decrease.After screening for barnyardgrass-inhibition of REMI mutants of Trichoderma spp.,eight mutants were obtained.They are TA2,TB3,TB7,TE2,TE5,TH8,TJ6 and TJ9.Their culture filtrates（CFs） have bamyardgrass-inhibition activities,and are safe to rice.In Secondary experiments,the bioassays of the spore suspension,ultrasonic broken spore PBS suspension treatment and CFs without spores are conducting.Thus mutant TB3 was the best one.For analysing barnyardgrass-inhibition component of TB3,some extract experiments and protein precipitation were carried on.The result was that barnyardgrass-inhibition component from TB3 was neither gliotoxin nor viridiol,but protein.The protein could lose activity by acetone precipitation,but preserves activity by ammonium sulphate precipitation.Furthermore,ammonium sulphate of high saturation was applicable for the protein precipitation.We must study which kind of protein was the protein further.In purpose of expounding the phenotype change of Trichoderma mutants,protein profiles of T.koningii CICC 40144 and it’s mutant TK-2R-1 were detected by 2-DE. Eight unique spots were found and four of them were identified their functions by MS-TOF-TOF which were connected with growth and metabolization of Trichoderma spp.Furthermore,the frank fragment of TB3 and TK-2R-1,TK-2R-14,TK-2R-15 genomics DNA and restriction integration site characterization by REMI was separated and analyzed by plasmid rescue.But the sequence of function gene had not been found in Blast database.Therefore they were unknown amino acid sequences possibly.Searching ORF in ORF Finder,enough big ORF of code protein wasn’t found among these sequences.The effect of these sequences would been studying further.更多还原