【作者】 钟耀华；

【导师】 汪天虹；

【作者基本信息】 山东大学 ， 微生物学， 2007， 博士

【摘要】 瑞氏木霉(Trichoderma reesei，anamorph；Hypocrea jecorina)是具有重要经济价值的工业丝状真菌，可以用于生产纤维素酶、半纤维素酶、淀粉酶以及蛋白酶等多种酶类，这些酶类已广泛应用于纺织、造纸、制浆等工业领域。同时，由于具有很强的蛋白分泌能力，该菌已被开发成为表达同源蛋白和异源蛋白的良好真核宿主。多样化的代谢能力、对人没有毒性以及生长快、无性繁殖等竞争性生长是瑞氏木霉的重要特征。目前，瑞氏木霉全基因组测序工作已经完成，这对深入了解这个经济重要的工业菌株具有重大意义，同时也为鉴定那些与该菌生长、代谢相关的关键基因提供了良好平台。利用分子标签技术获得突变体己成为进行基因克隆和遗传学分析最有效的方法之一。因此，建立瑞氏木霉基因标签的插入突变体库是功能基因组学发展的趋势，而对不同表型突变体的正、反向遗传学分析可以促进揭示瑞氏木霉生长、代谢的分子机理。本论文研究的目的在于建立和优化根癌农杆菌介导的瑞氏木霉转化系统，利用T-DNA随机插入突变建立瑞氏木霉的突变体库；采用反向遗传学和正向遗传学方法相结合的策略对突变株进行变异性状的分析和基因水平的研究，鉴定那些与突变性状相关的新基因；选择那些控制瑞氏木霉生长、发育或重要代谢途径的关键基因发生插入突变的突变株进行生理、生化和分子遗传学的研究，深入了解基因的功能和阐明相关的分子机理。1．农杆菌介导的瑞氏木霉转化及T-DNA插入突变分析本文用农杆菌AGL1成功介导转化了丝状真菌瑞氏木霉QM9414，T-DNA随机插入到瑞氏木霉染色体中，并得到了一批瑞氏木霉T-DNA插入突变体。将pAN7-1质粒的HPH基因表达盒插入到Ti质粒pBI121中，获得了双元载体pBI-hph。该载体导入农杆菌AGL1后，在乙酰丁香酮的诱导下，瑞氏木霉原生质体或孢子与农杆菌共培养产生了具有潮霉素抗性的转化子，转化效率较高，并筛选到了稳定的转化子。从随机挑选的9个转化子的PCR和杂交分子验证表明，T-DNA上的HPH基因已插入到瑞氏木霉染色体中。借助于TAIL-PCR方法，从转化子中成功克隆到了T-DNA插入位点侧翼序列，序列分析证明T-DNA是随机插入到瑞氏木霉染色体中的。目前已获得分子验证的突变菌株200余株，以平板为单位收集的抗性稳定的瑞氏木霉转化子数十板，初步建立起小范围的瑞氏木霉T-DNA标签的插入突变体库。实验结果表明，农杆菌介导转化(AMT)瑞氏木霉是一种有效的插入突变策略，对于瑞氏木霉功能基因组学的研究具有重要意义。2．瑞氏木霉生长发育异常突变子的筛选和分析对瑞氏木霉T-DNA标签的突变子库进行表型筛选，发现了7株形态异常的突变子。在这些突变子中，有两个突变子的T-DNA插入位点恰好位于同一个基因的不同位点。序列比对和系统分析发现，该基因编码类胡萝卜素双加氧裂解酶(CCD，carotenoid cleavage dioxygenase)，命名为TrCCD1，该基因位点的突变产生了与形态发生和生孢相关的异常表型。序列分析和实验验证表明，这两个突变子的T-DNA分别位于TrCCD1的开放阅读框(ORF)和启动子区(promotor)，因此，分别命名为ccdO和ccdP。突变子ccdO和ccdP与出发菌株QM9414相比，在MM平板上生长缓慢，生长速率仅为出发菌株的1／2左右，同时菌落相对疏松；当在含0.2％Triton X-100的MM平板上生长时，突变子菌落形成了一些长的气生菌丝，导致菌落蓬松；菌丝的显微观察发现，突变子的菌丝顶端与菌丝中间区域相比明显变化，菌丝顶端颜色变暗；突变子PDA平板培养4d后仅有中间部位产生稍许绿色孢子，而周边都是白色菌丝，与QM9414形成鲜明对比生孢比较困难。为了确定菌株类胡萝卜素的变化情况，利用丙酮对分泌到培养基的类胡萝卜素进行吸光度检测和β胡萝卜素的高效液相色谱(HPLC)测定。测定445nm处类胡萝卜素吸光度值，发现QM9414的类胡萝卜素吸光度值仅为突变菌株的1／2左右；高效液相色谱法测定451nm处β胡萝卜素的色谱，根据峰面积和β胡萝卜素浓度关系，计算β胡萝卜素含量。结果发现，突变子分泌到PDA培养基中的β胡萝卜素含量比出发菌株QM9414低的多(约为1／2)。本实验的结果表明，TrCCD1基因的突变与类胡萝卜素的含量直接相关，TrCCD1在类胡萝卜素的代谢中具有重要作用。由此推测，它的突变可能影响了某种具有重要信号作用的类胡萝卜素分子的生成，因而导致了瑞氏木霉菌丝生长和孢子发育的异常，这也说明属于CCD家族成员的TrCCD1基因在生长发育过程中起到了重要的调控作用。表型筛选获得的另外5株形态异常的突变子为：PM2、PM48、HP7、HP13和HPL1。对这些突变子的一般形态进行了分析并利用TAIL-PCR对突变菌株染色体T-DNA插入位点侧翼序列进行了克隆。5株突变子的菌落形态各异，与出发菌株有明显区别，菌丝延伸较慢，有的菌落成明显辐射状，有的菌落白色，根本没有生孢迹象等。尤其值得关注的是PM2，它的菌丝顶端弯曲缠绕，形成了一个个卷曲成团的头部。目前，TAIL-PCR已经扩增到PM48和HPL1的插入位点序列。PM48的插入位点在一个编码细胞周期相关蛋白的基因ORF区。HPL1的插入位点基因编码的蛋白可能是一个多聚腺苷酸结合蛋白，该基因的突变引起了纤维素降解能力的变化。3．纤维素降解突变菌株的筛选和分析利用CF11平板透明圈筛选法，对200余株经分子验证的T-DNA插入突变子进行了筛选，结果发现了4株纤维素降解突变子：PM3、PM23、HPL1和36H-6。其中，突变子PM3和PM23的透明圈增大了30％左右，而HPL1和36H-6的透明圈减小了40％左右，并且36H-6形不成完整的透明圈。通过纤维素酶活力测定发现，各菌株的FPA活力在18h至24h之间达到高峰期，其中PM3和PM23的FPA最高活力比出发菌株高30％以上，且PM3比其它菌株提前达到酶活最大值，PM23的FPA活力持续时间最长的。另外两个突变子HPL1和36H-6的FPA活力始终低于QM9414，尤其是HPL1的FPA活力一直处于较低水平，最高酶活比出发菌株低了近2／3，而36H-6的最高酶活与出发菌株相差不大，但其酶活力随着时间延长下降很快，48h时就几乎丧失了活力。各菌株的CMCase都是在24h达到酶活高峰，PM3和PM23的最大CMCase活力比出发菌株高，而HPL1和36H-6比出发菌株低，但活力最高的是PM23而不是PM3。尤其值得注意的是，HPL1的FPA和CMCase活力都始终处于较低水平。利用TAIL-PCR从HPL1菌株的T-DNA右端RB区扩增到了T-DNA侧翼基因组序列，经与数据库的比对分析发现，该突变基因编码的蛋白可能是多聚腺苷酸结合蛋白，突变位点位于基因上游-26bp处。该基因的功能与RNA加工和修饰有关。4．绿色荧光蛋白标记的双元载体构建及农杆菌转化本文也构建了两个不同Ti质粒载体系统的绿色荧光蛋白为标记的双元载体，并进行了初步的农杆菌介导转化瑞氏木霉的实验。pBI-gfp载体是一个中间质粒，它是由四片段连接构建成的，含有EGFP表达盒。然后将该表达盒经过酶切和末端平滑化后插入到携带潮霉素的Ti质粒pBI-hph中，以此实现了双标记载体pBI-gh的构建。但是，从农杆菌转化瑞氏木霉结果看，没有检测到绿色荧光。在转化过程中也出现过一些问题，例如pBI-gh在转化到农杆菌后在扩增EGFP基因时得到的DNA片段总是减小等，我们纯化了农杆菌菌株这一问题得到了解决，但仍然没有检测到绿色荧光，其原因不清楚。本文又利用另一Ti质粒pCMBIA1301为骨架和pIG1783质粒的绿色荧光蛋白表达盒构建了新的双元载体pCB-hg。由于该GFP已经成功地在一些真菌中得到了表达，所以由该表达盒构建成地pCB-hg双元载体是有希望获得荧光蛋白表达的载体。该双元载体的转化农杆菌已经完成，农杆菌介导真菌转化的工作正在进行。更多还原

【Abstract】 Trichoderma reesei (anamorph: Hypocrea jecorina) is a biotechnically important filamentous fungus known as an efficient producer of enzymes and proteins. As one of the best known cellulolytic organisms, T. reesei can produce readily and in large quantities a complete set of extracellular cellulases for the degradation of cellulose and hemicellulose. Because of its capacity to secrete enzymes in high yields, T. reesei has been exploited as an industrial host for homologous and heterologous protein production. Knowledge gained from the genetic sequence of T. reesei will give researchers important insights into the synthesis and regulation of important enzymes, which will accelerate the development of bioprocesses for a range of industries and applications. The genome sequence of T. reesei, as the first member of Trichoderma species, has been completed and available freely (http://genome.jgi-psf.org/Trire2/Trire2.home.html). This work has significantly advanced the understanding of this biotechnically important organism and offered an opportunity to identify important new genes critical to its growth and development. Therefore, developing high-through-put methods to work out the biological function of these genes is of great significance. Genetic approaches, producing a large mutant population by disruption or replacement of genes and then studying the related fungal phenotypes, are powerful tools for deciphering gene function.1. Agrobacterium-mediated transformation (AMT) of T. reesei as an efficient tool for random insertional mutagenesisIn this study, filamentous fungus T. reesei QM9414 was successfully transformed with Agrobacterium tumefaciens AGL-1 for random integration of transforming DNA (T-DNA). The T-DNA binary vector pBI-hph was constructed by introducing the hygromycin B phosphotransferase gene (hph) isolated from pAN7-1 into pBIN121 between the right and left borders of T-DNA. Co-cultivation of T. reesei conidia or protoplasts with A. tumefaciens containing pBI-hph in the presence of acetosyringone resulted in the formation of hygromycin B-resistant fungal colonies with high transformation frequency. Nine randomly selected resistant clones were proved to be stable through mitotic cell division. The integration of the hph gene into T. reesei genome was determined by PCR and Dot blot analysis. TAIL-PCR successfully rescued the T-DNA borders and the flanking genome sequences from the putative transformants. Sequence analysis of the nine recombination junctions from TAIL-PCR revealed that no homology was found among them. The results showed that T-DNA inserts occurred evidently by fusing DNA at T-DNA borders via random recombination. It suggests that Agrobacterium-mediated transformation is a potentially powerful tool towards tagged mutagenesis and gene transfer technology for T. reesei. A population of T-DNA tagged T. reesei mutants has been obtained from the AMT transformation.2. Screening and characterization of the morphological and developmental mutants in T.reeseiWe screened the T-DNA tagged insertional lines of T. reesei and found seven strains with abnormal phenotypes. Among these strains, two mutants had T-DNA insertion at the TrCCD1 gene locus with different locations, designated ccdO and ccdP, respectively. ccdO had a T-DNA insertion into the open reading frame of the gene TrCCD1 and ccdP had a insertional mutagenesis in the promoter region of the same gene. Sequence analysis showed that TrCCD1 gene encodes a carotenoid cleavage dioxygenase, which belongs to RPE65 family found in fungi and higher organisms. Disruption of TrCCD1 caused several dramatic phenotypes. The hyphae from wild-type QM9414 colony extend rapidly while those from the disruptants radiate slowly and tend to form an incompact colony. The mutants are particularly distinctive in producing slow-growing hyphae and the colony growth rates of the mutants and wild-type strains were compared on minimal agar over a period of several days. The colony growth rate of each mutant strain was almost uniform; however, it was only one-half to three-fifth that of the parent, QM9414. When the strains grew on the MM with 0.2% Triton X-100, colonies from disruptants form some long aerial mycelia, in contrast to wild type. ccd mutants are also defective in sporulation. Though colonies from disruptants form some long aerial mycelia, they are difficult to form conidiophores. Wild type QM9414 strain produced green spores dispersing the Petri dishes, and the ccdO and ccdP disrupts only displayed a bit green in the center of medium after four days of incubation. On the same time, color displayed in mutants’ medium was quite different from that in wild-type’s medium, and carotenoid content in the former medium was higher than that in the later. These results suggeset that CCDs are important for the hyphal growth and conidiophore development of filamentous fungi.The other five mutants selected from phenotype screening were: PM2, PM48, HP7, HP13 and HPL1. They displayed different morphology. In contrast to the wild type, their hyphae extended slowly, some of them had radiant colony or white colony, and even some couldn’t perform sporulation. Most interestingly, PM2 had curved hyphal tips and their mycelium showed many small "heads". TAIL-PCR successfully rescued the T-DNA borders and the flanking genome sequences from the mutants PM48 and HPL1. In PM48, T-DNA was inserted into the ORF of the gene encoding a cell cycle-associated protein. While in HPL1, the gene disrupted by T-DNA insertion probably encoded a polyadenylate-binding protein, and the mutation of this gene reduced the cellulolytic ability of T. reesei.3. Screening and characterization of the cellulolytic mutants in T.reeseiAbout two hundred T. reesei AMT transformants, which had been proved to be inserted by T-DNA on the molecular level, were screened by the sizes of the clearing zones on cellulose CF11 agar plates. Among them, four mutants showed different cellulolytic ability: PM3, PM23, HPL1 and 36H-6. The clearing zones of PM3 and PM23 were about 30% bigger than that of QM9414, while HPL1 and 36H-6 had the smaller zones, and even 36H-6 couldn’t form the complete clearing zone. The T. reesei FPA activity reached the maximal value between 18h and 24h. The rnaximal FPA activities of PM3 and PM23 were both 30% higher than that of wild type, and PM3 reached the maximal activity ahead of the wild type and PM23 had the longer high FPA activity. Another two mutants HPL1 and 36H-6 had the lower FPA activity than that of QM9414, and even HPL1 had only 1/3 activity of QM9414 at the maximal value. The T. reesei CMCase activity reached the maximal value at 24h. The maximal CMCase activities of PM3 and PM23 were both higher than that of wild type, while HPL1 and 36H-6 had the lower CMCase activity. It is noticeable that the FPA and CMCase activites of HPL1 always keep the lower level. In HPL1, the gene disrupted by T-DNA insertion probably encoded a polyadenylate-binding protein, which involved in RNA processing and modification.4. Construction of the binary vector containing GFP reporter gene and transformation of AgrobacteriumTwo other binary vectors containing both GFP and HPH as double-selective markers based on different Ti plasmids were constructed. In order to construct double-selective-marker vector, an intermediate plasmid pBI-gfp was obtained from the ligation of four fragments, which contained GFP expression cassette with GFP gene joined to the trpC terminator and controlled by the gpdA promoter from A. nidulans. Then this cassette was isolated from pBI-gfp and inserted into pBI-hph between the RB and LB of T-DNA. Thus, the first double-selective-marker binary vector pBI-gh came into being. After co-cultivation between A. tumefaciens and T. reesei, the HPH-resistance transformants was selected. PCR analysis established that the gfp and hph gene also was incorporated into the genome, but we were not able to confirm GFP fluorescence in an examination of the selected transformants. With the available data, we cannot draw conclusions about the expression of this transgene in T. reesei but note that it has been problematic in other organisms owing to aberrant mRNA processing and codon preference. Another binary vector, designated pCB-hg, consisted of a pCAMBIA1301 backbone containing the hph and gfp genes. This GFP gene was isolated from pIG1783 plasmid, which had successfully expressed GFP in different filamentous fungi. pCB-hg vector had been introduced into A. tumefaciens. The AMT of T. reesei and the selection of the transformants are undergoing experimental procedures.更多还原