【作者】 胡国库；

【导师】 张义正；

【作者基本信息】 四川大学 ， 遗传学， 2006， 博士

【摘要】 黄孢原毛平革菌（Phanerochaete chrysosporium）属于白腐担子真菌，是研究木质素生物降解的模式生物，因其具有出色的降解木质素的能力，而且对环境中的有毒物质、爆炸污染物等也有降解作用，因此P. chrysosporium在造纸工业、环境保护等方面具有重要的应用前景。研究表明，P．chrysosporium降解木质素是由众多的胞外过氧化物酶参与的复杂的生物过程，LiP和MnP是主要的2类过氧化物酶。Northern blot和RT-PCR分析表明。这2类酶均受到C、N、O等因素的调节，暗示在其基因的5’-端可能存在顺式调控元件与反式作用因子相互作用调控基因的转录。已有的研究表明，在lipC 5’-端存在2个蛋白质结合元件——PBE1和PBE2。本研究在这一工作的基础上做了如下两方面的研究工作。首先，分别以PBE1、PBE2和PBE1+PBE2为钓饵，利用酵母单杂交技术对低氮培养基中培养3d P. chrysosporium的eDNA文库进行筛选，研究结果表明：以PBE1为钓饵筛选到9个cDNA序列；以PBE2为钓饵筛选到的6个序列；以PBE1+PBE2为钓饵所筛选到10个序列。对这25个eDNA序列进行了较系统的生物信息学分析。结果表明，只有4个序列编码的蛋白质能定位在细胞核中，编号是PBE1-A20.3、PBE1-A12.4、PBE2-A5.1和PBE1-2-A27.1，因此对这4个克隆进行了较深入的分析。序列分析表明，PBE1-A20.3编码一种未知蛋白；PBE2-A12.4编码的蛋白质与多功能蛋白14-3-3有较高的同源性，而14-3-3蛋白参与细胞凋亡、细胞分裂、信号转导、蛋白跨膜转运、基因转录等众多重要生命活动过程，14-3-3蛋白主要以蛋白质一蛋白质相互作用的方式发挥功能。在动物、植物以及酵母中，14-3-3还可以与十字型DNA结合调节DNA复制。功能位点分析表明，PBE2-A5.1编码蛋白质含有1个ATP结合结构域和1个亮氨酸拉链结构，很多调控蛋白质中含有亮氨酸拉链结构，包括CCATT盒及增强子结合蛋白、cAMP应答元件（CRE）结合蛋白、酵母一般调控蛋白GCN4、octamer-结合转录因子2（Oct-2／OTF-2）等。PBE1.2-A27.1编码的蛋白质含HIT（histidine triad）结构域，含该结构的蛋白可以与核酸结合。为进一步鉴定酵母单杂交筛选到的阳性克隆与顺式元件的结合作用，利用pET-28a载体对筛选到的14-3-3蛋白编码序列进行原核生物表达，SDS-PAGE分析表明，14-3-3蛋白在Escherichia．coli BL21中以部分包涵体和部分可溶性形式存在，分子量为33 kD。通过Ni柱纯化得到E.coli中可溶表达的14-3-3蛋白，并用透析和PEG20000浓缩该蛋白用于体外结合实验（GMSA）。表达的14-3-3蛋白与³²p标记的PBE1+PBE2探针混合后，经低离子强度聚丙烯酰胺凝胶电泳、放射自显影表明，14-3-3蛋白电泳有明显的延迟现象，当加入40倍的冷探针后，延迟现象减弱，加大14-3-3蛋白量后，延迟现象增强，表明14-3-3蛋白可以与该探针特异结合。在上述研究工作基础上，以14-3-3蛋白为钓饵，利用酵母双杂交技术筛选P.chrysosporium 3d eDNA文库，在SD／-Ade／-His／-Leu／-Trp缺陷培养基平板上共获得了约601个单克隆。进一步分析发现，只有少部分克隆可激活3个报告基因。分离自这些克隆中的质粒DNA转化大肠杆菌，利用HaellI和Sau3AI限制酶切分析可将其分为9类。序列测定和同源分析结果表明，测定的6个序列均具有多个潜在的磷酸化位点，也是14-3-3蛋白潜在的结合位点。其中有2个序列具有WD结构域，表明14-3-3蛋白在P.chrysosporium中也是一个多功能蛋白质。Y2H147与转录因子有一定相似性。高级结构分析显示，P.chrysosporium中的14-3-3蛋白与其它物种中的一样，含有9个Helix结构，且可以形成杯状的同源二聚体结构。蛋白质．蛋白质预测结果显示，14-3-3蛋白可以与Y2H147、389、412号克隆结合，推测14-3-3蛋白在P.chrysosporium中参与多个细胞过程，尤其在基因转录调控过程中发挥某种作用。另外，RT-PCR分析表明14-3-3基因在P.chrysosporium不同代谢阶段没有明显的转录差异。更多还原

【Abstract】 Tie white-rot basidiomycete-Phanerochaete chrysosporium is an excellentmodel organism for studying the mechanism of lignin biodegradation. Due to theexcellent ability to degrade lignin, as well as related compounds found in explosivecontaminated materials, P.chrysosporium is greatly potential in bio-pulping andenvironmental protection. The extracellular ligninolytic system is a complicatedprocess and comprises many enzymes, including lignin peroxidases and manganeseperoxidases. Northern blot and RT-PCR analyses revealed that the transcription oflips and mnps are regulated by nutrients such as carbon and nitrogen, etc. Theprevious investigations showed that two nuclear protein binding sites, named PBE1and PBE2, located upstream of the transcription initiation site of the lipC gene.Therefore, it is very important to clone the genes encoding the transcriptionalregulators of lip genes expression.First, the yeast one-hybrid was performed by screening 3-day cDNA library ofP. chrysosporium, using PBE1, PBE2 and PBE1+PBE2 as bait, respectively. Theresults suggested that there were 9, 6 and 10 candidate clones related to regulatinglipC transcription by targeting PBE1, PBE2 and PBE2+PBE2, respectively.Bioinformaties analysis revealed that only 4 clones are localized in nucleus, namedPBE1-A12.4, PBE1-A20.3, PBE2-A5.1 and PBE1-2-A27.1. Sequence analysesrevealed that PBE1-A20.3 is an unknown protein. PBE1-A12.4 has high homologywith 14-3-3 protein. The 14-3-3s are important in the regulation of such crucialcellular processes as apoptosis, cell division, signal transduction, protein trafficking,transcription. The 14-3-3s usually play roles via direct protein-protein interactions.Previously, 14-3-3 proteins were characterized a cruciform DNA-binding activity inhuman, plant and yeast. The interactions are involved in DNA replication. Functionsites analysis suggested that the clone PBE2-A5.1 contains an ATP binding site and a leucine zipper pattern. The pattern is present in many gene regulatory proteins, suchas: the CCATT-box and enhancer binding protein （C/EBP）, cAMP response element（CRE） binding proteins, etc. PBE1-2-A27-1 encoded a protein containing, histidinetriad （HIT） domain. Proteins in the HIT superfamily are conserved asnucleotide-binding proteins.To identify the interactions between the positive clones and cis-element, theORF of 14-3-3 was then subcloned and expressed in Escherichia.coli using pET28aexpression vector. SDS-PAGE analysis showed that the recombinant protein exist asinclusion body and soluble fraction, with molecular weight 33 kD. Then the solubleproduct was purified with Ni-colunm. The purified protein could bind toPBE1+PBE2, labeled with ³²p by gel mobility shift assay （GMSA）. The resultsshowed that the shifted band was enhanced when increase 14-3-3 protein and theshifted bands disappeared when 40×cold probe was added. These results indicatedthat 14-3-3 protein was able to specifically bind to the PBE1+2 probe.Secondly, the 14-3-3 gene was used to screen the 3-day library by yeasttwo-hybrid and more than 600 clones were obtained on the SD/-Ade/-His/-Leu/-Trpplates, but a few of positive recombinants could activate three reporter genes.Plasmids isolated from yeast transformants were successfully transformed into E.coli. These clones could be classified into 6 types on the basis of the digestionpatterns with HaeⅢand Sau3AI as well as their sequences. A PROSITE searchrevealed multiple potential phosphorylation sites in the 6 proteins, which arepotential binding sites of 14-3-3 protein. Two proteins containing predicted WDdomain could interact with 14-3-3. The amino acid sequence of Y2H147 is 27%similar to transcription initiation factor TFIID. The three-dimensional model of14-3-3 suggested that it contains 9 helix structures and may form homodimer in P.chrysosporium. Protein-protein prediction indicated that 14-3-3 protein could bindwith Y2H147, Y2H389 and Y2H412. These results suggested that 14-3-3 in P.chrysosporium may function in different cellular processes, especially in genetranscriptional regulation.更多还原