【作者】 魏宁；

【导师】 樊军锋；

【作者基本信息】 西北农林科技大学 ， 林木遗传育种， 2013， 硕士

【摘要】 油松在松属中的地位非常突出，油松分布范围广，适应能力强，是华北、西北以及东北部分地区荒山造林的先锋树种。对于北方地区水土的保持，水源的涵养以及大气的净化发挥着重要的作用。长期以来，油松一直受到病虫害的侵袭，一定程度上制约了北方地区荒山植被面积的扩大。苯丙氨酸解氨酶（phenylalanine ammonia-lyase， PAL）是苯丙烷类代谢过程中的关键酶和限速酶，其代谢产物参与黄酮、木质素和植保素等物质的合成，这些化合物对植物的生长发育、抗病虫以及抗逆性等具有重要作用。本文以油松作为研究对象，克隆了其苯丙氨酸解氨酶基因（TaPAL），并在大肠杆菌中成功表达，以期为将来的油松转基因工程育种提供一定的理论依据，同时也为深入研究苯丙氨酸解氨酶结构和功能奠定了分子基础。本研究主要取得的结果如下：1.油松PAL基因CDS区的克隆及生物信息学分析。利用同源克隆的方法，分别扩增TaPAL的cDNA和DNA序列。对得到的序列分析表明，该cDNA序列全长2157bp，编码718个氨基酸，包含有一个完整的ORF（Open reading frame），命名为TaPAL（Genbank登录号：JX280460）。预测的TaPAL蛋白分子量为78.24Kd，理论等电点PI为6.04，GRAVY值为-0.171（亲水性蛋白）。使用SOPMA软件对TaPAL氨基酸序列进行二级结构预测，结果表明，该序列包含57.1%的α-螺旋，5.57%的β-转角，29.39%的无规卷曲和7.94%的延伸链。SignalP3.0分析表明，该序列不含信号肽，说明该蛋白不属于胞外分泌蛋白。TMPred在线工具预测TaPAL跨膜结构域表明在第261-286氨基酸序列可能存在一个由内到外的跨膜区。以欧芹PAL蛋白为模型，利用Swissmodel对TaPAL蛋白进行三维建模，并通过Pymol软件分析。预测的TaPAL氨基酸序列与水稻、玉米等含有相同的脱氨基位点和催化活性位点。核苷酸序列多重比较表明：TaPAL基因与其他植物PAL基因高度同源，其中与马尾松PAL基因同源性达到99%。PAL系统进化树显示，TaPAL与松科类PAL聚为一个大类。另外对比发现，TaPAL的DNA和cDNA序列完全一致，即TaPAL无内含子。2．油松PAL基因原核表达载体的构建及表达分析。利用基因重组技术，以油松PAL基因（TaPAL）CDS区(2157bp)作为外源基因的同源重组片段，构建了pET-28a-TaPAL重组表达载体，在大肠杆菌BL21（DE3）中融合表达并进行分析。SDS-PAGE检测表明，在分子量80KD处产生一条特异性蛋白条带，与预测蛋白大小一致。对蛋白表达条件优化表明，在温度28℃，IPTG浓度1.5mmol·L-1,时间3h条件下，重组质粒的表达量最大，但重组蛋白主要以包涵体形式存在。粗酶活性测定表明，粗酶（在20℃、IPTG浓度1mmol·L-1条件下诱导10h）比活力为55.3μmol/min﹒g pro,即55.3U/g，略大于欧芹重组PAL的比活力（53.99μmol/min﹒g），低于大豆重组PAL的比活力（211.7μmol/min﹒g）更多还原

【Abstract】 The status of Pinus tabulaeformis is very prominent in Pinus, it is the pioneer tree speciesfor barren hill afforestation in northwest and northeast of China because of its widedistribution range and strong ability to adapt. Pinus tabulaeformis plays an important role inair purification、soil and water conservation in northern area. The Pinus tabulaeformis hasbeen invading from pets and diseases for a long time, which restricts expansion of the area ofbarren hill vegetation in a certain time. Phenylalanine ammonia-lyase is a key andrate-limiting enzyme in phenylpropanoid pathway, and it’s metabolite take part in synthesis offlavone、lignine、phytoalexins and so on, which have an important part in plant growth、stressresistance、 pets and disease resistance. In this syudy, the gene of phenylalanineammonia-lyase(TaPAL) was cloned from Pinus tabulaeformis, and the recombinant proteinwas successfully expressed in BL21(DE3), the study provided some theoretical foundation forgenetic engineering of Pinus tabulaeformis and established the molecular basis for furtherstudy on the structure and function of TaPAL. The results were as followed:1.Molecular clone and bioinformatics analysis of PAL Gene(CDS) from Pinustabulaeformis. The DNA and cDNA sequences of PAL were obtained by the method ofhomology cloning. Bioinformatic analysis showed the cDNA sequence was2157bp in length,containing a complete open reading frame and718amino acids were encoded, designated asTapal (Accession number：JX280460). The molecular weight was predicted to be78.24Kdwith an isoelectric point at6.04, and the GRAVY value was-1.171, which was supposed to bethe hydrophilic protein. Secondary structure of TaPAL amino sequence was predicted usingSOPMA showed that: the sequence contained57.1%of α-helix,5.57%of β-fold,29.39%ofrandom coil and7.94%extended strands. Singnal3.0analysis indicated there was no signalpeptide in the sequence, so the protein would not be secreted to the outside of the cell.TMPred analysis suggested that a transmembrane domains from inside to outside might beexisted in261-286amino acid. The3D modeling of TaPAL protein was calculated takingadvantage of Swissmodel based on the model of Parsley PAL’s protein. The domination sites and catalytic active sites appeared in PAL protein of Oryza and Zea mays were also found inthat of inferred protein sequence. The TaPAL gene sequences was highly homologous to otherPALs’ from the result of multiple comparisons of nucleotide sequence and was99%homologous with Pinus massoninana’s PAL. Phylogenetic tree analysis suggested that all thePAL of Pinaceae clustered into a major category. Moreover, this gene was found to beintronless because the cDNA and genomic DNA sequence of TaPAL were completely thesame.2. Construction and expression analysis of prokaryotic expression vector of TaPAL gene.The recombinant expression vector pET-28a-TAL was constructed by recombinant-DNAtechnique using the CDS fragment (2157bp) of TaPAL and transformed E.coli BL21(DE3) forfusion expression.It turns out: SDS-PAGE Gel electrophoresis indicated that an anticipatedprotein molecular weight of about80KD was detected. The best expression conditions is28℃、1.5mmol·L-1and3h. Most of pET-28a-TAL protein exists in the form of inclusion bodyat the optimal condition. The specific activity of crude enzyme(20℃、1mmol·L-1IPTG、10h)is55.3μmol/min﹒g pro, the date was slightly larger than Parsley’s(53.99μmol/min﹒g) butlower than soybean’s(211.7μmol/min﹒g).更多还原