【作者】 武敏；

【导师】 马文丽；

【作者基本信息】 山西大学 ， 动物学， 2012， 硕士

【摘要】 盐肤木(Rhus chinensis)是漆树科(Anacardiaceae),盐肤木属(Rhus)落叶小乔木,它作为中国经济价值极高的树种,可供制药、榨油、观赏和做工业染料的原料等。目前对盐肤木的研究主要在其成分及药用价值、形态特征、栽培方法和技术等方面,而有关其体内防御酶的研究很少。苯丙氨酸解氨酶(phenylalanine ammonia-lyase, PAL)是苯丙烷代谢途径中的关键酶和限速酶,植物的初级代谢和苯丙烷类代谢就是被它所连接,PAL是与植物的抗逆性密切相关的酶,是一种防御酶。本论文以盐肤木为研究材料,对其PAL基因进行分子克隆,构建重组表达载体,经原核表达系统得到其重组蛋白,进一步对其性质和功能进行研究。主要研究结果如下：1.本研究提取得到的盐肤木总RNA,28SrRNA和18SrRNA的电泳条带清晰,没有拖尾现象,完整性很好,能用于后续分子克隆等实验。2.首次得到盐肤木PAL基因(RcPAL)的ORF序列。RcPAL基因全长cDNA的长度为2491bp,其中2124bp的ORF编码707个氨基酸,5’端有一段197bp的非编码区(5’UTR),3’端有一个170bp的非编码区(3’UTR)。RcPAL全长cDNA序列是漆树科中首次被报道的。3.构建成功重组表达载体pET-28a-PAL,长度约为7500bp。在大肠杆菌中,此载体被诱导后得到约77kDa的重组蛋白。经50%咪唑洗脱液得到纯度及酶活性最高的RcPAL酶液。4. RcPAL的最适pH为9.0,最适温度为45℃,Ea为8.03kcal/mol。其动力学曲线呈双曲线型,底物L-苯丙氨酸浓度增大,RcPAL的酶活性也增强,并从底物浓度为20mM开始酶活性趋于平稳。RcPAL的Km为7.9mM, Kcat为52.31s-1。5.对3个重要位点进行定点突变,成功得到3个突变体,Phe126→His126(TTC→CAC)、Ser194→Trp194(TTC→CAC)、Asp374→Ala374(TTC→CAC),突变体M1、M2、M3的PAL酶活性为对照样品的25%、2%、20%,证明这3个位点是酶活性位点。将Phe126突变为His126后,突变体M1的PAL相对酶活性仅为对照的25%,而突变体M1的TAL相对酶活性却为对照的22倍,证明Phe126是此酶的底物特异性位点。更多还原

【Abstract】 Rhus chinensis belonging to Anacardiaceae, Rhus, a small deciduous tree, is a major economic tree species in China. This tree can be used to make pharmaceuticals, extract oil and serve as the raw materials of industrial dye. At present the researches of the Rhus chinensis were about its composition, medicinal value, morphological characteristics, cultivation method and technique, but little attention has been paid to its defense enzyme.PAL is the key enzyme that connects the plant primary metabolism and benzene propane metabolism, catalyzes the first step reaction of benzene propane metabolic, also is the key enzyme of phenylpropanoid pathway. It is directly related to plant resistance, and is a defense enzyme.This paper used Rhus chinensis as research material to clone its PAL gene, build a recombinant expression vector, obtain its recombinant protein by the prokaryotic expression, and further research its function and quality. The main results were as follows:1. The study got the total RNA of Rhus chinensis,28S rRNA and18S rRNA had clear stripe, and showed no degradation phenomenon, suggesting it can be used in the subsequent molecular cloning.2. The study acquired the ORF sequence of RcPAL for the first time. The full-length cDNA of RcPAL was2491bp, including2124bp open reading box, which encoded707amino acids. Its5’end noncoding region was197bp (5’UTR) in length, while the3’end noncoding region was170bp (3’UTR). The cDNA sequence of Re PAL was the first report in Anacardiaceae.3. The study built the recombinant expression vector pET-28a-PAL, with a length was7500bp and encoding a77kDa protein. We got the pure and high activity RcPAL enzyme by50%imidazole elution fluid.4. RcPAL’s optimum pH was9.0, the optimum temperature was45℃, and Ea was8.03kcal/mol. Its dynamics curve was hyperbolic. Its activity increased with increasing concentration of the substrate L-phenylalanine. Its Km was7.9mM, and Kcat52.31s-1.5. The study got three mutants, Phe126→His126(TTC→CAC) Ser194→Trp194(TCG→TGG). Asp374→Ala374(GAT→GCT). The activity of mutants M1, M2, M3is25%,2%,20%of CK, which proved that the three sites in catalytic reaction played an important role. Phe126→His126, mutant M1’s PAL activity was only25%of CK, but its TAL activity was22times as much as that of CK. So Phe126was important substrate specificity site.更多还原