独一味苯丙氨酸解氨酶基因的克隆与表达分析

以独一味叶片为材料,采用RT-PCR和RACE方法克隆了独一味苯丙氨酸解氨酶基因（PAL）的全长cDNA,命名为LrPAL基因。测序结果表明,LrPA L基因全长2 298 bp,含有1个2 145 bp的完整开放阅读框（ORF）,编码714个氨基酸。蛋白序列分析表明,其包含典型的PAL活性中心序列（GTITASGDLVPLSYIA）,与其他植物的PAL蛋白有很高的同源性。系统进化树分析表明,独一味LrPAL与唇形科植物的PAL蛋白聚为一类,说明两者的亲缘关系较近。用 Real-Time PCR方法检测发现,LrPAL基因在独一味的叶中表达量最高,茎中表达量最少。研究结果推测,从独一味中克隆获得的苯丙氨酸解氨酶基因（LrPAL）是典型的PAL家族成员,在独一味各组织发育过程中具有重要功能。     

粘红酵母产L-苯丙氨酸解氨酶发酵培养基的优化

通过单因子和正交试验 ,对粘红酵母产 L -苯丙氨酸解氨酶 ( PAL )培养基进行优化 ,L-苯丙氨酸的积累浓度可以从 2 .0 g/1 0 0 ml提高到 3 .3 g/1 0 0 ml,最终得到了 L-苯丙氨酸解氨酶发酵的最适条件     

油木奈果实苯丙氨酸解氨酶基因的分离与表达分析

该试验从(木奈)褐变果实均一化全长cDNA文库中获得一个苯丙氨酸解氨酶全长基因,命名为PsPAL,并对该基因进行了生物信息学分析和表达模式的研究.结果表明:(1) PsPAL基因全长2 497 bp,开放阅读框为2 154bp,编码718个氨基酸,蛋白质分子量为78 kD,理论等电点为6.6.(2)系统进化树比对分析表明,PsPAL蛋白与蔷薇科甜樱桃PaPAL属于同簇,具有苯丙氨酸解氨酶-组氨酸解氨酶(PAL-HAL)和苯丙氨酸解氨酶(PAL)保守区域.(3) P.sPAL在(木奈)果实发育的前期表达量较高,在花后50 d表达量最高,随后开始下降,在成熟果中表达较弱.(4)荧光定量PCR分析表明,在响应机械损伤和低温处理后,与对照相比,PsPAL呈明显的上调表达趋势;高温和无氧处理后PsPAL呈先上升后下降的趋势;乙烯处理后,PsPAL呈上调-下调-上调的变化趋势.     

丝瓜苯丙氨酸解氨酶基因PAL克隆及表达分析

苯丙氨酸解氨酶(PAL,phenylalanine ammonia-lyase)是苯丙烷途径代谢的关键酶,在果蔬褐变中发挥着重要作用。为了探究丝瓜中PAL基因的功能,本研究以丝瓜果实为材料,采用RACE和RT-PCR技术从丝瓜果肉中分离获得丝瓜PAL基因,命名为Lc PAL,Gen Bank登录号为:KP341758。该基因全长2406 bp,具有完整的开放阅读框(ORF),共2145个碱基;预测编码715个氨基酸,理论分子量(Mw)为77.69 k D,等电点(p I)为6.11,编码的蛋白与黄瓜和香瓜的同源蛋白相似性均在93%以上,显示其高度的保守性。系统进化树分析显示,丝瓜与同为葫芦科的黄瓜、香瓜的PAL蛋白的亲缘关系较近,聚为同一类。Motif Scan分析显示,Lc PAL编码的蛋白包含有PAL-HAL(60~525位)、PLN02457(8~715位)及phe_aml_yase(24~705位)3个保守结构域和1个酶活性中心序列(197~212位),属于典型的Lyase_I_Like超家族。Wolf Psort预测其亚细胞定位于叶绿体或内质网中。实时荧光定量PCR分析显示,Lc PAL基因在普通丝瓜果实、花、茎、叶和根中均有表达。Lc PAL在所选的8个丝瓜品种中表达存在一定差异,在普通丝瓜中的表达量均高于有棱丝瓜;在普通丝瓜品种闽丝3号鲜切及采后储藏褐变过程中,Lc PAL初期表达上调,后期表达量受到抑制,且与其酶活性的变化趋势基本一致。普通丝瓜Lc PAL表达与果肉褐变的发生过程高度相关,表明Lc PAL在普通丝瓜果肉褐变中发挥着作用。     

大蕉苯丙氨酸解氨酶基因的克隆、鉴定和表达分析

为了研究苯丙氨酸解氨酶基因与大蕉(Musa ABB cv. Dongguandajiao)抗枯萎病的关系,利用 RT-PCR 和 RACE技术克隆了大蕉苯丙氨酸解氨酶基因全长 cDNA。此 cDNA 长 1 300 bp,包含一个长为 1 191 bp,编码 397 个氨基酸的完整开放阅读框(ORF),推导的氨基酸序列与水稻 PAL 基因氨基酸序列同源性达 89%,将此基因命名为 M-PAL。Southern杂交结果表明大蕉中存在一个包含 4-5 个 PAL基因的基因家族,将此基因克隆到大肠杆菌表达载体 pET32(a )中,表达的蛋白质分子量大小与推导的相一致,并且表达的蛋白质表现出 PAL 酶活性。对接种香蕉枯萎病菌 4 号生理小种(Fusarium oxysporumf. sp. cubense (FOC) race 4 )后大蕉叶片中 M-PAL基因的转录谱进行研究表明,在接种枯萎病菌后,M-PAL基因在叶片中的转录水平提高,因此推测 M-PAL基因的表达可能与香蕉枯萎病抗性相关。     

苯丙氨酸解氨酶

苯丙氨酸解氨酶由四个亚基组成,含两个脱氢丙氨酸残基。植物酶具有内在不稳性,由多种同工酶组成。酶催化过程中发生构象变化,底物经过负碳离子中间体完成反应。该酶并非是二单体负协同变构酶。一级结构表明,酶以无规则卷曲结构为主。酵母基因约2.7kb,有六个内含子,编码75kD_a肽。植物酶由多基因编码,有一个内含子,编码78kD_a肽。启动子部位有两个富含A、C碱基的序列,为胁迫作用基因活化因子结合部位。     

植物苯丙氨酸解氨酶基因的研究进展

苯丙氨酸解氨酶(phenylalanineammonia-lyase,PAL)是连接植物初级代谢和苯丙烷类代谢、催化苯丙烷类代谢第一步反应的酶。综述植物PAL基因的研究进展,主要包括PAL基因的结构特点、表达特点和PAL基因表达的调控机制,并指出今后对PAL基因的研究方向。     

奉节脐橙果实苯丙氨酸解氨酶活性及其基因表达与果皮褐变的关系

柑橘果皮褐变严重影响果实的商品价值和耐贮性．以奉节脐橙（Citrus sinensis Osbcck）果实为材料,通过采后常温、涂蜡、低温、机械损伤等处理研究了果实果皮褐变率、苯丙氨酸解氨酶（PAL）活性及PAL上基因在果皮褐变过程中表达水平的变化。结果表明,涂蜡、损伤处理均极显著地提高果实的果皮褐变率,而低温贮藏可显著降低其发生率;贮藏期问各处理的PAL活性均呈上升趋势,损伤处理PAL活性显著高于对照。果实发生褐变或受到机械损伤后,PAL2、PAL6基因的表达均比对照明显增强。结果首次表明脐橙果实PAL活性变化以及PAL2基因的表达与果皮褐变具有非常密切的关系．     

粘红酵母产L-苯丙氨酸解氨酶的条件和酶法合成苯丙氨酸的研究

研究了粘红酵母(Rhodotorula glutinis)中L-苯丙氨酸解氨酶(PAL)(EC4.3.1.5)的产酶条件及用此酶把反式肉桂酸转化成苯丙氨酸的条件.结果表明,在下列培养基(g/L)及培养条件下PAL的活力较高:酵母膏10.0,蛋白胨10.0,NaCl5.0,KH_2PO_4 0.5,苯内氨酸0.5,(NH_4)_2SO_41.0,葡萄糖5.0,pH6.0—6.5,培养温度为30℃.转化过程中,[NH_4~+]对初速度的影响符合米氏方程,其K_m和V_(max)分别为16.85mol/L和5.96 g·L~(-1)·h~(-1),最适pH为10.0.底物肉桂酸对反应初速度的影响,在低浓度时有激活作用,在高浓度下则有抑制作用.肉桂酸转化为苯丙氨酸的转化率在60.0%以上.     

宁夏枸杞苯丙氨酸解氨酶基因的cDNA克隆及其表达分析

为探讨宁夏枸杞(Lycium barbarum)苯丙氨酸解氨酶基因(LbPAL)的表达特征,采用PCR法克隆了宁夏枸杞LbPAL基因的cDNA,并用实时定量PCR法分析了其表达特征。结果表明:宁夏枸杞的LbPAL基因的全长cDNA为2321 bp,包含2163 bp、编码720个氨基酸的开放阅读框;LbPAL与茄科其他物种的PAL氨基酸序列及三维结构具有较高保守性;与茄科物种的PAL聚类在同一个分支中。LbPAL在叶、花瓣、S1期果实的表达量较高,而在根及S2~S5期果实的表达水平较低。在NaCl胁迫处理下,LbPAL在根和茎中的表达量均有下调的趋势;而在叶片中,LbPAL表达量先急剧增加而后急剧下降并趋于稳定。这为解析宁夏枸杞中类黄酮化合物的生物合成调节及生理功能提供了参考。     

Control of phenylalanine ammonia-lyase gene promoters from pea by UV radiation

The gene fusion system was used to study UV light-control of PS PAL1 and PS PAL2 genes encoding phenylalanine ammonia-lyase of pea. The induction of pea PAL promoters was analysed in transgenic tobacco plants. Binary plasmids (derivatives of pBI101.2 vector) containing 5′ regulatory fragments of PS PAL1 and PS PAL2 linked to reporter genes (GUS,LUC) were constructed. The analyses were performed with the use of single constructs (containing one variant of PS PAL promoter and one reporter gene) and dual constructs (containing both PS PAL1 and PS PAL2 promoters connected with different reporter genes). The use of dual constructs enabled the evaluation of both PS PAL promoters activity in the same plant. The analyses of in vitro grown plants have shown that both PAL promoters are strongly induced in leaves subjected to UV radiation. In some cases, the UV-stimulated expression exceeded the exposed areas. This phenomenon was observed more often in the leaves of plants containing the PS PAL1::GUS than PS PAL2::GUS construct. Removal of boxes 2, 4, 5 from PS PAL1 promoter and deletion of its 5′ end region (-339 to -1394) decreases the level of gene expression but does not eliminate its responsiveness to UV.     

Transformation of Rhodosporidium toruloides

Rhodosporidium toruloides protoplasts could be transformed, in the presence of polyethylene glycol (PEG), at frequencies of approx. 1 X 10(3) transformants/micrograms of DNA. The plasmid used, pHG2, which contains the phenylalanine ammonia-lyase (PAL)-coding gene (PAL) of R. toruloides, could replicate as an unstable plasmid in the yeast, or could integrate at the PAL locus to give stable transformants. Plasmids that function in R. toruloides were constructed using either the PAL gene or LEU2 gene of Saccharomyces cerevisiae as dominant selectable markers. R. toruloides transformed with pHG8, which contains both genes, coinherited the two markers. It is also shown that the 2mu replicon of S. cerevisiae does not function in R. toruloides; neither is the PAL gene expressed in S. cerevisiae.     

苯丙氨酸解氨酶在诱导黄瓜幼苗抗寒性中的作用

为了探讨苯丙氨酸解氨酶(PAL)在诱导黄瓜幼苗抗寒性中的作用,采用喷施特异抑制剂(AOPP)的方法控制PAL活性,测定幼苗抗寒性的变化.结果表明： 低温可以诱导黄瓜幼苗叶片中PAL的基因表达和活性升高;喷施AOPP显著抑制了叶片中PAL活性,减少了酚类和类黄酮物质的积累.低温对黄瓜幼苗造成显著伤害,AOPP预处理加剧了低温对幼苗的损伤,幼苗抗寒性降低.与对照相比,幼苗叶片中相对电解质渗漏率和丙二醛(MDA)含量显著升高,PSII最大光化学效率(F_v/F_m)降低,光化学猝灭参数Y(NO)升高,胁迫相关基因(PR1-1a、COR47、P5CS、HSP70)的诱导表达受到抑制.低温导致黄瓜幼苗叶片中H₂O₂积累,还原型抗坏血酸(AsA)含量降低,脱氢抗坏血酸(DHA)含量升高,AsA∶DHA减小;喷施AOPP的幼苗中抗氧化酶(过氧化氢酶CAT、抗坏血酸过氧化物酶APX)活性显著低于对照,H₂O₂过量积累,AsA∶DHA更低.施用H₂O₂清除剂可以有效缓解喷施AOPP引起的低温损伤加剧,而施用CAT抑制剂的幼苗对低温胁迫更敏感.表明低温诱导了PAL活性升高,促进了苯丙烷类次生代谢产物的合成,提高了胞内抗氧化酶活性,可有效清除活性氧分子,维持AsA氧化还原状态,缓解低温引起的光损伤和氧化损伤.     

The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana

Phenylpropanoid derivatives are a complex class of secondary metabolites that have many important roles in plants during normal growth and in responses to environmental stress. Phenylalanine ammonialyase (PAL) catalyzes the first step in the biosynthesis of phenylpropanoids, and is usually encoded by a multi-gene family. Genomic clones for three Arabidopsis thaliana PAL genes containing the entire protein-coding region and upstream and downstream sequences have been obtained and completely sequenced. Two A. thaliana PAL genes (PAL1 and PAL2) are structurally similar to PAL genes that have been cloned from other plant species, with a single intron at a conserved position, and a long highly conserved second exon. Previously identified promoter motifs plus several additional sequence motifs were found in the promoter regions of PAL1 and PAL2. Expression of PAL1 and PAL2 is both qualitatively and quantitatively similar in different plant organs and under various inductive conditions. A third A. thaliana PAL gene, PAL3, differs significantly from PAL1 and PAL2 and other sequenced plant PAL genes. PAL3 contains an additional intron, and its deduced amino acid sequence is less homologous to other PAL proteins. The PAL3 promoter region lacks several sequence motifs conserved between A. thaliana PAL1 and PAL2, as well as motifs described in other genes involved in phenylpropanoid metabolism. A. thaliana PAL3 was expressed at very low levels under the conditions examined.     

Expression in E. coli of the gene encoding phenylalanine ammonia-lyase from Rhodosporidium toruloides

Summary The active sites of the enzyme phenylalanine ammonia-lyase (Pal) from Rhodosporidium toruloides contains a dehydroalanine residue that is believed to be essential for catalytic activity. Furthermore, the dehydroalanine is believed to be added post-translationally as part of a prosthetic group covalently attached to the enzyme. Perhaps for this reason no attempts to produce Pal in foreign host cells have been reported. We have inserted the entire uninterupted pal gene from R. toruloides into the Escherichia coli expression vector pKK 223-3. E. coli cells containing this vector synthesize a protein of the expected size, and extracts prepared from these cells contain a Pal-like activity. The potential implications of this finding are discussed.Offprint requests to: H. Ørum     

Expression of phenylalanine ammonia-lyase gene during tracheary-element differentiation from cultured mesophyll cells ofZinnia elegans L.

Expression of the phenylalanine ammonialyase (PAL) gene during tracheary-element differentiation was studied in mesophyll cell-suspension cultures ofZinnia elegans. Dose-response curves of benzyladenine (BA) and α-naphthaleneacetic acid (NAA) were obtained for the cultures in order to achieve the highest percentage of differentiation. The optimal concentrations of BA and NAA were 0.1 mg·l^-1 and 0.06 mg·l^-1, respectively, which normally stimulated about 40% differentiation by 96 h of culture. The effects of the same ratio but different amounts of BA and NAA on tracheary-element formation have been tested and the results indicate that the absolute amounts of BA and NAA rather than the ratio of them were important for tracheary-element formation in theZinnia cultures. The cells when cultured in the presence of 0.001 mg·l^-1 of BA and 0.06 mg·l^-1 of NAA expanded and divided but did not differentiate. The level of PAL activity, synthesis of PAL protein and the level of PAL mRNA peaked during 72 to 96 h when lignin was actively deposited. This indicated that the PAL gene was temporally and preferentially expressed in association with the lignification during tracheary-element differentiation and thus it can be regarded as a molecular marker for the process. We thank Bejing Agricultural University, the Lundgren Research fund (University of Cambridge) and an ORS award for financial support.