Phenylalanine ammonia-lyase gene families in cucurbit species: Structure, evolution, and expression

Phenylalanine ammonia-lyase （PAL）, the first enzyme of phenylpropanoid pathway, is always encoded by multigene families in plants. In this study, using genome-wide searches, 13 PAL genes in cucumber （CsPAL1-13） and 13 PALs in melon （Cm- PALl-13） were identified. In the corresponding genomes, ten of these PAL genes were located in tandem in two clusters, while the others were widely dispersed in different chromosomes as a single copy. The protein sequences of CsPALs and CmPALs shared an overall high identity to each other. In our previous report, 12 PAL genes were identified in watermelon （CIPAL1-12）. Thereby, a total of 38 cucurbit PAL members were included. Here, a comprehensive comparison of PAL gene families was performed among three cucurbit plants. The phylogenetic and syntenic analyses placed the cucurbit PALs as 11 CsPAL-CmPAL-CIPAL triples, of which ten triples were clustered into the dicot group, and the remaining one, CsPAL1-CmPAL8-CIPAL2, was grouped with gymnosperm PALs and might serve as an ancestor of cucurbit PALs. By comparing the syntenic relationships and gene structure of these PAL genes, the expansion of cucurbit PAL families might arise from a series of segmental and tandem duplications and intron insertion events. Furthermore, the expression profiling in different tissues suggested that different cucurbit PALs displayed divergent but overlapping expression profiles, and the CsPAL-CmPAL-CIPAL orthologs showed correlative expression patterns among three cucurbit plants. Taken together, this study provided an extensive description on the evolution and expression of cucurbit PAL gene families and might facilitate the further studies for elucidating the functions of PALs in cucurbit plants.

Activities of lipoxygenase and phenylalanine ammonia lyase in poplar leaves induced by insect herbivory and volatiles

A study was conducted to explore the defense response in woody plants after insect herbivory. The activities of two enzymes, lipoxygenase （LOX）, a key enzyme ofjasmonate （JA） pathway, and phenylalanine ammonia lyase （PAL）, a rate-limiting enzyme of phenyl- propanoid pathway, were measured in the leaves of one-year-old poplar （Populus simonii × P. pyramidalis ＇Opera 8277＇） cuttings after Clostera anachoreta larvae attack. The results show that the increased activities of LOX and PAL were found not only in the leaves wounded by C. anachoreta larvae but also in their tipper systemic leaves, indicating that JA and phenylpropanoid pathways were activated, and the defense response was stimulated systemically. The increase in LOX and PAL activities in neighboring intact poplar cuttings sug- gested that there exists the interplant communication between poplar plants mediated by the herbivore-induced volatiles. Methyl jasmonate （MeJA） was also proved to be an airborne signal to induce defense response in P simonii × P pyramidalis ＇Opera 8277＇ cuttings.

Genetic diversity and population structure analysis of Pistacia species revealed by phenylalanine ammonia-lyase gene markers and implications for conservation

Information on population genetic structure and crop genetic diversity is important for genetically improving crop species and conserving threatened species. The PAL gene sequence is part of a multigene family that can be utilized to design DNA marker systems for genetic diversity and population structure investigation. In the current study, genetic diversity and population structure of 100 accessions of wild Pistacia species were investigated with 78 PAL markers. A protocol for using PAL sequences as DNA markers was developed. A total of 313 PAL loci were recognized, showing 100% polymorphism for PAL markers. The PAL markers produced relatively more observed and effective alleles in Pistacia falcata and Pistacia atlantica, with a higher Shannon＇s information index and expected heterozygosity in P. atlantica, Pistacia vera and Pistacia mutica. Pairwise assessment of Nei＇s genetic distance and genetic identity between populations revealed a close association between geographically iso- lated populations of Pistacia khinjuk and Pistacia chinensis. The accessions of wild Pistacia species had more genetic relationship among studied groups of species. Analysis of molecular variance indicated 19% among- population variation and 81% within-population variation for the PAL gene based DNA marker. Population structure analysis based on PAL revealed four groups with high genetic admixture among populations. The results establish PAL markers as a functional DNA marker system and provide important genetic information about accessions from wild populations of Pistacia species.

Enhancement of Phenylalanine Ammonia Lyase,Polyphenoloxidase,and Peroxidase in Cucumber Seedlings by Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae) Infestation

In this study, the activities of phenylalanine ammonia lyase （PAL）, polyphenoloxidase （PPO）, and peroxidase （POD） were assayed in cucumber seedlings （Cucumis sativus L.） at 0, 6, 12, 24, 48, 72, and 96 h after they were infested by Bemisia tabaci （Gennadius） using spectrophotometric analysis. The results indicated that herbivore infestation increased the activities of PAL, PPO, and POD. The enzymes showed different activity levels at different times after the infestation. The PAL activity reached the first high peak by 23.1% at 6 h and the highest peak by 29.1% at 48 h compared to the control. The PPO activity reached the first high peak by 22.7% at 6 h and the highest peak by 52.6% at 24 h, and the POD activity reached the highest peak by 213.2% at 6 h and another higher peak value by 135.2% at 96 h. The results suggest that the enhanced activities of the enzymes may contribute to bioprotection of cucumber plants against B. tabaci infestation.

Polyphenol Oxidase, Peroxidase and PhenylalanineAmmonium Lyase Induced in Postharvest Peach Fruitsby Inoculation with Pichia membranefaciensor Rhizopus stolonifer

Rhizopus rot of peach fruits could be significantly suppressed by Pichia membranefaciens. Polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonium-lyase (PAL) activities induced by inoculation with P. membrane faciens or R. stolonifer were studied in postharvest peach fruits. The activities of PPO and PAL in peaches increased significantly after being inoculated with P. membrane faciens + R. stolonifer by 24 h, the activities maintained at a high level throughout the experiment. Under the condition of infected with R. stolonifer alone, activity of PPO and PAL could also increased, but the levels were lower than those treated with P. membrane faciens+ R. stolonifer. However, fruits inoculaed with P. membrane-faciens + R. stolonifer or R. stolonifer alone did not stimulated POD activity. The results suggest that the activation of these defense enzymes is involved in the action of P. membrane faciens against R. stolonifer.

Expression of phenylalanine ammonia lyase as an intracellularly free and extracellularly cell surface-immobilized enzyme on a gut microbe as a live biotherapeutic for phenylketonuria

Phenylketonuria(PKU),a disease resulting in the disability to degrade phenylalanine(Phe)is an inborn error with a 1 in 10,000 morbidity rate on average around the world which leads to neurotoxicity.As an potential alternative to a protein-restricted diet,oral intake of engineered probiotics degrading Phe inside the body is a promising treatment,currently at clinical stage II(Isabella,et al.,2018).However,limited transmembrane transport of Phe is a bottleneck to further improvement of the probiotic’s activity.Here,we achieved simultaneous degradation of Phe both intracellularly and extracellularly by expressing genes encoding the Phe-metabolizing enzyme phenylalanine ammonia lyase(PAL)as an intracellularly free and a cell surface-immobilized enzyme in Escherichia coli Nissle 1917(EcN)which overcomes the transportation problem.The metabolic engineering strategy was also combined with strengthening of Phe transportation,transportation of PAL-catalyzed trans-cinnamic acid and fixation of released ammonia.Administration of our final synthetic strain TYS8500 with PAL both displayed on the cell surface and expressed inside the cell to the Pah^(F263S)PKU mouse model reduced blood Phe concentration by 44.4%compared to the control Ec N,independent of dietary protein intake.TYS8500 shows great potential in future applications for PKU therapy.

玉米苯丙氨酸解氨酶(PAL)家族基因分析及ZmPAL5的功能研究

【目的】挖掘玉米抗旱关键基因、揭示其抗旱分子机制,为培育抗旱玉米新品种提供基因资源和理论指导。【方法】采用转录组数据结合加权基因共表达网络(weighted gene co-expression network,WGCNA)与抗旱性生理生化指标的筛选方法,鉴定与抗旱和复水相关的ZmPAL基因。利用生物信息学方法对编码PAL的基因进行全基因组分析。运用实时荧光定量PCR(quantitative real-time polymerase chain reaction,qRT-PCR)检测ZmPAL基因在干旱处理条件下的表达情况,以及ZmPAL5在不同自交系间的表达特性和在不同组织中的表达模式。最后,利用遗传转化分析ZmPAL5在玉米中的抗旱功能,并借助CRISPR/Cas9技术对PAL5同源基因进行缺失型拟南芥突变体的抗旱性分析。【结果】鉴定了19个玉米ZmPAL基因,其中6个基因聚集在第5染色体上,其编码蛋白多为亲水性酸性蛋白,且PAL家族基因的进化相对保守。ZmPAL基因启动子区域含有大量与激素和非生物应激响应相关的顺式作用元件。确定了6个核心基因,其中4个基因在干旱处理后显著上调表达。尤其是ZmPAL5在干旱胁迫后表达量增加了8.57倍。在干旱胁迫和复水处理条件下,发现抗旱自交系郑8713中ZmPAL5的表达水平显著高于旱敏感自交系B73。同时,ZmPAL5是一个组成型表达基因,在幼茎中表现出高水平的表达。过表达ZmPAL5玉米在干旱胁迫下生长良好,其相对含水量、木质素、叶绿素、可溶性蛋白、脯氨酸含量,以及超氧化物歧化酶、过氧化物酶、过氧化氢酶和抗坏血酸过氧化物酶的活性分别是野生型的1.52、1.49、1.47、1.43、1.44、1.41、1.53、1.41和1.35倍,但丙二醛含量是野生型的0.65倍。PAL5缺失型拟南芥突变体对干旱敏感。在干旱胁迫下,其生理生化指标与过表达ZmPAL5玉米的指标变化趋势相反。【结论】筛选出6个响应干旱胁迫的核心基因(ZmPAL3、ZmPAL5、ZmPAL6、ZmPAL8、ZmPAL11和ZmPAL13),其中,ZmPAL5的表达量与抗旱性呈正相关。ZmPAL5通过影响渗透调节物质含量和抗氧化酶活性正向调节植物的抗旱性和恢复能力。

The phenylalanine ammonia-lyase gene family in Isatis indigotica Fort.:molecular cloning,characterization,and expression analysis

Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase(PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21(DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene(IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that Ii PAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of Ii PALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I.indigotica.

Effect of selenium application on phenylalanine ammonia-lyase(PAL)activity,phenol leakage and total phenolic content in garlic(Allium sativum L.)under NaCl stress

It is well known that salinity has badly effect on plant growth all over the world and greatly reduces crop production in the affected regions.Selenium can function as an antioxidant in plants and also in low concentration can promotes plant growth and produce tolerance against stress.This study was conducted in order to determine the effects of selenium(Se)application(0,4,8 and 16 mg L^-1)on phenylalanine ammonia-lyase(PAL)activity,phenol leakage and total phenolic content of garlic under salt stress(0,30,60 and 90 mM NaCl).The highest PAL activity was recorded at 60 and 90 mM NaCl salinity with application of 8mg Se L^-1.Also,when Se was added to the salt-stress garlic,the level of phenol leakage was decreased significantly at two levels of NaCl concentration(by 52%and 40%at 30 mM NaCl with application of 4 and 16 mg Se L^-1,and by 50%at 90 mM NaCl with application of 4mg Se L^-1,respectively)in comparison to the salt-stressed garlic without Se.The results showed that Se can increase the salt tolerance of garlic by protecting the cell membrane against lipid peroxidation.The highest concentration of phenols was recorded at 90 mM NaCl salinity level with application of 4 and 8 mg Se L^-1,that respectively produced 59%and 51%higher phenols than control treatment without Se.So,application of optimal Se level can increase the potential of garlic in a medium with relatively high level of NaCl.

茉莉花PAL基因家族的多基因组鉴定与表达分析

为深入探究茉莉花PAL基因家族的功能及在香气成分形成中的作用,本研究以福州单瓣茉莉花、双瓣茉莉花和重瓣茉莉花基因组数据为基础,利用生物信息学方法及实时荧光定量PCR技术,对JsPAL基因家族进行多基因组分析。结果表明,在单瓣茉莉花、双瓣茉莉花和重瓣茉莉花全基因组中分别鉴定出4个、1个和4个PAL家族基因,JsPAL家族成员均具有丙氨酸-丝氨酸-甘氨酸(Ala-Ser-Gly)组成的三肽活性中心。共线性分析发现单瓣茉莉花、重瓣茉莉花和拟南芥的PAL基因间的共线性关系与双瓣茉莉花相比更强;系统发育树分析结果显示,JsPAL基因家族分布在2个亚家族中。JsPAL基因启动子区域存在大量与胁迫响应、激素响应、光响应和植物生长相关的顺式调控元件。荧光定量分析结果表明,JsPAL在茉莉花组织中存在特异性表达,多数JsPAL在花中的相对表达量高于其他组织;大多数JsPAL基因在福州单瓣茉莉花中的相对表达量高于双瓣茉莉花;福州单瓣茉莉花中SP_JsPAL2、SP_JsPAL4、MP_JsPAL1和MP_JsPAL3在其花朵预开放期(F3)的相对表达量显著高于其他时期,双瓣茉莉花中DP_JsPAL1在F3阶段的相对表达量也较高;SP_JsPAL1和SP_JsPAL2与2-苯乙醇和苯乙醛含量呈显著正相关,其中SP_JsPAL1还与苯甲醛含量呈显著正相关。说明,JsPAL可能在茉莉花的发育中起重要作用,SP_JsPAL2、SP_JsPAL4、MP_JsPAL1、MP_JsPAL3和DP_JsPAL1可能对福州单瓣茉莉花、双瓣茉莉花的香气成分形成具有重要的调控作用,SP_JsPAL1可能对福州单瓣茉莉鲜灵香气的形成具有重要作用。

Effect of Chloride Ions and Dissolved Oxygen on the Deactivation of Phenylalanine Ammonia-lyase

With the method of synthesizing the L-phenylalanine by trans-cinnamic acid and ammonia in the presence of phenylalanine ammonialyase (PAL) under high pH and high concentration of cinnamic acid, it is not favorable for the conversion because of the deactivation of the PAL.For this reason, it is important to find out the suita-

利用新型红酵母苯丙氨酸解氨酶制备低苯丙氨酸酪蛋白

【目的】挖掘高活性、高稳定性的苯丙氨酸解氨酶(EC 4.3.1.24;penylalanine ammonia-lyase,PAL),为后续在制备无(低)苯丙氨酸特膳食品的应用奠定基础。【方法】从胶红酵母(Rhodotorula mucilaginosa)和双倒卵形红酵母(Rhodotorula diobovata)中克隆到基因RmPAL和RdPAL,并通过生物信息学分析两个酶的序列和结构特征;在大肠杆菌中异源表达纯化RmPAL和RdPAL蛋白,测定最适反应条件和底物特异性;通过高效液相色谱和苯丙氨酸试剂盒测定了RmPAL和RdPAL转化酸解酪蛋白(casein acid hydrolysate,CAH)中苯丙氨酸(L-phenylalanine,L-Phe)的能力。【结果】RmPAL和RdPAL是真菌来源的PAL,分别由3个结构域组成:MIO结构域(MIO domain)、核心结构域(core domain)和屏蔽结构域(shielding domain),活性中心具有催化氨基酸Tyr和底物特异性特征氨基酸His;RmPAL和RdPAL在溶液中均以四聚体形式存在,两个酶的最适pH和最适温度均为8.9和50℃,且具有较宽泛的pH和温度稳定性,优于黏红酵母来源的商用PAL酶;此外,两种酶均能催化L-Phe和酪氨酸(L-tyrosine,L-Tyr)反应,且对L-Phe的催化效率较高,约为L-Tyr的5倍,脱除酸解酪蛋白中L-Phe的转化率分别为88%和93%。【结论】RmPAL和RdPAL具有较强稳定性和L-Phe水解偏好性,可从食源蛋白中有效去除L-Phe。

不同丛枝菌根真菌在辣椒根系定殖形态及对植株抗病相关酶活性的影响

为揭示辣椒疫霉菌对丛枝菌根真菌(AMF)在辣椒根系定殖能力以及专性内生细菌(MRE)在宿主AMF促进辣椒抗病害胁迫中的潜在作用,选择摩西斗管球囊霉(Fm)、明根孢囊霉(Rc)和幼套球囊霉(Ce)3种与MRE共生的AMF和根内根孢囊霉(Ri)无MRE的AMF种类为试材,通过盆栽建立AMF与辣椒植株的共生定殖体系,然后采用挑战接种方法将辣椒疫霉菌接种于辣椒根系周围,并检测根系中几丁质酶和苯丙氨酸解氨酶(PAL)活性的变化。结果表明,辣椒疫霉菌能够影响AMF在辣椒根系的侵染定殖,不同种类AMF在辣椒根系侵染定殖能力不同,挑战接种辣椒疫霉菌6 d后,MRE共生的Fm、Rc、Ce对辣椒植株的侵染率显著高于无MRE共生的Ri,AMF定殖的辣椒根系中几丁质酶与PAL活性均较对照显著增强;挑战接种辣椒疫霉菌6 d后,接种MRE共生的Fm、Rc和Ce的辣椒几丁质酶活性比接种Ri的分别提高了10.54%、17.00%和13.20%,PAL活性则分别提高了12.49%、3.84%和10.03%。MRE与AMF共生并相互作用,可能是造成不同种类AMF在宿主植物根部侵染率以及诱导宿主抗病害胁迫等方面差异的潜在因素。

苯丙氨酸解氨酶合成L-2-氯苯丙氨酸的研究

L-2-氯苯丙氨酸要求旋光对映体过量(ee)≥98%,导致使用化学拆分法合成L-2-氯苯丙氨酸的收率不足50%。为提高收率,文章采用具有立体专一性的苯丙氨酸解氨酶(PAL),催化邻氯肉桂酸与氨合成L-2-氯苯丙氨酸,旋光对映体过量(ee)>99%。通过考察不同形态PAL酶的稳定性,研究得出全细胞冻融法制作的PAL酶稳定性较好,结合氮气保护,反应80 h依然能保留62%的酶活。同时,优化PAL酶催化反应的pH、温度、酶用量,确定最佳条件后,结合分段补料的方式进行酶催化反应,最终使L-2-氯苯丙氨酸的累积量达到了68.5 g/L,转化率达到了97.8%。该技术合成L-2-氯苯丙氨酸的产量和收率较高,有效降低了成本,提升了L-2-氯苯丙氨酸的市场价值。

药用植物PAL基因及其功能研究进展

苯丙烷代谢途径是植物主要代谢途径之一,可产生黄酮、酚酸和木质素类等物质,这些物质不仅参与调控植物生长发育和抗逆等生理活动,还被用于预防和治疗疾病,是决定药用植物品质的关键因素之一。苯丙氨酸解氨酶(Phenylalanine ammonia-lyase,PAL)负责催化苯丙烷类代谢途径中第一个反应,是该途径的关键酶和限速酶。本文就药用植物中PAL基因的基本特性、表达调控机制等方面的研究进行综述,为进一步阐明药用植物中PAL的功能提供参考。

Cis- and Trans-Cinnamic Acids Have Different Effects on the Catalytic Properties of Arabidopsis Phenylalanine Ammonia Lyases PAL1, PAL2, and PAL4

Abstract: Cis-cinnamic acid (CA) is a naturally occurring compound, presumably converted from trans-CA in higher plants. To investigate the effect of cis-CA on the activity of Arabidopsis phenylalanine ammonia lyase (PAL), AtPAL1, AtPAL2, and AtPAL4 genes were isolated using reverse transcription poly-merase chain reaction. These genes were fused to a glutathione S-transferase gene and overexpressed in a heterologous prokaryotic system of Escherichia coli. The purified PAL1, PAL2 and PAL4 enzymes were characterized biochemically to determine the effects of cis-CA on the kinetic parameter Km. The results showed that cis-CA is a competitive inhibitor for PAL1, but not PAL2 and PAL4, whereas trans-CA acts as a competitive inhibitor for all three PAL isomers, suggesting that cis- and trans-CA have different effects on the catalytic activity of PAL.

苜蓿霜霉病接种方法及染病植株体内酶活性

苜蓿霜霉病(Peronospora aestivalis)是苜蓿上的重要病害之一,严重限制了苜蓿产业的发展。为了探明苜蓿霜霉菌侵染对苜蓿体内保护酶活性的影响及优化苜蓿霜霉菌的接种方法,本研究以紫花苜蓿‘中草3号’(Medicago sativa‘Zhongcao No.3’)为研究对象,进行苜蓿霜霉菌的接种试验,并研究不同染病程度下过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和苯丙氨酸解氨酶(PAL)的酶活性,评价接种苜蓿霜霉菌对苜蓿体内保护酶活性的影响。结果表明:4种接种方法中病枝插入法不能获得成功接种的病株,病叶摩擦法、培养箱内喷雾法和培养箱外喷雾法能够得到接种的病株,发病率分别为11.1%、88.9%和25.6%,由此可以看出培养箱内接种法的接种效果最佳。接菌对苜蓿体内酶活性影响显著,苜蓿霜霉菌轻度侵染会导致CAT活性显著增强(P<0.05),较健康苜蓿,苜蓿霜霉菌侵染能够显著增强POD和SOD的活性(P<0.05),但是重度染病情况下与健康苜蓿相比SOD活性差异不显著(P>0.05);PAL活性表现为随着病原菌的侵染程度加深显著下降(P<0.05)。综上表明病原菌侵染会导致苜蓿体内酶活性的变化,本研究可为优化苜蓿霜霉菌的接种方法及通过苜蓿体内酶活性变化筛选抗性品种提供理论参考。

竹醋液对常温贮藏砂糖橘的保鲜效果

以不同浓度(50、150、250 mL/L)竹醋液(bamboo pyroligneous acid,BPA)和蒸馏水(对照)处理健康、机械造伤及接种指状青霉的砂糖橘,测定常温(25±1)℃贮藏过程中果实腐烂率、果实侵染率、病斑指数、果皮总类黄酮、总酚含量和苯丙氨酸解氨酶(phenylalanine ammonia lyase,PAL)、过氧化物酶(peroxidase,POD)活性等指标评价贮藏效果。结果显示,50、150 mL/L BPA处理能降低常温贮藏期间果实腐烂率(36 d时处理组比对照组低15%),提高果皮总类黄酮含量、PAL和POD活性(贮藏0~20 d),此外还降低了机械伤果实腐烂率。250 mL/L BPA处理能减缓指状青霉接种后砂糖橘的腐烂和病斑扩展,显著提高果皮总酚、总类黄酮含量和PAL活性(P<0.05)。综上,150 mL/L BPA可显著提高砂糖橘的常温贮藏性(P<0.05),而250 mL/L BPA抑制指状青霉的效果较好。

棉花PAL基因家族的全基因组鉴定及其在逆境胁迫下的表达分析

苯丙氨酸解氨酶(Phenylalanine ammonia lyase,PAL)是苯丙烷代谢的关键酶和限速酶,在植物的生长发育、抗病虫害和抗逆等方面发挥着重要作用。研究在全基因组水平上对棉花PAL基因家族进行了鉴定和分析,以了解棉花中PAL的功能,为后续深入研究棉花PAL功能提供一定参考。结果表明,在陆地棉(Gossypium hirsutum)、海岛棉(Gossypium barbadense)、草棉(Gossypium herbaceum)和雷蒙德氏棉(Gossypium raimondii)中分别鉴定出15、13、7、8个PAL基因。进化分析结果显示,棉花PAL基因家族可分为3个亚组。保守基序和基因结构分析显示,亲缘关系近的PAL基因之间的保守基序组成和基因结构也十分相似。启动子分析显示,GhPAL基因的启动子上存在多个与非生物和生物胁迫应答以及激素信号转导相关的顺式作用元件。组织表达模式分析表明,GhPAL基因主要在根、茎、花丝和胚珠中高表达。转录组和qPCR分析显示,部分GhPAL基因的表达量在PEG、盐、高温、低温和碱胁迫下显著提高,其中一些GhPAL基因能够对多种非生物胁迫作出响应。生物胁迫转录组数据显示,GhPAL1/GhPAL5/GhPAL13的表达在大丽轮枝菌处理条件下发生显著上调。GhPAL基因在逆境胁迫下的表达分析结果说明,PAL基因可能在棉花应答逆境胁迫的过程中发挥一定的功能。

松材线虫侵染对日本落叶松生理指标的影响

通过测定光合色素含量、渗透调节物质、苯丙氨酸解氨酶和抗氧化酶系统的动态变化,从生理生化角度探究日本落叶松对松材线虫侵染的响应规律。以3年生日本落叶松盆栽苗为实验材料,采用人工皮接法接入2 000条松材线虫,于接种后第0、5、10、20、30天时收集并测定针叶内叶绿素含量、可溶性蛋白含量、抗氧化酶[超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)]活性和苯丙氨酸解氨酶(PAL)活性等指标。结果表明:日本落叶松接种点上部和下部均检测到大量松材线虫,且下部线虫数极显著高于上部(P<0.01)。叶绿素a、b含量均在第10天时出现明显下降,随后开始上升,与对照组无显著差异。可溶性蛋白含量在接种10 d后开始逐渐上升,且显著高于对照组(P<0.05)。接种松材线虫后,SOD活性显著上升,各时间点均极显著高于对照组(P<0.01);CAT活性先小幅下降,后逐渐升高,于30 d时极显著高于对照组(P<0.01);POD活性表现为先降低后升高,且5 d后迅速升高,各时间点都显著高于对照组(P<0.05)。PAL活性呈现先升高后降低的变化规律,接种后10、20 d活性都显著高于对照组(P<0.05)。日本落叶松被松材线虫侵染后,叶绿素含量、可溶性蛋白含量、抗氧化酶和PAL活性变化与其抗病性存在密切关系,研究结果可以为日本落叶松对松材线虫抗性品系筛选和抗病机理提供参考。