枸杞LmP5CS基因的克隆及表达分析

目的:为进一步研究枸杞抗逆境胁迫的机制,并为转基因育种,提供理论依据。提高农作物的抗逆性提供优质的基因资源。方法:提选取盐胁迫后脯氨酸含量变化较大的耐盐植物枸杞为材料,用1.5%NaCl处理后,提取枸杞叶片总RNA,利用 RT-PCR 及3' RACE方法克隆获得吡咯啉-5-羧酸合成酶(delta 1-pyrroline-5-carboxylate synthetase,P5CS)基因的全长cDNA,命名为LmP5CS,构建pH7m24GW,3rc-LmP5CS植物表达载体。结果:LmP5CS基因的ORF长2 154 bp,编码1个等电点为6.07、分子量为 77.5kDa、由717个氨基酸组成的蛋白。枸杞在200 mmol/L NaCl 盐胁迫下, LmP5CS基因表达量随处理时间,有先升高后降低的趋势,9h基因表达量最高,脯氨酸含量变化与之一致。结论:LmP5CS基因在盐胁迫下脯氨酸含量的变化中起关键作用。     

大豆脯氨酸积累相关基因家族鉴定及干旱胁迫表达分析北大核心CSCD

脯氨酸是一种广泛存在的渗透调节物质,在植物生长发育以及响应干旱胁迫的信号途径中具有重要作用。吡咯啉-5-羧酸合成酶(P5CS)、鸟氨酸转氨酶(δ-OAT)、吡咯啉-5-羧酸还原酶(P5CR)、脯氨酸脱氢酶(Pro DH)、吡咯啉-5-羧酸脱氢酶(P5CDH)、脯氨酸转运体(Pro T)是影响植物体内脯氨酸积累的关键酶。但关于大豆脯氨酸积累相关基因家族成员的研究尚未见报道。本研究在大豆基因组中鉴定出7个Gm P5CS、2个Gm OAT、2个Gm P5CR、5个Gm Pro DH、3个Gm P5CDH及6个Gm Pro T基因,不均匀地分布在大豆20条染色体中的12条上,发生16对片段复制事件。系统进化树分析发现,大豆脯氨酸积累相关基因家族分为不同的进化分支,同一亚族间的基因结构和保守基序相似。顺式作用元件分析结果显示,脯氨酸积累相关基因家族含响应逆境胁迫及植物激素的顺式作用元件。干旱胁迫下的表达模式分析结果显示,脯氨酸合成代谢相关基因家族成员(Gm P5CS、Gm OAT、Gm P5CR)响应干旱胁迫,在干旱胁迫24 h时显著上调表达;大多脯氨酸分解代谢相关基因家族成员(Gm Pro DH、Gm P5CDH)下调表达,脯氨酸转运相关基因家族成员(Gm Pro T)在干旱胁迫24 h显著上调表达,其中Gm P5CS5、Gm OAT1、Gm Pro T2、Gm Pro T4及Gm Pro DH3~5基因在干旱胁迫下的脯氨酸积累中可能起到关键作用。大豆幼苗P5CS、OAT活性随干旱胁迫时间的延长呈显著上升的趋势,与脯氨酸的积累呈正相关:Pro DH活性随干旱胁迫时间的增长呈显著下降的趋势,与脯氨酸的积累呈负相关。本研究为进一步解析大豆脯氨酸积累相关家族基因响应干旱胁迫的功能提供了参考。     

外源ABA对低温胁迫下小桐子幼苗脯氨酸积累及其代谢途径的影响

本文以小桐子为材料,研究了外源脱落酸（ABA）对低温胁迫下小桐子幼苗脯氨酸积累的影响。结果表明,低温胁迫（5℃）可促进小桐子幼苗体内脯氨酸的积累,上调脯氨酸合成关键酶Δ¹-吡咯琳-5-羧酸合成酶（P5CS）和鸟氨酸转氨酶（OAT）的活性,上调P5CS基因（JcP5CS）的表达,及抑制脯氨酸降解酶脯氨酸脱氢酶（Pro DH）的活性。用外源ABA处理低温胁迫下的小桐子幼苗,发现150μmol·L^-1的ABA可显著提高其低温胁迫下的脯氨酸含量,上调P5CS的活性和JcP5CS的表达水平,及抑制ProDH的活性。表明外源ABA可通过活化脯氨酸合成的谷氨酸途径和抑制脯氨酸的降解途径来促进低温胁迫下小桐子幼苗脯氨酸的积累。     

野生大豆P5CS基因的克隆及对盐胁迫反应

逆境下植物大量积累脯氨酸是减轻胁迫伤害的一种自我保护机制。本研究应用同源克隆方法从NaCl处理的野生大豆中克隆获得一个脯氨酸合成酶(P5CS)基因,命名为GsP5CS。该基因核苷酸序列全长2.232 kb,含一个2148bp开放阅读框,编码715个氨基酸,包含有高等植物P5CS蛋白质的5个主要功能域,与菜豆PvP5CS1基因核苷酸序列相似性高达98.79%。Real Time PCR分析显示该基因受轻度盐胁迫诱导上调表达,根中表达高峰出现在200 mmol/L NaCl处理下,相对表达量为对照的5.83倍;叶片中表达高峰出现在300 mmol/L NaCl处理条件下,相对表达量为对照的12.78倍。并且该基因在根和叶片中的表达模式和脯氨酸含量的变化模式相同。上述结果说明,GsP5CS可能参与野生大豆脯氨酸合成。     

转拟南芥P5CS1基因增强羽衣甘蓝的耐旱性

为提高羽衣甘蓝的耐旱性,本文将拟南芥Δ1-吡咯啉-5-羧酸合成酶（P5CS1）基因经农杆菌介导转入羽衣甘蓝植株中,检测转基因株系与野生型植株在干旱胁迫下P5CS1 mRNA表达量、幼苗脯氨酸含量、株系根系性状、整株干重、鲜重和整株存活率。结果表明,在15%PEG6000渗透胁迫下,转基因植株的P5CS1基因mRNA表达量明显增加,转基因植株脯氨酸含量是野生型的2.4倍;主根长、最长侧根长、侧根数目、整株干重和鲜重均高于野生型,干重/鲜重则低于野生型,转基因植株的平均存活率为78%,极显著高于野生型。数据显示,AtP5CS1基因在羽衣甘蓝中的表达明显改善了转基因植株的耐旱性。     

外源水杨酸对盐胁迫下小桐子幼苗脯氨酸代谢的影响

以小桐子幼苗为材料,设置盐胁迫(200mmol·L-1 NaCl)和外源水杨酸处理(0~2.0mmol·L-1 SA)水培试验,通过检测幼苗叶片脯氨酸含量、脯氨酸代谢关键酶活性及相关代谢酶基因的表达水平,研究了外源水杨酸对盐胁迫下小桐子幼苗脯氨酸代谢机理的影响。结果显示:(1)外源0.9mmol·L-1 SA处理可显著提高盐胁迫下小桐子幼苗的脯氨酸含量,上调脯氨酸合成关键酶Δ1-吡咯琳-5-羧酸合成酶(P5CS)和鸟氨酸转氨酶(OAT)活性,以及上调JcP5CS和JcOAT基因的表达水平。(2)SA也显著抑制了脯氨酸降解酶ProDH的活性及JcProDH基因的表达水平。(3)SA处理还显著提高了盐胁迫下小桐子幼苗的组织活力,降低了叶片电解质渗漏率和丙二醛(MDA)含量。研究发现,外源SA可通过活化脯氨酸合成的谷氨酸途径和鸟氨酸途径,以及抑制脯氨酸的降解途径来促进盐胁迫下小桐子幼苗脯氨酸的积累;外源SA处理也可提高小桐子幼苗的耐盐性,且这种提高可能与SA诱导脯氨酸的积累密切相关。     

水稻中p5cs基因的存在及其在高脯氨酸变异系中的作用

用来自豇豆根瘤的脯氨酸合成酶△’-吡咯啉-5-羧酸合成酶（P5CS）cDNA作探针进行杂交分析,结果表明水稻中存在豇豆根瘤P5cs基因的同源序列,它在盐胁迫条件下转录水平提高。水稻高脯氨酸变异系杂交后代株系的脯氨酸含量和耐盐性均明显高于原始型。应用水稻高脯氨酸变异系为材料进行的试验,结果显示高脯氨酸特性和高耐盐性与此基因的存在及其转录水平的提高有相关性。     

石蒜LrP5CS基因的克隆与表达特性分析

Δ1-吡咯啉-5-羧酸合成酶(P5CS)是植物渗透胁迫下谷氨酸途径合成脯氨酸的关键酶。该研究以石蒜(Lycoris radiata)为材料,采用同源克隆、RACE方法结合RT-PCR技术克隆获得LrP5CS基因全长cDNA序列。序列分析表明,LrP5CS基因全长2 521bp,其中开放阅读框(ORF)为2 139bp,编码713个氨基酸,预测编码蛋白质的分子量为77.19kD,等电点为6.34;LrP5CS是1个稳定的疏水蛋白,不含信号肽,不具有跨膜结构,具有AAK超基因家族和ALDH-SF超基因家族的保守结构域。氨基酸序列比对和系统进化树分析发现,LrP5CS与植物其他P5CS蛋白具有较高的一致性,且与海枣PdP5CS及油棕EgP5CS聚为一类,亲缘关系最近。实时荧光定量PCR分析结果表明,LrP5CS在根、鳞茎和叶片中均有表达,其中在鳞茎中的表达量最高。LrP5CS在20%聚乙二醇(PEG)处理下的表达模式分析发现,LrP5CS受PEG胁迫处理的诱导表达,其基因相对表达量在处理后6h达到最高;随着处理时间的延长,LrP5CS基因相对表达量水平逐渐下调至对照水平。将LrP5CS连接到表达载体pET-28a上,转化获得LrP5CS编码基因的大肠杆菌BL21(DE3)工程菌,通过IPTG诱导表达,SDS-PAGE分析表达产物发现成功表达目的蛋白。该研究结果为进一步分析LrP5CS基因功能及石蒜抗逆分子育种奠定了基础。     

脯氨酸转运相关基因的研究进展

作为植物中普遍存在的一种逆境适应机制, 脯氨酸积累一直被认为是其合成和降解调控的结果。然而越来越多的研究表明, 脯氨酸转运也可能在其积累过程中起重要作用。在植物中, 有多个氨基酸转运蛋白家族, 如氨基酸通透酶家族(AAPs)、赖氨酸组氨酸转运蛋白家族(LHTs)和脯氨酸转运蛋白家族(ProTs)参与脯氨酸在各个器官间的运输。该文对参与脯氨酸运输的基因家族成员的表达模式、生理功能及表达调控进行了综述, 以期为脯氨酸运输与积累在植物抗逆方面的研究提供参考。     

外源一氧化碳对小麦幼苗耐盐性以及根中脯氨酸含量的影响

小麦幼苗经饱和度为50％的一氧化碳（C0）溶液预处理24h可以缓解随后以200mmol·L^-1NaCl处理所导致的小麦幼苗生长的受抑程度和相对含水量的下降。CO预处理还可有效提高盐胁迫下小麦幼苗根中吡咯啉-5-羧酸合成酶（P5CS）活性及其基因的表达,同时抑制脯氨酸脱氢酶（ProDH）活性,从而诱导脯氨酸的大量合成,缓解盐胁迫对小麦幼苗的伤害。     

Improved salt tolerance of melon (Cucumis melo L.) by the addition of proline and potassium nitrate

A pot experiment was carried out under glasshouse conditions with melon (Cucumis melo) cv. “Tempo F1” in a mixture of peat, perlite and sand (1:1:1) to investigate the effects of external proline and potassium nitrate applications to salinity-treated (150 mM) plants with respect to fruit yield, plant growth, some physiological parameters and ion uptake. Treatments were—(i) control (C): plants receiving nutrient solution, (ii) salinity treatment, as for control plus 150 mM NaCl. Salinity treatment was combined with or without either 5 mM supplementary KNO₃ or 10 mM proline. The salt treatment (150 mM NaCl) led to significant decreases in plant growth, fruit yield, relative water content (RWC), stomatal density, uptake of Ca²⁺, K⁺ and N, and chlorophyll a and b contents, accompanied by significant increases in Na⁺ uptake, proline concentration and membrane permeability. Supplementary KNO₃ and proline treatments significantly ameliorated the adverse effects of salinity on plant growth, fruit yield and the physiological parameters examined. This could be attributed to the effects of all the external supplements in maintaining membrane permeability, and increasing concentrations of Ca²⁺, N and K⁺ in the leaves of plants subjected to salt stress.     

高等植物脯氨酸代谢研究进展

很多植物在胁迫条件下可以通过增加合成、减少降解而在体内累积大量脯氨酸,这对于调节渗透平衡、防止渗透胁迫对植物造成伤害、清除自由基、保护细胞结构具有重要意义。脯氨酸合成、降解相关酶的编码基因大都已经克隆到,但对脯氨酸在植物发育中的具体作用、胁迫条件下脯氨酸累积的分子机理了解还比较少。概述了植物控制脯氨酸合成、降解相关酶的编码基因的研究进展情况。     

Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings

Abiotic stresses, such as high salinity or drought, can cause proline accumulation in plants. Such an accumulation involves proline transport into mitochondria where proline catabolism occurs. By using durum wheat seedlings as a plant model system, we investigated how proline enters isolated coupled mitochondria. The occurrence of two separate translocators for proline, namely a carrier solely for proline and a proline/glutamate antiporter, is shown in a functional study in which we found the following: (1) Mitochondria undergo passive swelling in isotonic proline solutions in a stereospecific manner. (2) Externally added l-proline (Pro) generates a mitochondrial membrane potential (ΔΨ) with a rate depending on the transport of Pro across the mitochondrial inner membrane. (3) The dependence of the rate of generation of ΔΨ on increasing Pro concentrations exhibits hyperbolic kinetics. Proline transport is inhibited in a competitive manner by the non-penetrant thiol reagent mersalyl, but it is insensitive to the penetrant thiol reagent N-ethylmaleimide (NEM). (4) No accumulation of proline occurs inside the mitochondria as a result of the addition of proline externally, whereas the content of glutamate increases both in mitochondria and in the extramitochondrial phase. (5) Glutamate efflux from mitochondria occurs at a rate which depends on the mitochondrial transport, and it is inhibited in a non-competitive manner by NEM. The dependence of the rate of glutamate efflux on increasing proline concentration shows saturation kinetics. The physiological role of carrier-mediated transport in the regulation of proline catabolism, as well as the possible occurrence of a proline/glutamate shuttle in durum wheat seedlings mitochondria, are discussed.Catello Di Martino, Roberto Pizzuto these authors contributed equally to the paper     

Arbuscular mycorrhizal fungi modulates dynamics tolerance expression to mitigate drought stress in Ephedra foliata Boiss

Arbuscular mycorrhizal fungi (AMF) are one of the most important drivers of soil ecosystem dynamics. AMF have the potential to improve plant growth and development by modulating key hormonal pathways, which result in decreasing the adverse impact of abiotic stress, such as drought. Pot experiments were conducted in this study to investigate the ability of AMF to ameliorate the adverse impact of drought in Ephedra foliate. Non-inoculated AMF E. foliate (Ef) plants, exhibited reduced growth in response to drought stress with a concomitant lowering of chlorophyll pigments, relative to non-stressed and AMF inoculated plant. AMF inoculated E. foliate showed improved nitrogen metabolism by positively regulating nitrate and nitrite reductase activity which results in greater ammonium availability for the synthesis of amino acids. Inoculation with AMF also increased antioxidant enzyme activity, ascorbic acid contents, and reduction in glutathione level. This resulted in significant amelioration of oxidative damage to plant membranes by restricting the excess generation of reactive oxygen species (ROS), such as hydrogen peroxide. Greater content of proline, glucose, and total soluble protein in AMF-inoculated plants provided further benefit to E. foliate plants and their ability to withstand drought stress, and also evident by a greater level of sucrose phosphate synthase activity. AMF significantly enhanced the uptake of essential nutrients like K, Mg, and Ca. Importantly, higher concentrations of plant hormones, including indole acetic acid (IAA), indole butyric acid (IBA), gibberellic acid (GA), and abscisic acid (ABA), were maintained in AMF-inoculated Ef plants. AMF inoculation also boosted phosphorous metabolism by increasing alkaline and acid phosphatase enzyme activity. In summary, AMF-inoculation of Ef plants significantly reduced the deleterious effect of drought stress by up-regulating the antioxidant defense system, synthesis of osmolytes, and maintaining phytohormone levels.     

Comparative Genomics of the Lipid-body-membrane Proteins Oleosin,Caleosin and Steroleosin in Magnoliophyte, Lycophyte and Bryophyte

Lipid bodies store oils in the form of triacylglycerols. Oleosin, caleosin and steroleosin are unique proteins localized on the surface of lipid bodies in seed plants. This study has identified genes encoding lipid body proteins oleosin, caleosin and steroleosin in the genomes of five plants: Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Selaginella moellendorffii and Physcomitrella patens. The protein sequence alignment indicated that each oleosin protein contains a highly-conserved proline knot motif, and proline knob motif is well conserved in steroleosin proteins, while caleosin proteins possess the Dx[D/N]xDG-containing calcium-binding motifs. The identification of motifs (proline knot and knob) and conserved amino acids at active site was further supported by the sequence logos. The phylogenetic analysis revealed the presence of magnoliophyte-and bryophyte-specific subgroups. We analyzed the public microarray data for expression of oleosin, caleosin and steroleosin in Arabidopsis and rice during the vegetative and reproductive stages, or under abiotic stresses. Our results indicated that genes encoding oleosin, caleosin and steroleosin proteins were expressed predominantly in plant seeds. This work may facilitate better understanding of the members of lipid-body-membrane proteins in diverse organisms and their gene expression in model plants Arabidopsis and rice.     

Phospholipase D is a negative regulator of proline biosynthesis in Arabidopsis thaliana

Accumulation of proline has been observed in a large number of plant species in response to drought and salt stresses, suggesting a key role of this amino acid in plant stress adaptation. Upstream components of the proline biosynthesis signal transduction pathways are still poorly defined. We provide experimental evidence that phospholipase D (PLD) is involved in the regulation of proline metabolism in Arabidopsis thaliana. The application of primary butyl alcohols, which divert part of PLD-derived phosphatidic acid by transphosphatidylation, stimulated proline biosynthesis even without hyperosmotic constraints. Moreover, application of primary butyl alcohols enhanced the proline responsiveness of seedlings to mild hyperosmotic stress. These data indicate that some PLDs are negative regulators of proline biosynthesis and that plants present a higher proline responsiveness to hyperosmotic stress when this regulator is abolished. We clearly demonstrate that PLD signaling for proline biosynthesis is similar to RD29A gene expression and different from the abscisic acid-dependent RAB18 gene expression. Our data reveal that PLDs play positive and negative roles in hyperosmotic stress signal transduction in plants, contributing to a precise regulation of ion homeostasis and plant salt tolerance.     

Proline induces calcium-mediated oxidative burst and salicylic acid signaling

Although free proline accumulation is a well-documented phenomenon in many plants in response to a variety of environmental stresses, and is proposed to play protective roles, high intracellular proline content, by either exogenous application or endogenous over-production, in the absence of stresses, is found to be inhibitory to plant growth. We have shown here that exogenous application of proline significantly induced intracellular Ca²⁺ accumulation in tobacco and calcium-dependent ROS production in Arabidopsis seedlings, which subsequently enhanced salicylic acid (SA) synthesis and PR genes expression. This suggested that proline can promote a reaction similar to hypersensitive response during pathogen infection. Other amino acids, such as glutamate, but not arginine and phenylalanine, were also found to be capable of inducing PR gene expression. In addition, proline at concentration as low as 0.5 mM could induce PR gene expression. However, proline could not induce the expression of PDF1.2 gene, the marker gene for jasmonic acid signaling pathway. Furthermore, proline-induced SA production is mediated by NDR1-dependent signaling pathway, but not that mediated by PAD4. Our data provide evidences that exogenous proline, and probably some other amino acids can specifically induce SA signaling and defense response.