外源脯氨酸对盐胁迫下甜瓜脯氨酸代谢的影响

为探明外源脯氨酸对盐胁迫下甜瓜脯氨酸代谢的影响,以甜瓜品种‘雪美’为材料采用营养液栽培,对盐胁迫(100mmol·L-1 NaCl)、盐胁迫下添加外源脯氨酸(100mmol·L-1 NaCl+0.2mmol·L-1 Proline)以及对照3种处理后甜瓜幼苗叶片脯氨酸(Pro)含量、吡咯啉-5-羧酸合成酶(P5CS)、鸟氨酸转氨酶(OAT)和脯氨酸脱氢酶(ProDH)活性进行测定,并对OAT和ProDH基因进行克隆及半定量表达分析。结果显示:与对照相比较,盐胁迫条件下甜瓜幼苗叶片内Pro含量显著增加,P5CS活性增幅大于OAT活性,OAT基因表达量大部分时段内没有增加,ProDH活性下降,ProDH基因表达量减少;盐胁迫下添加外源脯氨酸进一步使幼苗叶片内Pro含量增加、OAT、ProDH活性提高、P5CS活性降低,并且使OAT基因表达量迅速增加、ProDH基因表达量先增加后回落。研究表明,盐胁迫条件下,甜瓜幼苗体内脯氨酸积累主要是通过增强脯氨酸的谷氨酸合成途径和抑制脯氨酸降解来实现;适量外源脯氨酸可以增强盐胁迫幼苗脯氨酸的鸟氨酸合成途径,但对谷氨酸合成途径有一定的抑制作用;通过调节合成和降解2种代谢途径进一步提高了脯氨酸含量,从而增强甜瓜幼苗耐盐胁迫能力。     

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。     

盐生植物盐爪爪的耐盐生理特性探讨

当前土壤盐渍化日益严重,是限制植物生长的一个主要环境因子,然而在盐碱自然环境中生长着许多耐盐植物,为更好地了解盐生植物的耐盐机理,该文从无机离子Na+,K+,Ca2+含量、脯氨酸水平、水势变化、丙二醛含量和盐胁迫的表型等生理参数以及半定量RT-PCR检测脯氨酸合成关键酶基因(P5CS)的表达规律等方面探讨盐胁迫下盐爪爪的耐盐特性。结果表明:(1)随着盐浓度的升高,Na+在根和肉质化的叶中显著地富集,且叶中积累的Na+比根中更多;(2)在盐胁迫条件下,随着盐浓度的增加,脯氨酸的含量和脯氨酸合成关键酶基因的表达显著地增强;(3)Na+和脯氨酸是植物有效的渗透调节剂,可使处于低水势的植物细胞仍能从细胞外高浓度的盐溶液中吸收水分;(4)在0和700 mmol·L-1Na Cl处理下,盐爪爪肉质化叶中丙二醛的含量较其它处理高,这表明植物在这两个处理下可能受到了氧化胁迫;(5)从盐胁迫3个月的生长表型来看,低盐环境中生长的盐爪爪植株的生物量更多,肉质化的叶嫩且绿。综上所述,结合对野外生境的调查和实验室长期的盐胁迫表型结果表明盐爪爪的生长是需盐的,相对低的盐浓度环境对盐爪爪的生长是顺境,而无盐或高浓度盐环境对于盐爪爪的生长来说都是逆境。该研究结果为全面深入研究盐爪爪的耐盐特性,以及更好地利用盐爪爪的生物和基因资源改良土壤和提高作物和林木的耐盐性奠定基础。     

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。     

外源茉莉酸甲酯对盐胁迫下小桐子幼苗渗透调节和脯氨酸代谢的影响

以小桐子幼苗为材料,采用人工气候箱内水培试验,设置不同浓度(0、20、40、60、80、100μmol·L^(-1))茉莉酸甲酯(MeJA)和150 mmol·L^(-1)NaCl胁迫处理,分析不同处理条件下小桐子幼苗叶片的组织活力、MDA含量、水势、含水量和叶片渗透调节物质脯氨酸、甜菜碱和可溶性糖的含量,以及脯氨酸代谢关键酶P5CS、OAT和甜菜碱合成关键酶BADH活性和相关基因表达水平,探讨外源茉莉酸甲酯对盐胁迫下小桐子幼苗渗透调节能力的影响及其机制。结果表明:(1)外源MeJA处理可提高盐胁迫下小桐子幼苗叶片的组织活力和叶片含水量,降低小桐子幼苗叶片的MDA含量和水势,且60μmol·L^(-1)浓度处理效果最佳。(2)外源MeJA处理可提高盐胁迫下小桐子幼苗叶片的脯氨酸、甜菜碱和可溶性糖的含量,且60μmol·L^(-1)MeJA处理显著提高了盐胁迫下小桐子幼苗内源茉莉酸、脯氨酸和甜菜碱的含量。(3)60μmol·L^(-1)MeJA也提高了盐胁迫下小桐子BADH、P5CS和OAT的活性,并上调了JcBADH、JcP5CS、JcOAT基因的表达水平,但MeJA降低了脯氨酸降解酶ProDH的活性,下调了JcProDH基因的表达。研究发现,在盐胁迫条件下,外源MeJA通过活化脯氨酸生物合成的谷氨酸和鸟氨酸途径,尤其是鸟氨酸途径,以及抑制脯氨酸降解途径来促进小桐子幼苗脯氨酸的积累,同时MeJA也激活了幼苗体内甜菜碱的生物合成过程,从而强化了盐胁迫下幼苗的渗透调节作用和耐盐性,表明MeJA诱导的渗透调节在小桐子耐盐性形成过程中发挥着重要作用。     

燕麦盐胁迫响应基因的差异表达与生理响应的关系

以耐盐燕麦品种VAO-9为材料,通过Illumina测序与数字基因表达谱技术对300mmol/L NaCl处理前后的叶片cDNA文库进行RNA-Seq与DGE分析,同时测定0(CK)、100、200、250、300mmol/L NaCl胁迫下VAO-9幼苗叶片的相对电导率、丙二醛含量和脯氨酸含量,探讨燕麦盐胁迫响应基因的差异表达与生理响应的关系。结果表明:(1)RNA-Seq分析得到Unigenes 65 801条,其基因表达呈现高度的不均一性和冗余性;若差异基因表达谱鉴定分析以log2Ratio≥2且FDR值≤0.001为选择标准,则发现上调和下调表达基因在胁迫0.5h时分别有306和64个,在胁迫3h时分别有639和290个,胁迫24h时分别有1 488和882个。(2)KEGG代谢分析显示,有23 652条Unigenes比对到KEGG中的128条代谢途径,包括与逆境胁迫相关的植物激素信号转导途径、ABC转运蛋白途径、肌醇磷酸代谢途径、渗透调节途径等。(3)在300mmol/L NaCl处理下燕麦叶片的相对电导率、丙二醛含量、脯氨酸含量等生理指标的变化与相关差异表达基因的变化趋势基本一致,说明基因差异表达量与生理反应密切相关。研究认为,在相同的栽培及胁迫处理条件下,可根据植物盐响应生理指标的变化判断耐盐基因的表达情况。     

植物脯氨酸合成酶基因工程研究进展

逆境条件下植物细胞积累脯氨酸是植物适应逆境的一种反应,有利于减轻逆境对植物的伤害。简要回顾了生物体脯氨酸代谢过程和逆境下植物积累脯氨酸的作用,重点总结了植物体内脯氨酸合成酶基因吡咯啉-5羧-酸合成酶(P5CS)的克隆、表达和转基因研究进展。研究认为,P5CS是一个逆境胁迫应答基因,通过基因工程方法调节P5CS基因的表达有利于提高植物体脯氨酸的积累量,改善植物的抗逆性。因此,目前可以在大田植物中开展利用提高脯氨酸来改善植物抗逆性的研究。     

茶树脯氨酸转运蛋白基因鉴定及表达分析

脯氨酸转运蛋白在植物体内脯氨酸的分配及响应多种非生物逆境胁迫过程中发挥着重要作用。为明确茶树体内脯氨酸转运蛋白家族情况,该研究从全基因组水平鉴定获得茶树脯氨酸转运蛋白家族成员,进行了系统进化关系、蛋白结构、基因表达特异性等分析。结果表明:(1)茶树中有6个脯氨酸转运蛋白基因,长度为1 326～1 725 bp之间,编码氨基酸数目在441～574 aa之间,蛋白质分子质量在48.5～63.0 kD之间,等电点为8.51～9.41,大部分为碱性蛋白,其结构中含有大量的α-螺旋和自由卷曲,少量的延长链和β-转角结构。(2)亚细胞定位分析结果显示,茶树CsProT1、CsProT2、CsProT4、CsProT5和CsProT6蛋白定位于细胞膜,CsProT3蛋白则定位于高尔基体。(3)CsProTs蛋白中含9～11个典型的跨膜结构域,其三级结构与保守基序特征均与拟南芥高度相似,具有高度的保守性,不同成员间氨基酸序列相似性达40.14%。(4)基因表达特异性分析显示,CsProT1,CsProT2和CsProT3基因在各个组织部位的表达量均较高,CsProT4、CsProT5和CsProT6表达量均较低,且CsProT1基因的表达量最高;除CsProT5基因外,CsProTs蛋白家族的基因均受到NaCl、干旱及冷胁迫的诱导表达。(5)蛋白相互作用分析结果显示,CsProTs蛋白可与脯氨酸氧化酶ERD5,脯氨酸生物合成限速酶P5CS1、P5CS2和δ-吡咯啉-5-羧酸脱氢酶ALDH12A1等脯氨酸合成,转运及降解有关的蛋白相互作用,共同调控茶树体内脯氨酸的含量。研究认为,茶树6个CsProTs蛋白可共同参与茶树体内脯氨酸的转运平衡及对多种非生物逆境胁迫响应的过程。     

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。     

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。     

Regulation of gene expression governing proline metabolism in <Emphasis Type="Italic">Thellungiella salsuginea</Emphasis> by NaCl and paraquat

Differential expression of the proline metabolism genes in Thellungiella salsuginea (Pall) E. Schulz was investigated under salinity (100 and 300 mM NaCl), upon the effect of paraquat (0.1 μM), and at their joint action. It was shown that, depending on the intensity of stress factor, expression of the P5CS1 gene was induced in the leaves (at 100 mM NaCl) or roots (at 300 mM NaCl). When the plants on control medium were treated with paraquat, the proline content changed only in the leaves. Time course of proline content in the leaves complied with the dynamic of P5CS1 gene expression, while expression of PDH gene essentially did not change. When the plants, which experienced salt stress, were treated with paraquat, the content of proline and the P5CS1 mRNA level increased even more. The obtained results suggest a complicated nature of signaling between the organs of the halophyte Th. salsuginea causing expression of the proline biosynthesis genes in the leaves and roots under the effect of salinity, paraquat, or upon their joint action. The proline catabolism in these plants was maintained essentially unchanged, which is probably related to the participation of proline and/or the products of its degradation in the pathways of other metabolite biosynthesis. We suggested that proline took part in ROS scavenging process and proline level was under strong control in Th. salsuginea.     

Characterization of the gene for Δ1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L.

A cDNA for ¹-pyrroline-5-carboxylate (P5C) synthetase (cOsP5CS), an enzyme involved in the biosynthesis of proline, was isolated and characterized from a cDNA library prepared from 14-day-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the P5CS protein (OsP5CS) from O. sativa exhibited 74.2% and 75.5% homology to that of the P5CS from Arabidopsis thaliana and Vigna aconitifolia, respectively. Northern blot analysis revealed that the gene for P5CS (OsP5CS) was induced by high salt, dehydration, treatment of ABA and cold treatment, while it was not induced by heat treatment. Simultaneously, accumulation of proline was observed as a result of high salt treatment in O. sativa. Moreover, the levels of expression of OsP5CS mRNA and content of proline under salt stress condition were compared between a salt-tolerant cultivar, Dee-gee-woo-gen (DGWG) and a salt-sensitive breeding line, IR28. It was observed that the expression of the P5CS gene and the accumulation of proline in DGWG steadily increased, whereas those in IR28 increased slightly.     

Correlation between the induction of a gene for Δ1-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress

The isolation and characterization is reported of a cDNA for Δ¹-pyrroline-5-carboxylate (P5C) synthetase (cAtP5CS), an enzyme involved in the biosynthesis of proline, from a cDNA library prepared from a dehydrated rosette plant of Arabidopsis thaliana . Southern blot analysis suggested that only one copy of the corresponding gene ( AtP5CS ) is present in A. thaliana . The deduced amino acid sequence of the P5CS protein (AtP5CS) from A. thaliana exhibited 74% homology to that of the P5CS from Vigna aconitifolia . Northern blot analysis revealed that the gene for P5CS was induced by dehydration, high salt and treatment with ABA, while it was not induced by heat or cold treatment. Moreover, the simultaneous accumulation of proline was observed as a result of the former treatments in A. thaliana . A cDNA for P5C reductase (cAtP5CR) was also isolated from A. thaliana and Northern blot analysis was performed. The AtP5CR gene was not induced to a significant extent by dehydration or high-salt stress. These observations suggest that the AtP5CS gene plays a principal role in the biosynthesis of proline in A. thaliana under osmotic stress.     

Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobacco

To evaluate the physiological importance of cytosolic ascorbate peroxidase (APX) in the reactive oxygen species (ROS)-scavenging system, a full-length cDNA clone, named LmAPX, encoding a cytosolic ascorbate peroxidase was isolated from Lycium chinense Mill. using homologous cloning, then the expression of LmAPX under salt stress was investigated. After sequencing and related analysis, the LmAPX cDNA sequence was 965 bp in length and had an open reading frame (ORF) of 750 bp coding for 250 amino acids. Furthermore, the LmAPX sequence was sub-cloned into prokaryotic expression vector pET28a and the recombinant proteins had a high expression level in Escherichia coli. Results from a southern blot analysis indicated that three inserts of this gene existed in the tobacco genome encoding LmAPX. Compared with the control plants (wild-type and empty vector control), the transgenic plants expressing the LmAPX gene exhibited lower amount of hydrogen peroxide (H₂O₂) and relatively higher values of ascorbate peroxidase activity, proline content, and net photosynthetic rate (P_n) under the same salt stress. These results suggested that overexpression of the LmAPX gene could decrease ROS production caused by salt stress and protect plants from oxidative stress.     

Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress

Legume root nodule nitrogen-fixing activity is severely affected by osmotic stress. Proline accumulation has been shown to induce tolerance to salt stress, and transgenic plants over-expressing Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), which accumulates high levels of proline, display enhanced osmotolerance. Here, we transformed the model legume Medicago truncatula with the P5CS gene from Vigna aconitifolia, and nodule activity was evaluated under osmotic stress in transgenic plants that showed high proline accumulation levels. Nitrogen fixation was significantly less affected by salt treatment compared to wild-type (WT) plants. To our knowledge, this is the first time that transgenic legumes have been produced that display nitrogen-fixing activity with enhanced tolerance to osmotic stress. We studied the expression of M. truncatula proline-related endogenous genes M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 1 (MtP5CS1), M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 2 (MtP5CS2), M. truncatula ornithine delta-aminotransferase (MtOAT), M. truncatula proline dehydrogenase (MtProDH) and a proline transporter gene in both WT and transgenic plants. Our results indicate that proline metabolism is finely regulated in response to osmotic stress in an organ-specific manner. The transgenic model allowed us to analyse some of the biochemical and molecular mechanisms that are activated in the nodule in response to high salt conditions, and to ascertain the essential role of proline in the maintenance of nitrogen-fixing activity under osmotic stress.