水曲柳FmJAZ1基因在非生物胁迫中的响应模式和激素诱导表达分析

克隆FmJAZ1基因,明确其在低温和NaCl胁迫中的响应模式和激素诱导下的转录表达特性。通过基因克隆的方法得到水曲柳中的FmJAZ1基因,利用生物信息学软件对所得到的序列进行分析并构建系统进化树,对水曲柳FmJAZ1基因进行了时空表达特异性的分析,对根、茎、叶、芽、雄花、雌花、种子等7个部位以及在5-9月5个月份分别取样,对水曲柳进行低温(4℃)和盐胁迫(200 mmol/L NaCl)2种非生物胁迫处理以及脱落酸(ABA)、赤霉素(GA3)、生长素(IAA)、茉莉酸(JA)、水杨酸(SA)等激素信号诱导处理,然后对试验材料进行荧光定量分析。克隆出全长为684 bp的核苷酸序列。生物信息学软件分析得到JZA1基因具有完整的开放阅读框,编码227个氨基酸,JAZ1蛋白不含有信号肽,不属于跨膜蛋白,为不稳定亲水性蛋白。时空表达结果显示,FmJAZ1基因在茎中表达量最高,且在8月份表达量最高;非生物胁迫结果表明低温处理后FmJAZ1在6h、24h表达量较高;而NaCl处理后,在24 h表达量较高,且该基因响应低温胁迫较NaCl胁迫迅速;激素信号诱导结果显示,处理后不同时间,基因表达量变化较为明显,其中GA3处理后3h最为明显,为对照组的77.3倍,分析了FmJAZ1基因在低温、NaCl胁迫和激素诱导下的表达模式。FmJAZ1基因充分响应了逆境胁迫和激素信号诱导,通过蛋白和基因层面对逆境进行响应,JAZ蛋白在其中起到了桥梁的作用,并扮演了重要的角色。     

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。     

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。     

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。     

盐碱胁迫下碱地肤Na+/H+逆向转运蛋白基因KsNHX1表达分析

利用RACE技术得到碱地肤KsNHX1的3'cDNA序列,分子系统进化分析显示,KsNHX1为液泡膜Na+/H+逆向转运蛋白编码基因.通过半定量RT-PCR检测了该基因在盐碱胁迫下的表达,结果表明:200 mmol·L-1 NaCl胁迫2～24h,KsNHX1在叶片中表达量持续增加;200 mmol·L-1 NaCl处理10 h,KsNHX1在根、茎、叶和花中的表达都上调;不同浓度NaCl处理下,叶片中KsNHX1表达上调,160 mmol·L-1时达到最高;低于400 mmol·L-1浓度下,根中该基因的表达也都上调.经不同浓度Na2CO3胁迫,根中KsNHX1的表达变化趋势与相应浓度NaCl胁迫下的变化相同;但叶片中除160 mmol·L-1 Na,CO3处理下KsNHX1表达略有上调外,其他浓度下KsNHX1的表达都低于对照.KsNHX1的表达模式暗示,在不同盐碱胁迫下,碱地肤能够维持体内相对稳定的K+/Na+,其耐盐特性可能与Na+/H+逆向转运蛋白的作用密切相关.     

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。     

StP5CS基因转化结球甘蓝的研究

为研究StP5CS基因在结球甘蓝中的耐盐作用,以结球甘蓝下胚轴为外植体,采用农杆菌介导法将耐盐基因StP5CS和抗除草剂Bar基因导入结球甘蓝基因组中,在双丙氨膦的筛选下扩繁、生根,共获得了36株抗性植株。PCR扩增和Southern印迹杂交检测表明:目的基因StP5CS和Bar基因已经成功导入结球甘蓝基因组中。RT-PCR检测表明:StP5CS基因在转录水平也有表达。转基因植株耐盐试验结果显示:高浓度盐处理(400mmol/L NaCl)下,对照植株整株枯死,而转基因植株仍能正常生长;转基因植株的SOD活性、脯氨酸含量和相对膜透性均随盐浓度的升高呈上升趋势,均在400mmol/L NaCl处理下达到最大。结果表明转基因植株对高盐环境有一定的耐受性。     

沙棘HrTCP转录因子家族鉴定及其干旱胁迫下的表达分析

TCP家族是植物特有的响应高盐、干旱等非生物胁迫的重要转录因子。该研究基于沙棘转录组数据,利用生物信息学与qRT-PCR对HrTCP转录因子家族进行鉴定,预测其家族成员的结构和功能,为解析TCP转录因子调控沙棘抵御干旱胁迫的作用机制奠定基础。结果表明:(1)获得了11个HrTCP转录因子成员,并命名为HrTCP2/4/7/8/11/13/15/17/18/19/20,编码氨基酸序列长度在218～590之间,蛋白质相对分子量为23.44～61.78 kD;亚细胞定位预测发现,除HrTCP13/17/18蛋白定位于细胞质,其余8个蛋白均定位于细胞核。(2)在干旱(15%PEG-6000)和高盐(200 mmol/L NaCl)胁迫后HrTCP4/7/19/20基因表达量呈不同程度上升趋势,其中HrTCP20表达量极显著高于对照,分别是对照的24倍与23倍。(3)外源激素脱落酸(0.1 mmol/L ABA)和茉莉酸甲酯(0.1 mmol/L MeJA)处理后,HrTCP7/19/20基因表达量也均呈上升趋势,其中,ABA诱导下HrTCP19基因表达量达到最高,是对照的16倍,而MeJA诱导下HrTCP20基因表达量上升最高,是对照的5倍。研究发现,HrTCPs转录因子家族成员可受干旱、高盐和激素诱导表达,进而调控沙棘对干旱胁迫的响应。     

陆地棉GhCSN6A基因克隆及低磷胁迫下的表达

该研究基于陆地棉根部低磷胁迫基因表达谱芯片差异表达序列结果及基因组数据库，对表达差异序列ES816317进行克隆，利用生物信息学分析其核苷酸及蛋白序列，并通过qRT-PCR技术检测其组织表达模式和在低磷胁迫下的相对表达特征，为解析棉花GhCSN6A的生物学功能奠定基础，并为棉花磷高效基因工程育种提供基因资源。结果表明：(1)成功获得陆地棉GhCSN6A基因，该基因的开放阅读框全长为948 bp,编码315个氨基酸；GhCSN6A蛋白为COP9信号小体复合亚基6a,属于MOV34蛋白超家族，具有MPN＿CSN6结构域，定位于细胞核。(2)序列比对和进化分析显示，陆地棉GhCSN6A与木槿HsCSN6A、拟南芥AtCSN6A的相似性分别为95.87%和84.54%。(3)qRT-PCR分析表明，GhCSN6A基因在陆地棉的根、茎、叶、花中均有表达，且在叶中表达水平最高，但叶与根中的表达无显著差异；GhCSN6A基因在低磷处理24 h时根中相对表达量最低，但低磷处理72 h时根中的相对表达量最高达到了适磷(对照)处理的2倍。研究推测，陆地棉GhCSN6A基因在棉花响应低磷胁迫过程中具有重要作...     

盐胁迫对拟南芥AtPUB18基因的诱导表达及其启动子分析

用300mmol/L NaCl处理拟南芥幼苗,分别于处理后0、1、2、4、8、16、24、48h通过Northern Blot检测其AtPUB18基因的表达量。结果显示:拟南芥AtPUB18基因的表达量受高盐胁迫的诱导而升高,于处理后4h表达量达到最高,处理后16h表达量最低。采用PCR技术克隆AtPUB18的启动子,序列为1 974bp;序列分析发现启动子内含有大量与非生物胁迫相关的顺式作用元件,如HSE、LTR、MBS及ABRE;将启动子克隆到表达载体pCambia1300-221-GUS中,驱动报告基因GUS表达。组织化学染色结果表明,未经过高盐处理的幼苗中GUS基因表达水平很低;300mmol/L NaCl处理后GUS基因表达量显著升高。研究表明,AtPUB18的表达受高盐胁迫诱导,且AtPUB18基因的启动子是一个盐胁迫诱导型启动子。     

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。     

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.     

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.     

Effect of Salt Stress on Growth,Na+ Accumulation and Proline Metabolism in Potato (Solanum tuberosum) Cultivars

Potato (Solanum tuberosum) is a major crop world-wide and the productivity of currently used cultivars is strongly reduced at high soil salt levels. We compared the response of six potato cultivars to increased root NaCl concentrations. Cuttings were grown hydroponically and treated with 0 mM, 60 mM and 180 mM NaCl for one week. Growth reduction on salt was strongest for the cultivars Mozart and Mona Lisa with a severe senescence response at 180 mM NaCl and Mozart barely survived the treatment. The cultivars Desiree and Russett Burbank were more tolerant showing no senescence after salt treatment. A clear difference in Na⁺ homeostasis was observed between sensitive and tolerant cultivars. The salt sensitive cultivar Mozart combined low Na⁺ levels in root and stem with the highest leaf Na⁺ concentration of all cultivars, resulting in a high Na⁺ shoot distribution index (SDI) for Mozart as compared to Desiree. Overall, a positive correlation between salt tolerance and stem Na⁺ accumulation was found and the SDI for Na⁺ points to a role of stem Na⁺ accumulation in tolerance. In stem tissue, Mozart accumulated more H₂O₂ and less proline compared to the tolerant cultivars. Analysis of the expression of proline biosynthesis genes in Mozart and Desiree showed a clear reduction in proline dehydrogenase (PDH) expression in both cultivars and an increase in pyrroline-5-carboxylate synthetase 1 (P5CS1) gene expression in Desiree, but not in Mozart. Taken together, current day commercial cultivars show promising differences in salt tolerance and the results suggest that mechanisms of tolerance reside in the capacity of Na⁺ accumulation in stem tissue, resulting in reduced Na⁺ transport to the leaves.     

Contribution of putrescine degradation to proline accumulation in soybean leaves under salinity

Proline accumulation was studied in the leaves of Glycine max (L.) Merr. subjected to salt stress in the presence of aminoguanidine (AG, a specific inhibitor of diamine oxidase, DAO) and exogenous putrescine (Put). Both DAO activity and proline content were increased while endogenous Put content was decreased in soybean leaves under 50 to 150 mM NaCl. There was a negative correlation between proline accumulation and endogenous Put content. The addition of AG during NaCl stress inhibited DAO activity, caused Put accumulation and a 15 to 20 % decrease in proline content. Application of 1 mM Put to NaCl solution markedly increased proline content. The promotive effect of Put application could be alleviated by the treatment with Put plus AG. Moreover an application of AG had no effect on proline accumulation in soybean seedlings grown under normal condition. These results indicate that the quantitative contribution of Put degradation to proline formation is 15 to 20 %.