刚毛柽柳NAC24基因的表达及抗逆功能分析

【目的】NAC类转录因子是植物特有的最大的转录因子家族之一,广泛参与植物的生长发育过程,并在植物响应盐、干旱等多种非生物胁迫的过程中发挥至关重要的调控作用。本研究拟从盐生木本植物刚毛柽柳中克隆获得一个NAC转录因子基因,研究该基因的耐盐、抗旱功能,以期为研究木本植物NAC转录因子的抗逆分子机制奠定理论基础。【方法】在刚毛柽柳NaHCO_3胁迫转录组数据库中筛选获得一个NAC转录因子基因,将其命名为ThNAC24(GenBank登陆号:KF031949)。利用生物信息学工具将其与其他9个物种的NAC蛋白进行多序列比对,与拟南芥105个NAC蛋白进行进化树分析。分别用300 mmol·L^-1 NaCl和400 mmol·L^-1甘露醇对刚毛柽柳进行胁迫,在胁迫6、12、24和48 h后分别取刚毛柽柳根及叶组织。通过实时荧光定量RT-PCR(qRT-PCR)技术分析盐、干旱胁迫下ThNAC24基因在不同胁迫时间点及不同组织的表达情况,初步鉴定其是否响应盐、干旱胁迫。为进一步研究ThNAC24基因的抗逆功能,分别构建植物过表达(pROKⅡ-ThNAC24)及抑制表达(pFGC5941-ThNAC24)载体。利用农杆菌介导的高效瞬时遗传转化体系获得ThNAC24基因瞬时过表达(OE)、抑制表达(IE)及对照(Control)刚毛柽柳植株。在盐、干旱胁迫下分析比较了ThNAC24基因瞬时过表达、抑制表达及对照刚毛柽柳植株的二氨基联苯胺(DAB)和氯化硝基四氮唑蓝(NBT)染色情况,过氧化物酶(POD)和超氧化物歧化酶(SOD)活性,及电解质渗透率、失水率及丙二醛(MDA)含量,鉴定ThNAC24基因的耐盐、抗旱功能。【结果】ThNAC24基因的开放阅读框为1 023 bp,编码340个氨基酸。多序列比对结果显示ThNAC24在N端的氨基酸序列相似度比较高,具有NAC家族的序列特征;系统进化树分析结果显示ThNAC24与ANAC103和ANAC082的亲缘关系较近。qRT-PCR结果显示:盐胁迫下,ThNAC24基因上调表达,在根组织中胁迫12 h表达量最高,而叶组织中胁迫24 h的表达量最高;干旱胁迫下,ThNAC24基因上调表达,在根组织中胁迫6 h表达量最高,在叶组织中胁迫12 h的表达量最高。ThNAC24基因在刚毛柽柳根和叶组织中均有表达且响应盐和干旱胁迫。过表达ThNAC24基因显著降低了刚毛柽柳H_2O_2和超氧阴离子含量,增强了POD和SOD酶的活性,从而减少活性氧(ROS)的积累。过表达ThNAC24基因能够降低刚毛柽柳在逆境胁迫下的电解质渗透率、失水率及MDA的积累,从而保护细胞膜结构的完整性。【结论】刚毛柽柳ThNAC24基因能够响应盐、干旱胁迫,过表达ThNAC24基因植株通过增强POD和SOD活性,进而提高ROS清除能力,减少细胞受损或死亡,从而提高刚毛柽柳的耐盐及抗旱能力。     

盐分、干旱及其交叉胁迫对皂角幼苗生长性状的影响

对皂角在土壤盐分、干旱及其交叉胁迫下生长性状进行研究.结果表明:皂角的存活率、苗高、生物量以及根系比重在干旱、轻度盐分和中度干旱与盐分的交叉胁迫处理下均能维持在较高的水平;叶保水力在各个胁迫条件下均高于对照;盐旱交叉胁迫下水分含量越高,越有利于皂角在盐分下的生长;皂角在轻度干旱胁迫、中度干旱胁迫、轻度盐分胁迫、轻度干旱轻度盐分交叉胁迫、轻度干旱中度盐分交叉胁迫、轻度盐分重度干旱胁迫、中度干旱轻度盐分胁迫、中度干旱中度盐分胁迫胁迫下能正常生长.     

Overexpression of the ThTPS gene enhanced salt and osmotic stress tolerance in Tamarix hispida

Trehalose is a non-reducing disaccharide with high stability and strong water absorption properties that can improve the resistance of organisms to various abi-otic stresses.Trehalose-6-phosphate synthase (TPS) plays important roles in trehalose metabolism and signaling.In this study,the full-length cDNA of ThTPS was cloned from Tamarix hispida Willd.A phylogenetic tree includ-ing ThTPS and 11 AtTPS genes from Arabidopsis indicated that the ThTPS protein had a close evolutionary relationship with AtTPS7.However,the function of AtTPS7 has not been determined.To analyze the abiotic stress tolerance function of ThTPS,the expression of ThTPS in T.hispida under salt and drought stress and JA,ABA and GA3 hormone stimu-lation was monitored by qRT-PCR.The results show that ThTPS expression was clearly induced by all five of these treatments at one or more times,and salt stress caused par-ticularly strong induction of ThTPS in the roots of T.hispida.The ThTPS gene was transiently overexpressed in T.his-pida.Both physiological indexes and staining results showed that ThTPS gene overexpression increased salt and osmotic stress tolerance in T.hispida.Overall,the ThTPS gene can respond to abiotic stresses such as salt and drought,and its overexpression can significantly improve salt and osmotic tolerance.These findings establish a foundation to better understand the responses of TPS genes to abiotic stress in plants.     

Improved salt tolerance of Populus davidiana × P. bolleana overexpressed LEA from Tamarix androssowii

Development of transgenic plants with tolerance to environmental stress is an important goal of plant biotechnology. Late-embryogenesis-abundant(LEA) proteins accumulate in seeds during late embryogenesis, where they protect cellular membranes and macromolecules against drought. In this work, we transferred the Tamarix androssowii LEA gene into hybrids of Populus davidiana×P. bolleana. We compared relative rates of height growth, chlorophyll fluorescence kinetic parameters, and leaf Na+ levels of six TaLEA-containing lines with non-transferred plants(NT), all grown under 0.8% NaCl stress condition. Survival percentages of transgenic lines were all higher than for NT controls after rehydration and the survival percentage of SL2 was five-fold higher than for NT controls. Seedling height increased 48.7% in SL2(from the onset of induced stress to the end of the growing season), 31% more than for the NT controls. Chlorophyll fluorescence kinetic parameters showed a marked increase in photosynthetic capacity in SL2 and SL5. Na+ levels in young leaves of transgenic lines were lower than in control NT leaves, but higher in yellow and withered leaves, indicating improved salt tolerance in transgenic lines.     

Overexpression of AtNHX5 improves tolerance to both salt and drought stress in Broussonetia papyrifera (L.) Vent

Paper mulberry (Broussonetia papyrifera L. Vent) is well known for its bark fibers, which are used for making paper, cloth, rope, etc. It was found that, in addition to its well-documented role in the enhancement of plant salt tolerance, overexpression of the Na+/H+ antiporter (AtNHX5) gene in paper mulberry plants showed high drought tolerance. After exposure to water deficiency and salt stress, the wild-type (WT) plants all died, while the AtNHX5-overexpressing plants remained alive under high salt stress, and had a higher survival rate (>66%) under drought stress. Measurements of ion levels indicated that Na+ and K+ contents were all higher in AtNHX5-overexpressing leaves than in WT leaves in high saline conditions. The AtNHX5 plants had higher leaf water content and leaf chlorophyll contents, accumulated more proline and soluble sugars, and had less membrane damage than the WT plants under water deficiency and high saline conditions. Taken together, the results indicate that the AtNHX5 gene could enhance the tolerance of paper mulberry plants to multiple environmental stresses by promoting the accumulation of more effective osmolytes (ions, soluble sugars, proline) to counter the osmotic stress caused by abiotic factors.     

刚毛柽柳Thb HLH1转录因子识别的顺式作用元件的鉴定

[目的]本研究拟对Thb HLH1所识别的顺式作用元件进行鉴定,进一步揭示Thb HLH1调控抗逆基因表达的机理。[方法]利用以转录因子为中心的酵母单杂交系统鉴定Thb HLH1所识别的顺式作用元件;将元件与报告基因融合构建报告载体(p CAM-Cis),通过基因枪法将报告载体与效应载体35S∶Thb HLH1共转化烟草叶片,在盐、干旱胁迫下比较GUS酶活。[结果]鉴定出2段能够与Thb HLH1转录因子结合的DNA序列:分别为CCGAAA(LTRE1)和TGAC(WRKY710S)。[结论]在盐或干旱胁迫下,Thb HLH1通过与LTRE1或WRKY710S元件作用来激活基因表达。     

盐旱交叉胁迫对皂角幼苗保护酶活性的影响

以一年生皂角Gleditsia sinensis Lam.实生苗为材料,研究干旱和盐分交叉胁迫对其膜质过氧化和保护酶活性的影响.结果表明:盐旱交叉胁迫下,丙二醛(MDA)含量、膜透性呈上升趋势,超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性先上升后下降;在相同的处理下,随着处理时间的延长,膜透性和MDA含量呈上升趋势,SOD、POD活性均下降,CAT活性先上升后下降.分析认为皂角幼苗在盐旱交叉胁迫下表现出交叉适应性,适度的干旱胁迫可增加皂角的抗盐能力.     

Cloning of four DREB genes from Tibetan Sophora moorcroftiana and analysis of their expression during abiotic stress

Sophora moorcroftiana is an endemic, droughtresistant shrub that grows in Tibet and has some degree of resistance to salt, cold, heat, and drought. In the present study, four dehydration responsive element-binding(DREB) genes(Sm DREB1, Sm DREB2, Sm DREB and Sm DREB1) were isolated from S. moorcroftiana for the first time and their expression and proline content under abiotic stress were analyzed. Proline accumulated in seedlings under drought, salt, cold, and heat stress treatments. The four genes were variously expressed in response to the four abiotic stresses. Sm DREB1 was induced by drought, cold, and heat stresses; Sm DREB2 and Sm DREB4 were both induced by salt, cold, and heat stresses, whereas Sm DREB3 was induced by drought and heat stresses. Thus, these four genes may participate in conferring tolerance to these four abiotic stresses and are candidate genes for genetic engineering in the future.     

Improvement of paper mulberry tolerance to abiotic stresses by ectopic expression of tall fescue FaDREB1

Dehydration-responsive element binding/C-repeat-binding factors (DREB/CBF) control the activity of multiple stress response genes and therefore represent attractive targets for genetic improvement of abiotic stress tolerance. Paper mulberry (Broussonetia papyrifera L. Vent) is well known for its bark fibers and high levels of chalcone and flavonoid derivatives. Transgenic paper mulberry plants expressing a tall fescue (Festuca arundinacea Schreb.) FaDREB1 gene under the control of CaMV 35S were produced to examine the potential utility of FaDREB1 to increase the tolerance of paper mulberry plants to abiotic stress. The overexpressing FaDREB1 plants showed higher salt and drought tolerance than the wild-type plants (WT). After 13 days of withholding water, or 15 days in the presence of 250 mM NaCl, all the WT plants died, while the over-expressing FaDREB1 plants survived. The FaDREB1 plants had higher leaf water and leaf chlorophyll contents, accumulated more proline and soluble sugars, and had less ion leakage (which reflects membrane damage) than the WT plants had under high salt- and water-deficient conditions. The 35S promoter-driven expression of FaDREB1 did not cause growth retardation under normal growth conditions. Therefore, improved tolerance to multiple environmental stresses in paper mulberry might be achieved via genetic engineering through the ectopic expression of an FaDREB1 gene.     

代谢组学在植物逆境胁迫研究中的应用

代谢组学是继基因组学、转录组学、蛋白质组学之后新兴的一门交叉学科, 是系统生物学的一个重要分支, 近年来在研究植物逆境胁迫方面得到了广泛的应用。文中介绍了代谢组学的检测技术、数据处理及分析方法, 概述了其在植物逆境胁迫下的研究概况, 包括温度、盐分、干旱、病原菌和其他逆境, 以及多种逆境综合胁迫等, 展望了代谢组学的发展前景; 利用代谢组学分析植物在逆境条件下代谢物的变化, 推测应答胁迫植物对代谢途径的调整, 可为研究植物耐受性和适应逆境机理提供分析方法。     

盐胁迫下比拉底白刺差异表达基因的转录组分析

[目的]为了挖掘比拉底白刺耐盐相关基因,对其盐胁迫下差异表达基因进行筛选分析。[方法]以比拉底白刺幼苗为材料,用200 mmol·L^-1 NaCl对幼苗处理7 d,并对胁迫处理和对照植株叶片进行转录组测序及生物信息学分析。[结果]有效序列组装共得到应答盐胁迫的168463条unigenes和196个差异表达基因。通过差异基因GO和KEGG功能聚类,分别获得64个GO功能小类和25条KEGG通路。进一步基因相互作用网络分析发现,转录调控、氧化还原以及抗逆相关基因在比拉底白刺应答盐胁迫中发挥重要作用,其中,筛选到3个重要的节点基因,分别是热激同源蛋白基因、L型凝集素类受体激酶基因和Win类蛋白基因。[结论]本研究获得了盐胁迫下比拉底白刺的差异表达基因及功能注释信息,有助于理解其耐盐的分子机制,为后续开发耐盐分子标记及通过基因编辑改良植物耐盐特性提供了科学依据。     

Enhanced tolerance of photosynthesis to high-light and drought stress in Pseudotsuga menziesii seedlings grown in ultraviolet-B radiation

We investigated the effects of an ambient dose of ultraviolet-B (UV-B) radiation on chamber-grown Pseudotsuga menziesii var. glauca (Beissn.) Franco (Douglas-fir) seedlings, to determine if the presence of UV-B radiation in the growth light regime induces tolerance to environmental stresses such as high light and drought. Douglas-fir seedlings were grown without UV-B radiation or with 6 kJ m-2 day-1 of biologically effective UV-B, which is ambient for the intermountain regions of Idaho. Non-stressed seedlings grown with UV-B radiation had 35% lower seedling dry mass, 36% higher concentrations of UV-B absorbing compounds per unit leaf area, 30% lower stomatal frequencies, 25% lower light-saturated photochemical efficiencies of Photosystem II and 45% lower light-saturated stomatal conductance than non-stressed seedlings grown without UV-B radiation. After 4 days of high-light stress, seedlings grown with UV-B radiation had 32% higher light-saturated carbon assimilation rates (A(CO2)) than seedlings grown without UV-B radiation. After water was withheld from the seedlings for up to 15 days, seedlings grown with UV-B radiation had 50% higher A(CO2) and 40% higher seedling water potentials than seedlings grown without UV-B radiation. The results support the hypothesis that UV-B radiation can act as an environmental signal to induce tolerance to high-light and drought stress in Douglas-fir seedlings. Possible mechanisms for the enhanced stress tolerance are discussed.     

矿山型东南景天cDNA表达文库构建与耐镉基因筛选

以筛选出的镉超积累矿山型东南景天为材料,经镉胁迫后提取总RNA,纯化mRNA,利用改良SMART技术合成了全长cDNA,回收500 bp以上cDNA大片段克隆到改造过的大肠杆菌/酵母穿梭载体pYES2.0G中,建成东南景天镉全长cDNA文库,对随机挑取的阳性克隆进行PCR鉴定,插入片断大小在1 000 bp左右,说明所构建的文库达到了用于目的基因分离筛选和表达的建库要求,为将来筛选克隆与东南景天抗重金属相关基因奠定了基础。     

低温、干旱和盐胁迫下沙冬青幼苗脯氨酸含量的变化

沙冬青(Ammopiptanthus mongolicus.(Maxim)Cheng.f)为我国西北荒漠区唯一的常绿灌木,具有极强的抗性。本文对低温、干旱和盐胁迫下沙冬青幼苗中脯氨酸积累的变化进行研究,以期探讨不同胁迫下脯氨酸累积的共性和特异性,为进一步探讨逆境下脯氨酸积累的生理意义提供参考。     

Drought stress tolerance analysis of Populus ussuriensis clones with different ploidies

The selection of drought-tolerant plants is an important aspect of plant breeding.We studied physiological and biochemical mechanisms of different ploidies of Populus ussuriensis Kom.that relate to drought stress tolerance.We used a 5%(v/v)polyethylene glycol(PEG-6000)solution to simulate drought stress.We recorded leaf phenotypes including color,dry area and curl degree.We evaluated sequential variations in some drought stress tolerance-related physiological and biochemical indices and compared these among diploid clones(CK),triploid clones(T12)and tetraploid clones(F20).T12 leaves exhibited slightly more drought stress damage than CK and F20 leaves.CK leaves suffered the most severe drought stress damage.The physiological and biochemical indices of the different ploidies differed significantly 12 days after drought stress treatment.The activities of superoxide dismutase,peroxidase,catalase and proline in the triploid(T12)leaves were the highest.The relative electric conductivity and malondialdehyde content of T12 leaves were the lowest.The index values of F20 were between those ofthe diploid and triploid.In consideration of these results,the drought resistance of the three different ploidies of P.ussuriensis can be ranked as T12>F20>CK.We speculate that the gene expression patterns of polyploid clones of poplar will change after genome doubling and that some of the drought stress tolerance-related physiological and biochemical indices will be improved,resulting in greater drought tolerance of polyploid clones.     

蜡梅转录因子CpTAF10基因的克隆及功能分析

【目的】 TATA框结合蛋白相关因子TAF10作为基本转录因子之一,在生长发育和胁迫响应过程中发挥着广泛的、重要的生物学作用。对蜡梅中TAF10同源基因 CpTAF10的克隆与功能分析,有利于丰富对植物TAFs基因功能的认识,并为解析蜡梅抗逆形成的转录调节机理提供新的理论依据。【方法】以转录组数据库中获得的蜡梅TAFs家族基因序列,克隆得到 CpTAF10基因的cDNA序列,并对其编码蛋白进行序列特征和进化树分析。采用实时荧光定量PCR 技术分析 CpTAF10基因在蜡梅不同组织及花期中的表达特性,以及高温、低温、盐胁迫及ABA处理后的表达变化。同时,构建 CpTAF10基因的过表达载体,采用花序侵染法进行拟南芥遗传转化,对拟南芥转基因纯合株系进行表型观察和胁迫耐性分析。【结果】获得的 CpTAF10基因 cDNA序列为712 bp,包含405 bp的开放阅读框(ORF),编码134个氨基酸,蛋白理论分子量为15.21 kDa,预测的等电点pI值为5.19。CpTAF10蛋白序列与其他植物同源序列具有较高的同源性,蛋白多序列比对显示CpTAF10蛋白属于TAF10同源蛋白,并含有组蛋白折叠结构域。表达特性分析结果发现,CpTAF10基因在蜡梅的根、茎、子叶、幼叶、成熟叶和花6个不同组织中均有不同程度的表达,其中,在成熟叶中的表达量最高。 CpTAF10在蜡梅花朵的不同花期中,呈现出波动的表达模式,在衰老期表达量最高。在低温、盐胁迫和ABA处理的蜡梅叶片中均能被诱导表达,但其表达变化各不相同。在拟南芥中过表达 CpTAF10基因可提高盐胁迫下拟南芥种子的萌发率,相对于野生型植株,转基因植株的主根和侧根在盐胁迫下均表现出一定的生长优势。【结论】 CpTAF10基因能在低温、盐胁迫和ABA处理后诱导表达,可能参与蜡梅逆境胁迫耐性的分子调控。在拟南芥中过表达 CpTAF10基因显著提高了转基因拟南芥的萌芽率及主根和侧根的生长优势,在一定程度上可增强植物的盐胁迫耐性。     

聚乙二醇模拟干旱胁迫对榆树种子萌发影响

为探究干旱条件下榆树种子的发芽特性,以榆树种子为试验材料,采用滤纸发芽法,研究聚乙二醇(PEG)模拟干旱对其种子萌发的影响。结果表明:不同浓度的PEG干旱胁迫对榆树种子萌发有不同影响,5%浓度的PEG胁迫对榆树种子的萌发有明显的促进作用;榆树种子可耐5%~10%PEG浓度的干旱胁迫,而15%浓度的PEG胁迫明显抑制榆树种子萌发,20%浓度的PEG胁迫则对其种萌发有严重抑制作用。榆树的种子耐旱适宜范围、种子耐旱半致死浓度、种子耐旱极限浓度对应的PEG浓度分别为7.71%、12.33%、19.73%。     

等渗胁迫及其钙调节下青钱柳幼苗叶肉细胞中ATP酶的细胞化学定位及亚细胞变化

研究了等渗的盐和水分胁迫及其钙调节下,青钱柳幼苗叶肉细胞中ATP酶活性在亚细胞中的分布及其超微结构变化。在人工气候室中采用水培法,将青钱柳幼苗进行5个不同浓度处理：对照,85mMNaCl,85mMNaCl＋12mMCa（NO3）2,PEG（渗透势等于85mMNaCl）及PEG（渗透势等于85mMNaCI）＋12mMCa（NO3）2。结果表明：正常生长条件下,ATP酶活性较低并主要定位在细胞核中 等渗胁迫12d以后,ATP酶活性增大并以液泡中居多。等渗水分胁迫下出现的嗜锇颗粒较等渗盐胁迫下的多。由于外源钙的调节作用,等渗胁迫下ATP酶活性增加并主要转移至细胞核上,且在等渗水分胁迫下的嗜锇颗粒明显减少。ATP酶定位在细胞核中表明幼苗遭受胁迫伤害程度较轻,而定位在液泡中则表明受伤害程度较重。等渗处理4和20d后,等渗胁迫及其钙调节下青钱柳幼苗叶片超微结构被破坏程度较无钙调节处理,尤其是较等渗水分胁迫下的轻。初步认为,等渗盐胁迫下青钱柳幼苗遭受的胁迫伤害程度较等渗水分胁迫下的轻,而钙调节作用则以等渗水分胁迫下的效果较佳。