过量表达棉花GaSus3基因增强拟南芥的耐盐性

构建了植物过量表达载体p35S：：GaSus3,通过花序浸染法成功获得转GaSus3基因拟南芥植株。利用NaCl模拟盐胁迫处理,证实转基因拟南芥与野生型相比耐盐性明显增强。在盐胁迫下,转基因拟南芥受到的影响较小,而野生型则受盐害影响严重：转基因拟南芥具有更好的萌发率和主根长度,以保证植株正常生长;盐胁迫下转基因拟南芥能保持较多的绿色叶片,而野生型则过早黄化死亡。研究还发现,转基因拟南芥的过氧化氢酶活性在胁迫前后都高于野生型,这说明转GaSus3基因能够提高拟南芥抗氧化胁迫的能力。研究结果为进一步探讨GaSus3基因在棉花耐盐方面的功能奠定了基础。     

转柽柳晚期胚胎富集蛋白基因烟草T1代的耐盐性评价

目的:验证转柽柳晚期胚胎富集(LEA)蛋白基因烟草T1代的耐盐性。方法:采用盐胁迫方式,对转柽柳LEA蛋白基因烟草T1代的6个株系及非转基因对照烟草T1代进行不同浓度NaCl胁迫处理,分析了NaCl胁迫下转基因烟草的生长量、根系的发育及盐害程度。结果:各转基因烟草T1代组培苗在150mmol/L的NaCl培养基上根系生长良好,平均增重(鲜重)是非转基因对照的7.72倍,平均高生长是非转基因对照的3.51倍,盐害指数低于或等于50%;而非转基因对照烟草T1代组培苗生长缓慢,根系几乎不能生长发育,盐害指数达65%。结论:柽柳LEA蛋白基因的导入提高了T1代烟草的耐盐性。     

LeERF2基因对旱稻耐盐性的影响

采用T2代转LeERF2基因旱稻(旱297)为材料,研究分蘖期盐胁迫下植株光合性能和生理反应.结果表明,50 mM NaCl胁迫条件下,转基因植株(T)和野生型对照(WT)的各项光合参数差异不明显,但在100 mM NaCl胁迫条件下,转基因植株仍能维持较高的光合速率和气孔导度.随着NaCl胁迫浓度增加,野生型旱稻和转基因旱稻植株叶片SOD活性增加幅度加大,但都表现为转基因植株增幅更明显,而MDA含量则表现为野生型植株增幅更明显,表明LeERF2基因增强了旱稻盐胁迫下抗氧化能力,提高了耐盐性,能维持较高光合速率.     

小黑杨花药培养植株转化胆碱氧化酶基因提高耐盐性

以小黑杨（Populus simonii×p．nigra）花药培养植株无菌苗叶片为外植体,通过根癌农杆菌（Agrobacterium tumefaciens）介导法将胆碱氧化酶基因（codA）导入小黑杨中,共获得4株转化株系,PCR扩增和Southern杂交检测结果全部呈阳性,表明codA基因已整合到小黑杨花药培养植株基因组中。荧光定量RT-PCR检测证明,codA基因在小黑杨花药培养植株中获得表达。耐盐实验结果显示,各转基因株系在0．6％的NaCl浓度下能够生长,而非转基因对照小黑杨受盐害严重,说明codA基因的导入提高了转基因植株的耐盐性。     

转ZjAPX基因拟南芥对NaCl、干旱胁迫的耐性研究

旨在探讨枣树抗坏血酸过氧化物酶基因ZjAPX在植物渗透胁迫中的作用。将ZjAPX基因转入到模式植物拟南芥,以野生型(WT)、转ZjAPX拟南芥株系T2为试材,进行不同浓度NaCl胁迫和干旱胁迫。结果表明,转基因株系的种子萌发、植株生长均优于野生型株系;荧光定量PCR检测转基因拟南芥植株在干旱和盐胁迫处理10 d后目的基因ZjAPX的表达量显著高于野生拟南芥,表明ZjAPX的高表达明显提高了植株的抗旱和耐盐性。     

农杆菌介导的甜菜碱醛脱氢酶基因转化甘蓝的研究

为获得抗旱和耐盐性提高的甘蓝植株,通过农杆菌介导法将来自菠菜的甜菜碱醛脱氢酶（Betaine Aldehyde Dehydrogenase,BADH）基因导人甘蓝品系03079,并采用正交设计优化影响转化效率的参数,建立了甘蓝高效转化体系,即以侵染液为AA液体培养基、乙酰丁香酮200μmol L^-1、侵染时间20min、共培养天数2d为最佳转化参数,在该条件下转化率可达54．26％。转基因甘蓝植株经PCR检测初步说明BADH基因已导入甘蓝中,Southern杂交证明BADH基因已稳定整合到甘蓝基因组中。甜菜碱脱氢酶活性测定结果表明,经过聚乙二醇（PEG）、NaCI和干旱处理的转基因甘蓝植株的BADH酶的平均比活力范围在2．1Umg^-1～3．6Umg^-1之间,不同处理的转基因株系酶比活力显著高于相应的未转基因株系。膜的相对电导率测定结果说明,经过PEG、NaCl和干旱处理的转基因植株平均相对电导率在16．2％～32．6％之间,耐逆境胁迫处理后的绝大多数转基因株系相对电导率显著低于相应对照。多数转BADH基因甘蓝植株在干旱、盐胁迫和PEG胁迫条件下生长势强于未转基因植株,表现为大多数转基因株系株高增幅显著高于对照,说明BADH基因的导入能提高转基因甘蓝植株的抗旱和耐盐性。我们获得的抗旱和耐盐能力明显提高的转基因甘蓝植株,可作为培育耐盐、抗旱甘蓝品种的种质材料。     

转基因ABP9玉米株系的耐盐性分析

旨为分析转基因ABP9玉米的耐盐性,并进一步研究玉米耐盐的分子调控机制。通过对转基因ABP9玉米植株进行PCR、Southern blot和qRT-PCR分析鉴定;采用Hoagland营养液水培法,对转ABP9基因玉米及非转基因对照进行NaCl胁迫处理,分析二者在幼苗期的生理指标和基因表达差异。结果表明,转入的ABP9以单拷贝整合到基因组中,且能较高表达。与非转基因对照相比,转基因玉米幼苗NaCl胁迫下的叶绿素含量、Fv/Fm、渗透调节物质和抗氧化酶活性,以及盐胁迫后恢复过程中的叶片相对含水量均显著提高;而其丙二醛含量、相对电导率均显著降低。转录组测序和qRT-PCR分析显示,一系列盐胁迫应答基因在转基因玉米植株中上调表达。转入ABP9的增强表达提高了转基因玉米的耐盐能力。     

ptc-miR801人工microRNA表达载体构建及功能初步研究

在构建盐胁迫下青杨microRNA文库中发现了ptc-miR801,为探索植物在盐胁迫条件下ptc-miR801参与胁迫应答的机制,本实验构建了植物表达载体pCAM2300-ami801,经根癌农杆菌EHA105介导、花序侵染法获得拟南芥转基因植株。RT-PCR半定量结果显示ptc-miR801可以在转基因拟南芥中超表达且NaCl胁迫下ptc-miRNA801转基因植株种子萌发率和根长显著高于野生型,说明ptc-miR801超表达增强了转基因拟南芥耐盐性。该试验为进一步研究miR801在杨树胁迫应答机制中的作用奠定基础。     

羊草LcPIP基因遗传转化露地菊及其抗盐性鉴定

PIP是重要的水孔蛋白之一,与植物的抗逆性有关。本文用叶盘法将羊草LcPIP遗传转化露地菊火焰,通过常规PCR和GUS染色方法鉴定转基因株系,Real-time PCR检测盐胁迫下转基因和野生植株中CmPIP1和CmPIP2的相对表达量,并测定SOD、POD活性和MDA含量。结果显示,在转LcPIP基因的露地菊植株叶片中,CmPIP1和CmPIP2基因表达量均上升,是野生型植株的2倍;根部的CmPIP1与CmPIP2基因的表达量均上升,CmPIP1上升程度高于CmPIP2。在200 mmol·L~(-1) NaCl的胁迫下,野生型植株中CmPIP1基因表达量明显下降,而转基因植株中CmPIP1基因表达量在12~48 h出现明显的上升;在盐胁迫12 h后,转基因植株中CmPIP2基因表达量上升程度明显高于野生型植株。盐胁迫12 h后,转基因植株的SOD活性提高更为明显。在盐胁迫前期(0~24 h)转基因植株MDA含量增加幅度低于野生型植株;在盐胁迫后期(48~72 h)转基因露地菊POD活性出现明显上升,而野生型露地菊呈下降趋势。     

小黑杨花药培养植株转化胆碱氧化酶基因提高耐盐性

以小黑杨(Populus simonii ×P. nigra)花药培养植株无菌苗叶片为外植体, 通过根癌农杆菌(Agrob acteriumtumefaciens)介导法将胆碱氧化酶基因(codA)导入小黑杨中, 共获得4株转化株系, PCR扩增和Southern杂交检测结果全部 呈阳性, 表明codA基因已整合到小黑杨花药培养植株基因组中。荧光定量RT-PCR检测证明, codA基因在小黑杨花药培养植株中获得表达。耐盐实验结果显示, 各转基因株系在0.6%的NaCl浓度下能够生长, 而非转基因对照小黑杨受盐害严重, 说明codA基因的导入提高了转基因植株的耐盐性。     

转柽柳eIF1A基因烟草的耐盐性分析

目的:验证柽柳eIF1A基因的功能,为通过基因工程手段培育耐盐植物提供基础资料。方法:对转eIF1A基因的烟草和对照烟草进行不同浓度NaCl胁迫实验,测定其相对电导率、SOD活性和丙二醛含量,统计生根率、生长量和盐害程度。结果:转基因烟草的相对电导率、丙二醛含量均随盐浓度的增加而增大,但都较非转基因对照烟草低。SOD活性随着盐浓度的升高而升高,相同浓度NaCl胁迫下各转基因烟草的SOD活性均高于对照烟草的SOD活性。NaCl浓度为240mmol/L时,非转基因对照烟草不能生根,盐害指数高达67.7%;而转基因烟草均能生根,大部分转基因株系的生根率大于50%。结论:柽柳eIF1A基因的转化提高了烟草的耐盐性。     

Over-expression of Osmotin Induces Proline Accumulation and Confers Tolerance to Osmotic Stress in Transgenic Tobacco

Osmotin has been implicated in conferring tolerance to drought and salt stress in plants. We have over-expressed the osmotin gene under the control of constitutive CaMV 35S promoter in transgenic tobacco, and studied involvement of the protein in imparting tolerance to salinity and drought stress. The transgenic plants exhibited retarded leaf senescence and improved germination on a medium containing 200mM NaCl. Further, the transgenics maintained higher leaf relative water content (RWC), leaf photosynthesis and free proline content than the wild type plants during water stress and after recovery from stress. When subjected to salt stress (200mM NaCl), the transgenic plants accumulated significantly more proline than the wild type plants. These results suggest the involvement of the osmotin-induced increase in proline in imparting tolerance to salinity and drought stress in transgenic plants over-expressing the osmotin gene.     

Enhanced salt tolerance of transgenic poplar plants expressing a manganese superoxide dismutase from <Emphasis Type="Italic">Tamarix androssowii</Emphasis>

Superoxide dismutases (SODs) play important role in stress tolerance of plants. In this study, an MnSOD gene (TaMnSOD) from Tamarix androssowii, under the control of the CaMV35S promoter, was introduced into poplar (Populus davidiana × P. bolleana). The physiological parameters, including SOD activity, malondialdehyde (MDA) content, relative electrical conductivity (REC) and relative weight gain, of transgenic lines and wild type (WT) plants, were measured and compared. The results showed that SOD activity was enhanced in transgenic plants, and the MDA content and REC were significantly decreased compared to WT plants when exposed to NaCl stress. In addition, the relative weight gains of the transgenic plants were 8- to 23-fold of those observed for WT plants after NaCl stress for 30 days. The data showed that the SOD activities that increased in transgenic lines are 1.3–4-folds of that increased in the WT plant when exposed to NaCl stress. Our analysis showed that increases in SOD activities as low as 0.15-fold can also significantly enhance salt tolerance in transgenic plants, suggesting an important role of increased SOD activity in plant salt tolerance.     

Constitutive over-expression of AtGSK1 induces NaCl stress responses in the absence of NaCl stress and results in enhanced NaCl tolerance in Arabidopsis

GSK3/shaggy-like protein kinases have been shown to play diverse roles in development and signal transduction pathways in various organisms. An Arabidopsis homologue of GSK3/shaggy-like kinase, AtGSK1, has been shown to be involved in NaCl stress responses. In order to further clarify the role of AtGSK1 in NaCl stress responses in plants, we generated transgenic Arabidopsis plants that over-expressed AtGSK1 mRNA. These plants showed enhanced resistance to NaCl stress when assayed either as whole plants or by measurement of root growth on NaCl plates. In addition, AtGSK1 transgenic plants in the absence of NaCl stress showed phenotypic changes, such as accumulation of anthocyanin, that were similar to those observed in wild-type plants under NaCl stress. Transgenic plants accumulated 30-50% more Na+ than did wild-type plants when subjected to NaCl stress, and Ca2+ content was increased by 15-30% in the transgenic plants regardless of the NaCl stress level. Northern blotting revealed that AtGSK1 over-expression induced expression of the NaCl stress-responsive genes AtCP1, RD29A and CHS1 in the absence of NaCl stress. In addition, AtCBL1 and AtCP1 were super-induced in the NaCl-stressed transgenic plants. Taken together, these results suggest that AtGSK1 is involved in the signal transduction pathway(s) of NaCl stress responses in Arabidopsis.     

Over-expression of a barley aquaporin increased the shoot/root ratio and raised salt sensitivity in transgenic rice plants

Barley HvPIP2;1 is a plasma membrane aquaporin and its expression was down-regulated after salt stress in barley [Katsuhara et al. (2002) Plant Cell Physiol. 43: 885]. We produced and analyzed transgenic rice plants over-expressing barley HvPIP2;1 in the present study. Over-expression of HvPIP2;1 increased (1) radial hydraulic conductivity of roots (Lp(r)) to 140%, and (2) the mass ratio of shoot to root up to 150%. In these transgenic rice plants under salt stress of 100 mM NaCl, growth reduction was greater than in non-transgenic plants. A decrease in shoot water content (from 79% to 61%) and reduction of root mass or shoot mass (both less than 40% of non-stressed plants) were observed in transgenic plants under salt stress for 2 weeks. These results indicated that over-expression of HvPIP2;1 makes rice plants sensitive to 100 mM NaCl. The possible involvement of aquaporins in salt tolerance is discussed.     

转铜/锌超氧化物歧化酶和抗坏血酸过氧化物酶基因马铃薯的耐氧化和耐盐性研究

高盐等逆境可以加剧植物体内活性氧的产生,进而引起植物细胞死亡。为开发抗逆境作物,以置于氧化诱导型启动子下定位于叶绿体的转铜/锌超氧化物歧化酶（Cu/ZnSOD）和抗坏血酸过氧化物酶基因(APX)马铃薯为材料,研究了其对MV和 NaCl所引起的氧化胁迫的耐受性。结果表明, MV胁迫下,转基因马铃薯叶片膜的相对电导率明显低于对照; NaCl胁迫下,其叶绿素含量高于对照。 在含NaCl 的培养基上,转基因幼苗生根率明显大于对照。另外,NaCl胁迫下转基因马铃薯叶片的SOD和APX酶活性显著高于对照,与其耐盐性的提高相一致。这些研究表明,转入Cu/ZnSOD和APX基因的马铃薯清除活性氧的能力增强,抗逆性得到提高。本实验采用氧化诱导型启动子调控下的SOD和APX两个基因协同作用,使外源基因只有在逆境胁迫时才特异性表达,增强转基因植株的抗逆效果,为培育抗逆经济作物开阔了思路。     

AtNHX1基因对草木樨状黄芪的转化和耐盐性表达研究

应用RT-PCR技术从100mmol/LNaCl胁迫处理的拟南芥幼中克隆得到编码液泡膜Na /H 逆向转运蛋白的AtNHX1基因cDNA 编码ORF.并在该ORF上游分别插入CaMV 35启动子和TMV RNA5'UTR的Ω片段,而在下游插入NOS polyA构建真核表达盒,进而将该表达盒插入双元植物表达栽体pNT质粒的T-DNA区构建了携带AtNHX1 基因的植物表达载体质粒pNT-AtNHX1.将pNT-AtNHX1 导入农杆菌LBA4404,用农杆菌介导法将AtNHX1 基因导入豆科牧草草木樨状黄芪中,共获得103株Kan抗性再生植株.通过对农杆菌茵液浓度、侵染时间和乙酰丁香酮浓度等影响转化效率的因素进行优化,初步建立了稳定的草木樨状黄芪农杆菌转化体系.经过PCR检测、Southern杂交和RT-PCR检测表明,AtNHX1 基因已被成功整合到草木樨状黄芪基因组中,并且能够正常转录.野生型和转基因株系诱发的愈伤组织进行耐盐生长实验,结果显示相同盐胁迫条件下,转基因愈伤组织的相对生长率显著高于野生型愈伤组织.施加梯度NaCl胁迫后,植株叶片K ,Na 含量和叶片相对电导率测定结果显示,转基因植物叶片比野生型积累更多的Na 和K ,维持较高的K /Na ;转基因株系叶片相对电导率显著低于野生型.上述结果表明,AtNHX1 基因的导入和表达在提高草木樨状黄芪耐盐性的同时减轻了盐胁迫对植物细胞膜的伤害.关键词: AtNHX1 草木樨状黄芪农杆菌遗传转化耐盐性.     

Enhanced drought and salt tolerance by expression of AtGSK1 gene in poplar

A GSK3/shaggy-like kinase (AtGSK1) has been implicated in the regulation of drought and salt tolerance. We transferred AtGSK1 from Arabidopsis thaliana to a hybrid poplar (Populus alba × P. tremula var. grandulosa) to determine the effect of the transgene expression in the transgenic trees. The results from northern blot and RT-PCR analyses showed that the expression level varied among the transgenic lines. During their culture on tissue culture media, the transgenic poplars formed vigorous growing roots even in the presence of 125 mM NaCl and callus in the presence of 150 mM NaCl. When the transgenic poplars were growing in pots and provided with NaCl solution, they stayed much healthier than did nontransgenic poplars, showing higher rates of photosynthetic rates, stomatal conductance, and evaporation rates under the stress. Whereas the total level of leaf Na⁺ level increased dramatically in transgenic poplars under severe saline conditions (150 mM NaCl), that of leaf K⁺ decreased in the same plants under the same conditions. Total root Na⁺ level increased in nontransgenic poplars under severe saline conditions. In contrast, total root K⁺ level decreased in the same plants under the same conditions. The chloride content and relative electrical conductivity of the transgenic poplars after salt stress treatment were lower than those of nontransgenic poplars. The transgenic poplars were also tolerant to up to 20 % PEG remaining significantly healthy when compared with nontransgenic poplars with necrosis and chlorosis symptoms. Another dramatic feature of the transgenic poplars was wilting tolerance for prolonged drought treatment up to 2 weeks. The results provide evidence that the expression of AtGSK1 gene conferred drought and salt tolerance in the transgenic poplars.