过表达ZmSKIP基因提高烟草抗低温胁迫的能力

以野生型和过表达ZmSKIP基因烟草为试材, 研究了低温胁迫下过表达ZmSKIP对烟草抗氧化能力的影响。测定了不同低温处理时间下过表达ZmSKIP转基因烟草T3代植株和野生型植株抗氧化酶如超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）活性和丙二醛（MDA）含量以及相对电导率, 结果表明, 低温下, 相对于野生型植株, 转基因烟草具有较高的抗氧化酶活性和较低的相对电导率和MDA含量, 说明过表达ZmSKIP提高了转基因植株的耐低温胁迫能力。     

小黑杨PsnHB22基因在烟草中的遗传转化研究

HD-Zip转录因子在光信号转导、非生物胁迫、叶片发育等方面发挥重要的作用，HB22转录因子是HD-ZipⅠ亚家族的成员之一。为研究PsnHB22基因的功能，从小黑杨（Populus simonii×P.nigra）cDNA中克隆PsnHB22基因并构建植物表达载体进行烟草（Nicotiana tabacum）的遗传转化，以获得该基因过量表达的转基因株系。对转基因株系进行PCR、qRT-PCR分子检测后观察表型，结果显示在营养生长时期，转基因烟草叶片窄小并且株高显著低于野生型对照。测定转基因烟草及野生型叶片的叶绿素含量，发现转基因烟草叶绿素含量显著高于野生型。由此推测PsnHB22基因在植株高生长、光合作用及叶片的形态建成等过程中起着重要的作用。     

转PvP5CS2基因烟草对干旱胁迫的反应

为了验证普通菜豆脯氨酸合成酶基因PvP5CS2在改善作物抗旱性方面的作用,构建了普通菜豆PvP5CS2基因的烟草表达载体pCAPE2-PvP5CS2,利用农杆菌将PvP5CS2导入烟草,获得15株阳性转PvP5CS2基因植株。干旱胁迫条件下,转基因烟草植株和野生型植株叶片中脯氨酸含量、叶片相对含水量和萎蔫叶片数的差异均达到极显著水平。转基因烟草的脯氨酸含量、叶片相对含水量分别是野生型的2．30倍和1．26倍,而萎蔫叶片数只相当于野生型的90％。表明超表达PvP5CS2基因的烟草植株在干旱胁迫条件下能积累较多的脯氨酸,抗旱性得到初步改善。     

异源过表达胡杨PeDWF4基因提高烟草对非生物胁迫的耐性

本研究以烟草为材料异源过表达了胡杨PeDWF4基因,选取T1代的转基因阳性植株和野生型烟草幼苗水培培养50 d后对其分别施加100 mmol/L Na Cl、100 mmol/L甘露醇和100μmol/L Cu SO4进行处理,通过测定其植株生物量、叶绿素含量、抗氧化酶系统酶SOD和POD活性,渗透调节物质(脯氨酸,可溶性糖和可溶性蛋白)含量等,研究在不同非生物胁迫下PeDWF4转基因烟草对胁迫的生理生化响应及过量表达PeDWF4基因对其抗逆方面的影响和作用。结果表明,(1)异源过表达PeDWF4基因可显著提高转基因烟草的植株鲜重和生物量;(2)在3种胁迫下,转基因烟草通过减少叶片内叶绿素的降解,增加抗氧化系统酶SOD和POD活性以及提高渗透调节物脯氨酸等的含量等方式来减少非生物胁迫对自身的伤害。以上结果表明,异源表达PeDWF4基因可能是通过提高了转基因烟草內源的BR含量,从而提高了烟草对于以上3种非生物胁迫的耐性。     

白桦BpERF98基因的遗传转化及非生物胁迫应答反应

非生物胁迫严重影响植物的生长发育,植物通过内部的分子调控机制抵御这种伤害,其中转录因子发挥了至关重要的作用。从野生型白桦（Betula platyphylla）叶片克隆BpERF98基因,通过农杆菌介导法获得过表达BpERF98的转基因白桦植株。测量转基因白桦和野生型白桦在低温、冻害和盐胁迫下的生理指标并进行差异性分析。通过分析发现,在非生物胁迫下转基因白桦的丙二醛含量及相对电导率均低于野生型株系,且转基因株系SOD和POD活性的增长明显高于野生型株系。结果表明,过表达BpERF98基因可以提高白桦对非生物胁迫的抵御能力,这为研究白桦在非生物胁迫下形成的分子机制及白桦抗性分子育种提供了依据。     

大豆SbPRP3基因表达及转基因烟草非生物胁迫鉴定

为研究大豆(Glycine max L.)细胞壁脯氨酸富集蛋白基因(SbPRP3)在逆境胁迫中的作用,利用实时荧光定量PCR,对SbPRP3在高盐、干旱和低温处理下的表达情况进行检测。结果显示,SbPRP3在高盐处理下表达量升高,在干旱和低温处理下表达量先升高后降低。将SbPRP3构建到植物表达载体pRI101-AN上并转化烟草,获得阳性转基因烟草3株。对转基因烟草植株进行高盐、干旱和低温胁迫处理。结果表明,与对照相比,高盐和低温处理下转基因烟草脯氨酸积累量增加,而丙二醛产生量降低。但干旱处理后转基因烟草脯氨酸和丙二醛的含量与对照相比没有显著差异。由此推测SbPRP3可以提高转基因烟草的耐盐性和耐寒性。     

无苞芥锌指蛋白基因OpZFP提高烟草耐盐性的研究

旨在研究C2H2型锌指蛋白在植物生长发育、非生物胁迫信号转导过程中的作用。前期从新疆无苞芥中克隆的一个单锌指基因Op ZFP,利用叶盘法将Op ZFP基因转入普通烟草中。半定量RT-PCR表明,在转基因植株中Op ZFP基因能够高效表达。烟草耐盐性分析显示,在高盐胁迫下,转基因植株的根长要长于野生型植株,且转基因烟草丙二醛(MDA)的含量要明显低于野生型植株;并且高盐胁迫处理,野生型烟草离体叶片叶绿素降解率高于转基因植株。这些结果表明,过量表达Op ZFP的转基因植株可以提高植物对盐胁迫的抗性。     

过量表达菊花DmDREBa基因提高转化烟草耐低温能力

各种环境因素,如干旱、高盐、激素和低/高温等非生物胁迫对植物的生长发育造成很大影响。DREB转录因子在植物抵抗非生物胁迫中起到关键作用。本研究通过根癌农杆菌介导的叶盘转化法将菊花DmD REBa基因导入烟草中并进行了耐低温能力分析。研究利用PCR的方法鉴定出了43株转基因阳性植株。随机选取其中9株转基因植株,有7株在RNA转录水平能够表达。Southern杂交检测表明,DmD REBa基因以1~3个拷贝形式随机插入到烟草基因组中。胁迫处理结果表明,DmD REBa基因明显增强了转基因烟草抵抗低温能力。通过叶片上下表皮气孔密度检测,发现转基因烟草的蒸腾失水量远远低于对照野生型。进一步对低温胁迫下转基因烟草的丙二醛含量进行测定分析,发现转基因烟草丙二醛含量比野生型烟草低22.29%。综上结果表明,DmD REBa基因能够提高转基因烟草对低温的耐受能力,为菊花DREB转录因子的深入研究提供理论依据,并为进一步解析菊花DREB基因功能奠定基础。     

陆地棉GhCDPK1基因响应干旱胁迫的功能初探

钙依赖性蛋白激酶(CDPKs)是一类重要的钙信号感受蛋白和响应蛋白,在植物干旱、低温、盐碱等非生物胁迫应答中起着重要的调控作用。为探讨陆地棉GhCDPK1基因在干旱胁迫下所起的作用,该研究利用实时荧光定量PCR技术分析了PEG模拟干旱胁迫下该基因的表达量,发现GhCDPK1基因受干旱胁迫诱导。通过构建植物表达载体pCAMBIA2300-GhCDPK1,采用农杆菌介导的叶盘法转化模式植物烟草,发现干旱胁迫下转基因植株保水能力明显高于野生型植株,叶绿素、脯氨酸、可溶性蛋白含量及POD、SOD活性也高于野生型植株,而丙二醛含量低于野生型植株。研究结果表明,GhCDPK1基因作为正向调控因子响应干旱胁迫诱导,过表达GhCDPK1基因可以使植株积累更多的渗透调节物质、增强抗氧化系统酶的活性和维持细胞膜的稳定性来提高植物抵御外界干旱胁迫的能力。     

向日葵转录因子HB-12的定位分析及过表达验证

向日葵转录因子HB-12属于HD-Zip I类转录因子家族,在植物逆境胁迫应答中发挥着极其重要的作用。该研究构建HB-12基因瞬时表达载体并进行亚细胞定位分析,表明HB-12基因定位于细胞核。构建HB-12基因植物超表达载体转化野生型烟草获得了转基因植株。进行了Na Cl胁迫对转基因烟草耐盐性检测、生理生化指标测定和胁迫相关基因的表达分析。结果表明,Na Cl胁迫条件下,转基因烟草叶色失绿程度较野生型的轻,分化情况较野生型的好,生长速度和生根率均较野生型的高;转基因烟草叶绿素和脯氨酸含量及POD和SOD活性均高于野生型烟草;与野生型烟草相比,转基因烟草P5CS、POD、Mn SOD和Gu Zn SOD相对表达水平显著提高。这说明,向日葵HB-12在转基因烟草中的过量表达可抑制叶绿素降解酶的活性,降低叶绿素的分解,还可诱导脯氨酸合成酶基因P5CS及抗氧化相关基因POD、Mn SOD和Gu Zn SOD的上调表达,促进脯氨酸的生物合成,增强POD和SOD的活性,提高烟草抵抗盐害的能力。该研究结果将为进一步探讨植物的耐盐机理及改良作物的耐盐性状奠定基础。     

甘菊DlNAC1转录因子基因提高烟草耐高温能力

研究针对从甘菊中克隆获得的DlNAC1基因（GenBank登录号为EF602305）进行生物信息学分析,并利用根癌农杆菌介导的叶盘转化法将该基因在烟草中进行过表达研究。结果发现DlNAC1蛋白具有较高亲水性,二级结构中占比最高的为无规则卷曲,并具有N糖基化位点等6类潜在的模体结构和典型的由一个扭曲的反平行β片层和α螺旋组成的NAC结构域。将DlNAC1基因在烟草中过表达后,通过PCR方法从55株转化植株中鉴定出36株为阳性植株,并且转基因烟草T₀代植株在45℃高温胁迫后,转35S：DlNAC1基因阳性植株生长状况良好,而对照植株发生萎蔫,并且转基因植株叶片含水量显著高于对照植株。然而,在4℃低温胁迫后,发现转基因烟草T₁代植株没有提高耐低温能力。甘菊DlNAC1基因能够提高烟草植株耐高温能力,为今后菊花抗逆育种提供了科学依据。     

过表达胡杨XTH基因能够提高烟草抗旱性

胡杨是典型的抗旱树种。挖掘和鉴定胡杨的耐旱基因对于提高植物抗旱性具有重要意义。木葡聚糖内转糖苷酶/水解酶（XTH）是植物细胞壁重构过程中的关键酶,在植物逆境胁迫响应中发挥重要作用。我们前期已从胡杨叶片中克隆了PeXTH基因。本文利用Real-time PCR检测PeXTH基因在干旱胁迫下的表达水平。在此基础上,构建植物表达载体pMDC85-PeXTH,通过农杆菌介导法将PeXTH基因转入烟草,分析过表达PeXTH基因烟草的抗旱性。研究发现,胡杨叶片中PeXTH基因的表达受干旱胁迫诱导。干旱处理后,转PeXTH基因烟草的萌发率明显高于野生型烟草;与野生型植株相比,转基因植株的叶片失水速率明显降低。干旱胁迫下,转基因烟草的气孔开度仅为野生型烟草的51.2%～53.6%。结果表明,过表达PeXTH基因能够提高烟草的抗旱性。本研究丰富了对胡杨PeXTH基因功能的认识,为植物抗旱分子育种提供了重要的基因资源。     

<Emphasis Type="Italic">SlHDA5</Emphasis>, a Tomato Histone Deacetylase Gene,Is Involved in Responding to Salt,Drought, and ABA

Histone deacetylation catalyzed by histone deacetylases is an important type of histone modification. Histone deacetylases affect various processes of plant development and involve in responding to hormones and biotic and abiotic stresses. Here, we report a tomato PRD3/HDA1 histone deacetylase gene, SlHDA5, which is expressed ubiquitously in different tissues and development stages. Expression profiles in hormone treatments showed that SlHDA5 was induced by abscisic acid (ABA) and methyl jasmonate (MeJA). Seedlings growth of SlHDA5-RNAi lines were more inhibited on the medium containing salt compared with wild type (WT). Under salt stress, chlorophyll in mature leaves degraded earlier in transgenic leaves than that in WT, and transgenic plants displayed wilting earlier and more severe than WT. After drought treatment, transgenic plants wilted and dehydrated earlier than WT, which was confirmed by lower water and chlorophyll content, and higher malondialdehyde (MDA) content in transgenic plants manifesting that the tolerance of transgenic plants to drought receded. Under the treatment of ABA, root length of transgenic seedlings was more strongly repressed by contrast with WT, suggesting repression of SlHDA5 increased seedling sensibility to ABA. Our study indicated that silencing of SlHDA5 resulted in decreasing tolerance to salt, drought, and ABA.     

Expression of insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers the cold tolerance to transgenic tobacco

To elucidate the function of antifreeze protein from Microdera puntipennis dzhungarica for freezing stress tolerance in plant, the construct of MpAFP149 gene with the signal peptide sequence responsible for secreting the native MpAFP149 into the apoplast space under control of a cauliflower mosaic virus 35S promoter was introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. The observation of immunogold localization by TEM (transmission electron microscope) showed that the heterologous MpAFP149 protein was mainly distributed on the cell wall in apoplast of the transgenic tobacco plant. T1 generation transgenic tobacco plants displayed a more frost resistant phenotype and kept the lower ion leakage ratio and MDA (malondialdehyde) content in the leaves compared with wild-type ones at -1 degrees C for 3 days. The results showed that MpAFP149 provided protection and conferred cold tolerance to transgenic tobacco plant during freezing stress.     

Allene Oxide Cyclase from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Improves Tolerance Against Low Temperature and Salt Stress in Tobacco and Bacteria

Allene oxide cyclase (AOC, E 5.3.99.6) is an essential enzyme in jasmonate (JA) biosynthetic pathway. An AOC gene (defined as CaAOC, Database Accession No. AY863428) had been isolated from Camptotheca acuminata in previous work. Real-time quantitative PCR analysis indicated that mRNA expression of CaAOC was induced by salt stress (120 mM NaCl) and low temperature (4 degrees C). In order to further investigate the role of AOC gene in the processes, CaAOC was introduced into tobacco via Agrobacterium tumefaciens, and the transgenic lines were subjected to the examination of tolerance against salt stress and low temperature. Under salt stress, the chlorophyll content in transgenic tobacco was higher than that of in the wild plants. The electrolyte leakage test revealed that transgenic tobacco plants were more resistant to low temperature over control. Furthermore, 5'-truncated CaAOC was inserted into pET30 and then expressed in Escherichia coli strain BL21DE3 (pLysS). Interestingly, the transformants could grow on 2YT agar containing 400 mM NaCl. Although these mechanisms are not clear yet, this study suggested that CaAOC could not only be a potential target gene in the engineering of plants and bacteria for improved endurance against salt stress, but also be quite useful in enhancing plant tolerance to cold.     

Improvement of cold tolerance of the half-high bush Northland blueberry by transformation with the <Emphasis Type="Italic">LEA</Emphasis> gene from <Emphasis Type="Italic">Tamarix androssowii</Emphasis>

Previous studies have shown that the late embryogenesis abundant (LEA) gene of Tamarix androssowii can enhance the drought tolerance of transgenic tobacco. In this study, the cloned LEA gene was transformed into half-high bush Northland blueberry in order to enhance its ability to tolerate cold stress. The cephalosporin antibiotics ceftriaxone, cefotaxime and cefazolin were used to optimize transformation of leaf explants, and kanamycin sulfate was used to select for transgenic shoots. PCR and Southern blot analysis confirmed 8 transformants with LEA gene copy numbers ranging from 1 to 7. The LEA chimeric gene was found to be normally transcribed in 6 transgenic lines by RT-PCR. The 8 transgenic lines were tested for cold tolerance by measuring the activities of peroxidase (POD) and superoxide dismutase (SOD), malondialdehyde (MDA) content and relative electrolyte leakage (REL). Overexpression of the LEA gene enhanced the activity of both POD and SOD under low temperature stress conditions. Lipid peroxidation in the transgenic lines was significantly lower than in non-transgenic plants after cold stress, as determined by MDA content and REL. Thus, our findings indicate that the LEA gene confers increased cold tolerance in the Northland blueberry and implicate the metabolic pathways through which it exerts this effect.     

Transformation of tobacco with a gene for the thermophilic acyl-lipid desaturase enhances the chilling tolerance of plants

The desC gene for the acyl-lipid Delta9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus was introduced into Nicotiana tabacum under control of the 35S promoter. Expression of the desaturase was confirmed by Western blotting. Lipid analysis revealed that lipid content and the extent of fatty acid unsaturation significantly increased in leaves of transgenic plants. Chilling tolerance of those plants also increased, as estimated by the electrolyte leakage from the tissues damaged by cold treatments. Seeds of plants that expressed the desC gene imbibed at low temperatures demonstrated higher chilling tolerance than those of the control plants. The results demonstrate that the cyanobacterial thermophilic acyl-lipid desaturase was efficiently expressed in tobacco at ambient temperatures, and its expression resulted in the enhanced chilling tolerance of the transgenic plants.