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。     

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。     

转小麦冷胁迫蛋白截短基因烟草及其抗病性分析

TA3-13是克隆于小麦冷胁迫蛋白基因的截短片段。原核表达的TA3-13蛋白能够诱导烟草产生显著的抗烟草花叶病毒(TMV)的作用。文章将TA3-13基因片段克隆到植物表达载体pBI121上,构建成转基因重组体pB-3-13,通过冻融法转化农杆菌EHA105,构建成转基因侵染菌株。采用叶盘法将pB-3-13转化三生烟草,经卡那霉素抗性筛选,获得48株T0代再生植株。通过PCR检测,鉴定出33株转基因单株,收获了20株种子作为T1代株系。PCR-Southern杂交结果显示,PCR阳性条带与TA3-13探针有特异性杂交,说明外源基因被转化到烟草的基因组中。选取两个T1代株系的烟草植株用于各项测定。GUS组织化学活性鉴定和RT-PCR检测结果显示,外源基因可以成功地表达。接种TMV病毒后,转基因烟草抗TMV的能力较转空载体烟草提高3～5倍。转基因烟草具有抗TMV侵入和抗病毒病害发展的作用,同时转基因烟草可以抗细菌软腐病菌的扩展。     

羽衣甘蓝胁迫应答基因BoRS1转化烟草的初步研究

将克隆于羽衣甘蓝的胁迫应答基因BoRS1连入中间载体p35S-2300：：gus：：noster相应位点,成功地构建了含BoRS1基因的植物双元表达载体p35S-2300：：BoRS1：：noster,并通过农杆菌介导法对烟草进行了遗传转化。PCR检测结果表明目的基因BoRS1已成功地导入并整合到烟草基因组中。RT-PCR分析显示,在不同的转基因烟草植株中BoRS1表达量存在差异。转BoRS1烟草的耐干性和甘露醇胁迫研究表明,BoRS1基因的表达对提高植物抗干旱胁迫能力有一定的作用。     

农杆菌介导番茄铁载体蛋白基因(LeIRT2)转化烟草的研究

构建了含有目的基因(LeIRT2)的双元载体pYF840,并成功地将植物性内含子构建到筛选标记基因新霉素磷酸转移酶基因(nptⅡ)和gus报告基因中。利用农杆菌介导法,将铁载体蛋白(LeIRT2)基因导入革新一号烟草,PCR、PCR-Southern blotting及Southern blottlng检测结果显示,有3个烟草株系的基因组中整合了完整的铁载体基因(LeIRT2)。水培试验结果显示转基因株系1、3不但提高了植株的铁吸收能力,而且还促进了植株的生长,但转基因株系2却与对照无明显区别。     

低温下转昆虫抗冻蛋白基因烟草的亚显微结构变化

分析在-1℃温度中处理1、2和3d的转昆虫抗冻蛋白基因MpAFP149烟草和野生型烟草亚细胞显微结构变化的结果表明,转基因烟草和野生型烟草的超微结构有差异,尤其是细胞膜、叶绿体和线粒体的膜。转基因烟草细胞器的膜结构伤害程度明显低于野生型烟草,这提示低温条件下转基因烟草中的抗冻蛋白可能有保护细胞膜和细胞器膜结构的作用,由此降低了低温的伤害。     

转DREB基因烟草悬浮细胞系(BY-2)的建立及其几个与抗盐和抗渗透胁迫相关指标的检测

用基因枪法将DREB基因和诱导型启动子rd29B构建的载体pBAC128F转入烟草悬浮细胞。通过除草剂抗性筛选、PCR和PCR-Southern检测,获得转DREB基因的细胞系。以300mmol·L-1NaCl和15%PEG6000处理14d后,转基因细胞系存活并生长,而野生型则接近死亡。转基因细胞系的脯氨酸含量和超氧物歧化酶活性普遍高于野生型的,而丙二醛含量则相反。     

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。     

羽衣甘蓝胁迫应答基因BoRS1转化烟草的初步研究

将克隆于羽衣甘蓝的胁迫应答基因BoRS<、I>1连入中间载体p35S 2 30 0 ::gus ::noster相应位点,成功地构建了含BoRS1基因的植物双元表达载体p35S 2 30 0 ::BoRS1::noster,并通过农杆菌介导法对烟草进行了遗传转化。PCR检测结果表明目的基因BoRS1已成功地导入并整合到烟草基因组中。RT PCR分析显示,在不同的转基因烟草植株中BoRS1表达量存在差异。转BoRS1烟草的耐干性和甘露醇胁迫研究表明,BoRS1基因的表达对提高植物抗干旱胁迫能力有一定的作用。     

Transgenic Tobacco Plants Overexpressing a Grass PpEXP1 Gene Exhibit Enhanced Tolerance to Heat Stress

Heat stress is a detrimental abiotic stress limiting the growth of many plant species and is associated with various cellular and physiological damages. Expansins are a family of proteins which are known to play roles in regulating cell wall elongation and expansion, as well as other growth and developmental processes. The in vitro roles of expansins regulating plant heat tolerance are not well understood. The objectives of this study were to isolate and clone an expansin gene in a perennial grass species (Poa pratensis) and to determine whether over-expression of expansin may improve plant heat tolerance. Tobacco (Nicotiana tabacum) was used as the model plant for gene transformation and an expansin gene PpEXP1 from Poa pratensis was cloned. Sequence analysis showed PpEXP1 belonged to α-expansins and was closely related to two expansin genes in other perennial grass species (Festuca pratensis and Agrostis stolonifera) as well as Triticum aestivum, Oryza sativa, and Brachypodium distachyon. Transgenic tobacco plants over-expressing PpEXP1 were generated through Agrobacterium-mediated transformation. Under heat stress (42°C) in growth chambers, transgenic tobacco plants over-expressing the PpEXP1 gene exhibited a less structural damage to cells, lower electrolyte leakage, lower levels of membrane lipid peroxidation, and lower content of hydrogen peroxide, as well as higher chlorophyll content, net photosynthetic rate, relative water content, activity of antioxidant enzyme, and seed germination rates, compared to the wild-type plants. These results demonstrated the positive roles of PpEXP1 in enhancing plant tolerance to heat stress and the possibility of using expansins for genetic modification of cool-season perennial grasses in the development of heat-tolerant germplasm and cultivars.     

OsPHGPx基因的过量表达增强水稻抗氧化能力

植物在生物或非生物胁迫下会通过表达磷脂氢谷胱甘肽过氧化物酶(PHGPx)来抵御胁迫引起的氧化损伤,但是PHGPx在植物体内抗氧化途径中所扮演的生理角色目前尚不完全清楚。利用农杆菌遗传转化技术,构建了过表达Os PHGPx基因的转基因水稻,并对转基因水稻进行了PCR、实时定量PCR以及Western blot等检测分析,结果表明Os PHGPx基因已成功转入水稻并正常表达。与野生型水稻相比,这些过量表达Os PHGPx的转基因水稻抵御百草枯氧化伤害的能力提高。     

转铁蛋白基因烟草中内源铁蛋白基因的差异表达及含铁量的变化

铁蛋白是一种由24个亚基组成的高分子贮藏蛋白质,可以储存多达4500个铁原子,在动植物及微生物的新陈代谢中起着非常重要的作用。有研究表明,外源铁蛋白的大量表达可以提高植物储存铁离子的能力。为了明确外源铁蛋白基因转化植物中内源铁蛋白基因差异表达与植物含铁量的关系,本研究在成功获得2个烟草铁蛋白基因的全长cDNA克隆NtFerl（登录号：ay083924）和NtFer2（登录号：ay141105）的基础上,以烟草品种SR-1（Nicotiana tabacum cv．Petit Havana SR-1）为受体,培育了转铁蛋白基因烟草。将双元载体pBI121中的GUS基因用来自大豆的铁蛋白基因SoyFer1（登录号：m64337）置换,利用农杆菌介导法转化烟草叶盘,获得在CaMV35S启动子驱动表达的大豆铁蛋白基因转化烟草植株。Northern杂交和Western杂交分析表明外源铁蛋白基因在转基因烟草中得到了正确表达。比较转基因烟草和非转基因烟草的内源铁蛋白基因表达强度、叶片铁含量、根系铁还原酶活性、株高和鲜重表明,外源铁蛋白基因不但促进了NtFer1的表达,提高转基因植株的储存铁的能力和根系铁还原酶活性,而且促进植株的生长速度。以上结果说明,外源铁蛋白基因转化烟草中内源铁蛋白基因的表达、铁离子的还原吸收及光和作用都得到了进一步的提高。     

Differential Expression of Endogenous Ferritin Genes and Iron Homeostasis Alteration in Transgenic Tobacco Overexpressing Soybean Ferritin Gene

For studying the effects of endogenous ferritin gene expressions (NtFer1, GenBank accession number ay083924; and NtFer2, GenBank accession number ay141105) on the iron homeostasis in transgenic tobacco (Nicotiana tabacum L.) plants expressing soybean (Glycine max Merr) ferritin gene (SoyFer1, GenBank accession number m64337), the transgenic tobacco has been produced by placing soybean ferritin cDNA cassette under the control of the CaMV 35S promoter. The exogenous gene expression was examined by both Northern- and Western-blot analyses. Comparison of endogenous ferritin gene expressions between nontransformant and transgenic tobacco plants showed that the expression of NtFer1 was increased in the leaves of transgenic tobacco plants, whereas the NtFer2 expression was unchanged. The iron concentration in the leaves of transgenic tobacco plants was about 1.5-folds higher than that in nontransformant. Enhanced growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weights significantly greater than those in the nontransformant. These results demonstrated that exogenous ferritin expression induced increased expression of at least one of the endogenous ferritin genes in transgenic tobacco plants by enhancing the ferric chelate reductase activity and iron transport ability of the root, and improved the rate of photosynthesis.     

Alteration of Hormone Levels in Transgenic Tobacco Plants Overexpressing the Rice Homeobox Gene OSH1

The rice (Oryza sativa L.) homeobox gene OSH1 causes morphological alterations when ectopically expressed in transgenic rice, Arabidopsis thaliana, and tobacco (Nicotiana tabacum L.) and is therefore believed to function as a morphological regulator gene. To determine the relationship between OSH1 expression and morphological alterations, we analyzed the changes in hormone levels in transgenic tobacco plants exhibiting abnormal morphology. Levels of the plant hormones indole-3-acetic acid, abscisic acid, gibberellin (GA), and cytokinin (zeatin and trans-zeatin [Z]) were measured in leaves of OSH1-transformed and wild-type tobacco. Altered plant morphology was found to correlate with changes in hormone levels. The more severe the alteration in phenotype of transgenic tobacco, the greater were the changes in endogenous hormone levels. Overall, GA₁ and GA₄ levels decreased and abscisic acid levels increased compared with wild-type plants. Moreover, in the transformants, Z (active form of cytokinin) levels were higher and the ratio of Z to Z riboside (inactive form) also increased. When GA₃ was supplied to the shoot apex of transformants, internode extension was restored and normal leaf morphology was also partially restored. However, such GA₃-treated plants still exhibited some morphological abnormalities compared with wild-type plants. Based on these data, we propose the hypothesis that OSH1 affects plant hormone metabolism either directly or indirectly and thereby causes changes in plant development.     

Factors Affecting the Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Overexpressing Manganese Superoxide Dismutase in the Chloroplasts

Two varieties of tobacco (Nicotiana tabacum var PBD6 and var SR1) were used to generate transgenic lines overexpressing Mn-superoxide dismutase (MnSOD) in the chloroplasts. The overexpressed MnSOD suppresses the activity of those SODs (endogenous MnSOD and chloroplastic and cytosolic Cu/ZnSOD) that are prominent in young leaves but disappear largely or completely during aging of the leaves. The transgenic and control plants were grown at different light intensities and were then assayed for oxygen radical stress tolerance in leaf disc assays and for abundance of antioxidant enzymes and substrates in leaves. Transgenic plants had an enhanced resistance to methylviologen (MV), compared with control plants, only after growth at high light intensities. In both varieties the activities of FeSOD, ascorbate peroxidase, dehydroascorbate reductase, and monodehydroascorbate reductase and the concentrations of glutathione and ascorbate (all expressed on a chlorophyll basis) increased with increasing light intensity during growth. Most of these components were correlated with MV tolerance. It is argued that SOD overexpression leads to enhancement of the tolerance to MV-dependent oxidative stress only if one or more of these components is also present at high levels. Furthermore, the results suggest that in var SR1 the overexpressed MnSOD enhances primarily the stromal antioxidant system.     

过量表达番茄类囊体膜抗坏血酸过氧化物酶基因（StAPX）提高了烟草种苗的抗氧化能力

为了探讨叶绿体类囊体膜抗坏血酸过氧化物酶（tAPX）与其抗氧化性的关系,从番茄叶片中分离了叶绿体类囊体膜抗坏血酸过氧化物酶基因（StA跚并转入到烟草中。以野生型（WT）、转正义StAPX烟草株系T3-3和T3-6为试材,测定了外源过氧化氢诱导的氧化胁迫条件下APX酶活性、过氧化氢酶（CAT）活性、过氧化氢（H2O2）含量、叶绿素荧光参数及叶绿素含量等。Northern杂交显示StAPX因的表达受外源H2O2氧化胁迫的诱导。氧化胁迫下转基因烟草的APX酶活性和清除H2O2的能力都显著高于野生型,并且转基因烟草比野生型具有更高的PSII最大光化学效率及叶绿素含量。结果表明,．刚尸舶勺过量表达有助于提高外源H2O2诱导的转基因烟草的抗氧化能力。     

NaCl胁迫对转基因杨树外源基因表达的影响

以具高抗虫性的转抗虫基因‘741杨’及在此基础上转入了发根农杆菌Ri质粒T-DNA株系的组培苗为材料,研究了转基因株系BtCrylAc抗虫基因和发根基因的表达及其对NaCl胁迫的反应。结果表明,转入Ri质粒T- DNA上的rol基因后,导致苗木根系数目增加,根系长度减小,IAA和GA含量显著提高,抗虫BtCrylAc基因编码的毒蛋白的表达量降低;随着NaCl胁迫强度的增加,苗高、根系数量、叶绿素含量及IAA、GA含量逐步降低,而根系的长度加大,Bt毒蛋白含量显著提高,表明NaCl胁迫使转基因杨外源Bt毒蛋白基因的表达增强,而发根农杆菌Ri质粒T-DNA的表达下降。