导入Ghabp1基因对烟草叶细胞发育的影响

将棉花生长素结合蛋白基因cDNA与CaMV35S启动子和NOS终止子融合,构建了一个新的表达载体pGABP1-121,采用农杆菌介导法转化烟草,经过分化、筛选和再生,得到了具有卡那霉素抗性的植株。抗性植株经PCR及Southern杂交检测,证明外源目的基因已经整合到烟草基因组中。扫描电镜观察发现转基因烟草与对照相比叶细胞增大,结果表明,棉花生长素结合蛋白基因的表达影响了烟草叶细胞的发育。     

iaaM基因在烟草花粉中的表达及其在花粉发育中的作用

试验利用花粉特异表达的启动子(Lat52)和绒毡层特异表达的启动子(TA29)引导外源生长素合成代谢基因(iaaM)在烟草花粉中表达以研究生长素在花粉发育中的作用。转Lat52-iaaM基因或转TA29-iaaM基因烟草在形态上表现出变异,如从茎上形成不定根,叶呈卷曲状等典型的生长素过量表达的性状。另外,与对照相比,转基因烟草花药中IAA水平显著增加,且植株矮化,开花期推迟,有的转基因烟草未能开花。上述现象表明:Lat52和TA29启动子的表达并不仅限于花粉或绒毡层,或者说这两个启动子的表达有些泄漏。转基因烟草的花药形状有较大的变异,早期的每个花药中花粉数明显减少,但这些花粉可被醋酸一洋红染色。所有能开花的转基因烟草均可收到种子,但收自某些转基因株系的种子不能萌发。所有这些结果表明生长素在花粉发育过程中起重要作用,过量的生长素会导致花粉发育的异常。     

iaaM基因在烟草花粉中的表达及其在花粉发育中的作用

试验利用花粉特异表达的启动子（Lat52)和绒毡层特异表达的启动子（TA29）引导外源生长素合成代谢基因（iaaM)在烟草花粉中表达以研究生长素在花粉发育中的作用。转Lat52-iaaM基因或转TA29－iaaM基因烟草在形态上表现出变异,如从茎上形成不定根,叶呈卷曲状等典型的生长素表达的性状。另外,与对照相比,转基因烟草花药中IAA水平显著增加,且植株矮化,开花期推迟,有的转基因烟草未能开花。上述现象表明：Lat52和TA29启动子的表达并不仅限于花粉或绒毡层,或者说这两个启动子的表达有些泄漏。转基因烟草的花药形状有较大的变异,早期的每个花药中花粉数明显减少,但这些花粉可被醋酸－洋红染色。所有能开花的转基因烟草均可收到种子,但收自某些转基因株系的种子不能萌发。所有这些结果表明生长素在花粉发育过程中起重要作用,过量的生长素会导致花粉发育的异常。     

生长素结合蛋白cDNA的克隆及其在烟草中的表达

基于拟南芥内质网生长素结合蛋白基因的ｃＤＮＡ序列,设计合成了Ａｐ５和Ａｐ３两个引物,应用ＲＴ－ＰＣＲ技术扩增了拟南芥的ＡＢＰ基因。将该基因克隆在植物表达载体ｐ３５ＳＳＩＮ的３５Ｓ启动子必Ｎｏｓ３‘端之间,得到植物表达载体ｐ３５ＳＥ。通过农杆菌介导的方法对烟草ＳＲ１进行了转化,由分子杂交等检测证明,生长素结合蛋白基因已在烟草中表达,同时转基因烟草后代对生长素的敏感性明显增加。     

转异源abp基因烟草的叶外植体不同培养中的形态发生

以转正义和反义abp基因[生长素结合蛋白（ABP）的基因]烟草及对照的叶外植体为材料,在附加2mg／L或1mg／L的6－BA和不同浓度的NAA的MS培养基上进行不定芽分化试验。在较低NAA浓度条件下,转正义abp基因烟草（SE2）的叶外植体分化的不定芽数高于对照（SR1）和转反abp基因烟草（AS3）,说明SE2对生长素的敏感性比SR1和AS3高;而在较高NAA浓度条件下,AS3分化的不定芽数大于SE2和SR1,说明AS3对生长素的敏感性比SE2和SR1低。在培养基中加入生长素极性运输抑制剂HFCA后,不定芽分化受到抑制,分化不定芽中部分叶的形态由两侧对称性生长转变为形成不对称的喇叭状叶。在HFCA浓度为0－7．5mg／L条件下,SE2的喇叭叶发生频率明显高于SR1和AS3,其中SE2和SR1的喇叭叶发生频率分别在HFCA浓度为6．5和7．5mg／L时达到最高以后保持平稳,而AS3的喇叭叶发生频率在HFCA处理浓度直到15mg／L时仍保持上升趋势。这些结果说明ABP具有结合外源生长素,从而促进芽器官分化的功能,同时可能作为生长素受体参与了生长素的极性运输。     

转正义和反义abp基因烟草原生质体质膜超极化特性及其培养

以转生长素结合蛋白 (ABP)基因烟草为材料 ,通过原生质体培养和测定原生质体的跨膜电位 ,研究了abp基因转移与生长素诱导的烟草原生质体质膜超极化特性、原生质体培养过程中对生长素的敏感性三者之间的关系 .结果表明 :转正义abp基因烟草达到最大超极化的NAA浓度小于对照 ,原生质体培养中对生长素的敏感性增加 ;而转反义abp基因烟草达到最大超极化的NAA浓度则大于对照 ,原生质体培养中对生长素的敏感性降低 .这为ABP在内质网上合成后需运输到细胞膜上发挥作用提供了证据     

转异源abp基因烟草的叶外植体不同培养中的形态发生

以转正义和反义ａｂｐ基因「生长素结合蛋白的基因」烟草及对照的叶外植体为材料,在附加２ｍｇ／Ｌ或１ｍｇ／Ｌ的６－ＢＡ和不同浓度的ＮＡＡ的ＭＳ培养基上进行不定芽分化试验。在较低ＮＡＡ浓度条件下,转正义ａｂｐ基因烟草的叶外植体分化的不定芽数高于对照和转反义ａｂｐ基因烟草,说明ＳＥ２对生长素的敏感性比ＳＲ１和ＡＳ３高;而在较高ＮＡＡ浓度条件下,ＡＳ３分化的不定芽数大于ＳＥ２和ＳＲ１,说明ＡＳ３对生长素的敏     

aroA-In融合基因载体的构建、表达及对烟草的转化

PCR扩增突变的5’-烯醇丙酮酸莽草酸-3-磷酸合成酶(5’-enolpyruvylshikimate-3-phosphate synthetase,EPSPS)cDNA全长序列,插入pLitmus28得到pLEPSPS,进而通过反向PCR在EPSPS235／236aa之间将其打断为无功能的片段。选用人工构建的具有顺式和反式剪接功能的mini型蛋白内含子Ssp DnaB和Rma DnaB,插入被打断的aroA(抗除草剂基因),构建了质粒pLEBC、pLEBT、pLERC和pLERT。将4种重组质粒中的aroA-In(蛋白内含子Intein插入aroA)融合基因插入pET-32得到表达载体pETLEBC、pETLEBT、pETLERC和pETLERT,lPTG诱导后,SDS-PAGE分析显示其能在DE。中有效表达并发生相应的蛋白剪接。将aroA-cis Ssp DnaB和aroA-cis Rma DnaB融合基因分别插入pLYM中进一步构建成植物表达载体,农杆菌叶盘法转化烟草。基因组PCR分析表明融合基因整合入植物核基因组;RT-PCR分析显示其可在高等植物细胞中成功表达。结果说明蛋白内含子基因可以转化高等真核细胞,蛋白剪接技术可应用于高等植物细胞,从而为防止植物转基因扩散提供了一条新的途径。     

转基因雄性不育烟草花药绒毡层及花粉发育的特点

转基因雄性不育烟草花药绒毡层及花粉发育的特点罗玉英１李怀军２刘玉乐３李胜国３田波３（１首都师范大学生物系,北京１０００３７）（２北京市蔬菜研究中心,北京１０００８１）（３中国科学院微生物研究所,北京１０００８０）近年来,通过基因工程创建植物雄性...     

烟草初生胚胎乳细胞在微室培养中的发育

用酶解－研磨法分离出烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）受精后胚囊和初生胚乳细胞进行微室饲养培养。培养基为Ｋｍ８Ｐ附加各种其他成分,饲养细胞为分裂旺盛的烟草叶肉原生质体,在２５℃下静止暗培养。培养３天后,初生胚乳细胞开始第一次分裂,继续分裂至１４天时形成大的细胞团。首次报道了双子叶植物初生细胞的离体发育。     

Agrobacterium-mediated transformation and regeneration of cotton plants

Cotton (Gossypium hirsutum L.) was transformed by the EHA101 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and β-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue, and its segments taken from in vitro growing seedlings were used as explants. Explants were cultured in a Murashige and Skoog (MS) medium containing various hormone combinations to induce shoot regeneration. The highest frequency of shoot formation was obtained from the shoot meristem. After selection in the MS medium containing kanamycin (50 mg/l), these tissues were tested by histochemical GUS assay. Shoots regenerated from excised shoot meristems or their halves were cultured for 4–6 weeks to obtain rooted plants, which then produced fully-developed plants and seeds in pots. Genomic integration of the kanamycin-resistance gene was detected by the PCR analysis. Seed germination percentage was 95% after the F1 seeds of transgenic cotton plants were cultured on half-strength MS medium supplemented with 50 mg/l kanamycin. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cotton was optimized. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 462–467. The text was submitted by the authors in English.     

Natural deuterium and oxygen-18 enrichment in leaf water of cotton plants grown under wet and dry conditions: evidence for water compartmentation and its dynamics

Abstract. Significant differences in leaf water oxygen and hydrogen isotopic composition were observed between cotton plants grown under wet and dry conditions. The magnitude of the differences could be fully explained by the conventional model that describes the isotopic composition of an evaporating water pool under steady state conditions. The results indicate that leaf water isotopic composition is strongly influenced by transpiration rate via its effects on relative humidity adjacent to the leaf surface and on the isotopic composition of the air moisture. Our application of the model, however, provides evidence that leaf water must consist of a mixture of several isotopically distinct pools. These pools are suggested to reside in the symplast, in the cell walls and intercellular spaces and in the veins. A model is proposed suggesting that only the water residing in the cell walls and the intercellular spaces (the transpiration pool) interacts directly with the external environment. The large symplastic pool responds to the external environment to a limited extent via its relatively slow exchange with water in the transpiration pool. It is likely that the isotopic composition of water in the symplastic pool is strongly buffered against shortterm environmental variations, a possibility that would have important implications for the isotopic conditions under which organic matter biosynthesis occurs.     

Cotton Leaf Curl Virus: lonic Status of Leaves and Symptom Development

In the present study, the relationship between the nutritional status of leaves and the development of symptoms of cotton leaf curl virus (CLCuV) in two cotton (Gossypium hirsutum L.) cultivars (i.e. CIM-240 and S-12) was investigated. The incidence of disease attack was found to be 100% in the S-12 cultivar and 16% in the CIM-240 cultivar. Geminivirus particles in infected leaves were confirmed by transmission electron microscope examination of highly specific geminivirus coat protein antisera-treated cell sap. The CLCuV impaired the accumulation of different nutrients in both cultivars. A marked decrease in the accumulation of Ca2+ and K+ was observed in infected leaves. However, the disease had no effect on leaf concentrations of Na+, N, and P. It was observed that the curling of leaf margins in CLCuV-infected plants was associated with the leaf Ca2+ content; leaf curling was severe in plants with a significant reduction in Ca2+ content.Moreover, leaf K+ content was found to be associated with resistance/susceptibility to CLCuV infection.     

Cotton Leaf Curl Virus： Ionic Status of Leaves and Symptom Development

In the present study, the relationship between the nutritional status of leaves and the development of symptoms of cotton leaf curl virus （CLCuV） in two cotton （Gossypium hirsutum L.） cuItlvars （I.e. CIM-240 and S-12） was Investigated. The incidence of disease attack was found to be 100% In the S-12 cuItlvar and 16% in the CIM-240 cuItivar. Geminivirus particles in infected leaves were confirmed by transmission electron microscope examination of highly specific geminivirus coat protein antlsera-treated cell sap. The CLCuV Impaired the accumulation of different nutrients in both cuItivars. A marked decrease in the accumulation of Ca^2＋ and K^＋ was observed in infected leaves. However, the disease had no effect on leaf concentrations of Na^＋, N, and P. It was observed that the curling of leaf margins in CLCuV-Infected plants was associated with the leaf Ca^2＋ content; leaf curling was severe in plants with a significant reduction In Ca^2＋ content. Moreover, leaf K＆＋ content was found to be associated with resistance/susceptibility to CLCuV infection.     

转基因棉花的Bt基因流

通过两年实验检测转基因陆地棉品种间和海岛棉与陆地棉种间的Bt基因流.将转基因陆地棉国抗12号种植在12m×12m的样方内,周围分别种植非转基因陆地棉品种中棉所12号和海岛棉品种新海13号,在离转基因棉花不同距离选取样点,采集非转基因棉花种子,利用标记选择基因、Dot朎LISA和PCR扩增检测Bt基因流.结果表明在0～6m内陆地棉品种间显示较高频率的基因流,随着距离的增加Bt基因流降低,最大可达36m;提高样方内转基因棉花纯度,仅增加0～3m内基因流,对较远距离基因流无影响.海陆种间Bt基因流在0～6m内比陆地棉品种间低,但Bt基因流随距离增加下降幅度小,最远达72m.因此,在小规模转基因棉花环境释放实验,可选用同种不同品种棉花作为缓冲隔离带,并讨论了棉花转基因逃离至我国棉属种类的可能性.     

棉花LIM结构域基因(GhLIM1)的克隆和表达分析

LIM结构域蛋白是一个重要的发育调控因子,参与基因转录,细胞骨架建成和信号传导等许多发育调控过程,胞质骨架是形成和稳定细胞形态以及传递物质,能量和信息的重要成分。为研究棉花纤维细胞发育过程中胞质骨架的形成和作用机理,通过棉花纤维EST序列整合,从陆地棉徐州142胚珠（含纤维）中扩增并克隆出棉花LIM结构域基因的编码区段。该棉花LIM结构域基因（GhL1M1)长848bp,包含一个570bp的开放阅读框,推导的氨基酸序列（189个氨基酸）与拟南芥,烟草和向日葵的LIM结构域蛋白有极高的同源性,而且两个LIM结构域完整,RT-PCR和Northerm杂交分析表明,该基因（GhL1M1）在陆地棉的根,茎尖,上胚轴,叶片,花蕾,花药,胚珠和不同发育时期的陆地棉纤维（4DPA、12DPA、18DPA）以及海岛棉纤维（18DPA）和中棉纤维（12DPA）中均有表达,但GhL1M1基因在茎尖,纤维和有纤维的胚珠中表达量更高,因此GhL1M1基因应与棉花纤维发育有密切关系。     

Relationships Between Nitrogen Content and Net Gas Exchange Components of a Cotton Leaf During Ontogeny

Relationships between leaf nitrogen (N) content and leaf gas exchange components of a single cotton (Gossypium hirsutum L.) leaf subtending the fruit during ontogeny were investigated under field conditions. A 20-d old leaf exhibited the highest physiological activity characterized by net photosynthetic (PN) and transpiration (E) rates, stomatal conductances to CO2 exchange (gsCO2) and water vapor transfer (gsH2O), and nitrogen (N) content. With the advent of leaf senescence, the gas exchange rates declined as exhibited by the 30-, 40-, and 60-d old leaves. Regression analysis indicated close relationships between gsCO2 and PN, and gsH2O and E as the leaves advanced towards senescence. Both PN and gsCO2 were related to N as they declined with leaf age. Thus, the declines in PN were associated with stomatal closure and removal of N during leaf ontogeny.