Effects of Introducing the Chimaeric Gene TA29 Barnase on the Tapetum and Pollen Development in Tobacco

The development of tapetum and pollen in transgenic tobacco (Nicotiana tabacum L. ) harboring a chimaeric gene TA29-Barnase was compared with that of the wild-type plant. The specific expression of the exogenous genes in anther led to premature tapetal degradation, which started at the early stage of meiosis and terminated at the tetrad stage. In the wild-type anthers, tapetal degradation started at the early stage of bicellular microgametophyte and ended at the later stage of pollen development. The cytological changes of tapetal degradation in the transgenic plants were characterized by vacuolization of the tapetal cells, then nuclear condensation, and consequent massive degradation of tapetal cells. Meanwhile, the pollen mother cells gradually degraded and became destroyed along with the progress of meiosis, leaving only a few which could successfully complete their meiosis to form microspores. This observation also indicated that the TA29-Barnase gene in anther was not uniformly expressed. In addition, the structural difference between the male sterility induced by exogenous gene and the natural sterile was also discussed.     

Studies on Some Properties of the Apoplastic β-N-Acetyl-D-Hexosaminidase from Tomato Leaves

Some properties of the β-N-acetyl-D-hexosaminidase purified from intercellular fluid of tomato leaves after the plant was systematically infected by TMV (tobacco mosaic virus) were studied. When pNP β-D-GlcNAc (p nitrophenyl-N-aeetyl β-D-glucosaminide) or pNP β-D- GalNAc (p-nitrophenyl-N-acetyl-β-D galactosaminide) was used as the substrate, it showed the optical pH between 4. 8--5.0 and optical temperature between 44— 47℃. Studies of thermostabillty indicated that the enzyme had a biphasic denaturation curve. Using pNP-β-D-GIcNAc or pNP-β-D GalNAc as the substrate, the Km value of the enzyme was 0. 36 and 0. 67 mmol/L respectively. N acetyi-D glucosamine and N acetyl-D-galactosamine were competitive inhibitors of the enzyme activities. Ag+ and Hg2+ were sensitive inhibitors and Fe2+ . Fe3+ and Cu2+ were also inhibitors enzyme activities.     

Resistance to Potato Virus X Infection in Transgenic Tobacco Plants with Coat Protein Gene of Virus

A major commercial cultivar of tobacco was transformed via Agrobacterium mediated procedure. Tobacco leaves started to form shoots on shoot inducing medium containing kanamycin after infected by Agrobacterium containing the plasmid with PVX CP gene. Regenerated plants were obtained in two weeks on hormone-free MS medium containing kanamycin. The transgenic tobacco plants were identified with nopaline detection,enzyme-linked immunosorbent assay and western blot analysis, symptom appearance was significantly delayed and virus accumulation was either absent or reduced in PVX CP gene transformed plants. Progenies of transgenic tobacco plants also gained resistance to PVX infection to a certain degree. These experiments demonstrate that CP protection is effective against PVX.     

Studies on 6-benzyladenine Localization in Callus Cells of Tobacco

The localization of 6-benzyladenine in the callus cell of tobacco was studied by means of autoradiography. Electron microscope autoradiographs showed that the silver grains localized at not only cell membrane system such as plasma membrane, tonoplast, plastid membrane and mitochondrion membrane but also nucleus especially nucleolus. The results of the present study indicated that 6-benzyladenine receptor site associated with both cell membrane system and nucleus.     

烟草中央细胞离体受精过程中雌雄核融合生活动态的记录

受精作用一直是植物生殖发育生物学研究的热点课题。近年发展尤为迅速。特别是诸如偏向受精等新概念的提出更进一步推动了对双受精作用的寻微探秘,日益显现出这一过程的精巧与复杂。但限于体内研究的局限性,对其中一些关键环节,如雌雄配子间的识别;配子融合过程中的相互作用;雄核在雌性细胞内迁移的动力学及雌雄核融合的时间进程与机制等仍知之甚少。离体受精操作及相关技术的建立［１～３］为探讨上述问题提供了新途径。我们在过去工作的基础上以烟草为材料进行了离体双受精研究,以视频增差显微观察系统首次记录到在生活状态下精核进…     

矮牵牛花同源异型基因fbp2的克隆及其对烟草花形态的影响

以矮牵牛（Ｐｅｔｕｎｉａ　ｈｙｂｒｉｄａ　Ｌ．）栽培品种为材料,取开放前的花蕾分离ｍＲＮＡ,反转录合成ｃＤＮＡ,以ｃＤＮＡ为模板,通过ＰＣＲ扩增,对获得的目的片段进行序列分析。结果表明,分离的目的片段含有６８６个核苷酸（含有起始密码和终止密码）。核苷酸序列与文献报道相比,同源率为９９．６％,只有３个碱基发生改变,５’端的ＭＡＤＳ盒区域完全相同。将得到的矮牵牛花同源异型基因ｆｂｐ２的ｃＤＮＡ（ｙｆｂｐ２）与ＣａＭＶ３５５启动子和ＮＯＳ３’终止子融合,构建了表达载体ｐＢＢＰ２。表达载体通过农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ　ｔｕｍｅｆａｃｉｅｎｓ）ＬＢＡ４４０４（ｐＡＬ４４０４）介导转化烟草（ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ．）叶片,在含有１００ｍｇ／Ｌ卡那霉素的抗性培养基上再生成株。对抗性株进行总ＤＮＡＳｏｕｔｈｅｒｎ杂交和总ＲＮＡ的点杂交,证明目的基因已导入烟草细胞中,整合到烟草基因组上,并且在烟草细胞中转录。同源异型基因ｆｂｐ２导入烟草后导致烟草花型改变,在雄蕊上产生了花瓣。     

玉米19kD醇溶贮藏蛋白基因启动子种子特异性表达控制区段的分析

为研究玉米（Ｚｅａｍａｙｓ　Ｌ．）１９ｋＤ醇溶贮藏蛋白（ｚｅｉｎ）基因启动子种子特异性表达的控制区段,将全长６９４ｂｐ的启动子进行５’端缺失,共得到６个缺失突变体,长度分别为４８８ｂｐ、３７８ｂｐ、３０２ｂｐ、１５２ｂｐ、１２４ｂｐ和８５ｂｐ。将６个片段分别与报告基因ｇｕｓ连接构建成表达载体ｐＤＧＢ系列,经土壤农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ）介导转化,引入烟草。ＧＵＳ活性检测证明,４８８ｂｐ启动子片段能促使ｇｕｓ基因在种子中特异表达。３７８ｂｐ、３０２ｂｐ、１５２ｂｐ和１２４ｂｐ片段启动子引导的ｇｕｓ基因在烟草根、叶柄、种子中均可表达。     

水杨酸诱导烟草培养细胞的脂质过氧化和保护基因表达的研究

用外源水杨酸（ｓａｌｉｃｙｌｉｃ ａｃｉｄ,ＳＡ）处理烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）培养细胞,证实ＳＡ能诱导脂质过氧化,抑制过氧化氢酶活性并诱导ＰＲ－１基因的表达。对苯二酚和Ｈ２Ｏ２也能不同程度地诱导烟草培养细胞的脂质过氧化ＰＲ－１基因的表达,但不能抑制过氧化酶活性。     

废次烟叶提取液源木质素降解菌Bacillus subtilis SM降解特性

【目的】目前造纸法再造烟叶工艺已经成为我国重要的废烟叶处理和利用方式,该工艺中烟梗中高木质素的降解是个挑战性的需解决问题。从废次烟叶提取液(Tobacco waste extract,TWE)中筛选木质素的降解微生物用来直接处理烟梗或烟末提取液,可实现对木质素含量的调控。【方法】将废次烟叶提取液(TWE)浓缩液中分离出的Bacillus subtilis SM接种到以Kraft木质素为唯一碳源的无机盐培养基中,在pH 7.0、30°C培养基中培养4 d来检测菌株对木质素的降解效果。通过HPLC、TOC、GPC和色度来表征SM对木质素的降解,并采用烟梗无机盐培养基在pH 7.0、30°C培养4 d检测SM对烟梗木质素的降解。【结果】HPLC结果显示SM在以木质素磺酸钠为唯一碳源的无机盐培养基中可全部降解分子质量为534.5的木质素磺酸钠,而对Kraft木质素降解不明显,仅观察到组分的变化。脱色结果显示脱色率达到40.7%,但在对Kraft木质素矿化方面矿化率只能达到5.4%。SM在烟梗无机盐培养基中可使烟梗失重率分别达到50%以上(对照组为18.9%),烟梗中木质素含量减少了70%左右。【结论】来源于废次烟叶提取液(TWE)的Bacillus subtilis SM能够以Kraft木质素为唯一碳源生长,也能够有效降解烟梗中的木质素,可应用于烟草废弃物原料中木质素的降解。