转人α-乳清白蛋白基因烟草中半胱氨酸含量的提高

将人α-乳清白蛋白基因构建到植物表达载体上,通过农杆菌介导采用叶盘法将人α-乳清白蛋白基因导入到烟草中,经过PCR和Southern blot分析表明:人α-乳清白蛋白基凶已经整合到烟草基因组中;经过RT-PCR葙GUS组织化学染色证明:人α-乳清白蛋白基因得到了表达.测定了9株转基因烟草叶片半胱氨酸含量,大部分植株有着明显的提高,最高幅度达到了318.02%,半胱氨酸含量平均提高了166.40%.     

雪花莲凝集素基因（gna）的改造及其抗蚜性

用定点突变方法对编码雪花莲凝集素（Galanthus nivalis agglutinin,GNA)前体蛋白的DNA序列进行了改造和转基因烟草9Nicotana tabacum L.）抗蚜性的研究。结果表明,将GNA编码序列中含有的稀有密码子改造后,GNA的表达水平从占总可溶性蛋白的0.17％增加到0.25％,转基因烟草的抗蚜性也随之增强,从平均抑制桃蚜（Myzus per-sicae(Sulzer））虫口密度63.7％显地提高到71.0％。     

一种基于PCR技术鉴定单拷贝转基因烟草的方法

为了鉴定携带单拷贝外源基因的转基因烟草植株,以烟草核基因组上已知的单拷贝内源基因（RNR2）为内参,转基因烟草植株基因组DNA为模板,在同一PCR反应体系中扩增内源基因（RNR2）和外源目的基因（NPTⅡ）。反应产物在琼脂糖凝胶上电泳,获得了预期大小的两条特异性扩增条带。经ImageJ软件捕捉分析两条目的条带的灰度比,当T1代转基因烟草植株中外源基因与内源基因的扩增条带灰度比为1时,所检测植株即为单拷贝外源基因的转基因烟草植株。孟德尔经典遗传学方法证实了上述检测结果高度可信。     

An Insect-Resistant Transgenic Cabbage Plant with Cowpea Trypsin Inhibitor(CpTI) Gene

Cowpea trypsin inhibitor (CpTI) gene was transformed into Brassica oleracea var. capitara variety "Yingchun" and "Jingfeng" mediated by the Agrobacterium tumefaciens LBA4404(pRCL27). Transgenic plants were obtained from transformed calli or explants. It was shown from the ELISA assay that NPT Ⅱgene was expressed in the transgenic cabbage cells. The integration of the CpTI gene into cabbage genome DNA was confirmed by Southem blotting. Insect-tolerance of the transgenic plants to Pieris rapae L. was observed by bioassays on the transgenic plants in the laboratory.     

高效删除标记基因的Bhlea2植物表达载体的构建

为培育去除选择标记基因的耐旱转基因植物,同时利用Cre/Lox和FLP/frt系统,构建一个能够高效删除标记基因的Bhlea2基因植物表达载体.拟南芥rd29A启动子是在低温、干旱、高盐胁迫下的快速应答启动子,玉米ubiquitin启动子可有效驱动外源基因的转录,拟南芥pAB5启动子是花粉及胚胎等发育早期特异表达的启动子,利用上述启动子构建了表达Bhlea2基因并能够删除标记基因的植物表达载体.该表达载体包括重组酶表达元件pAB5-FLP、Bhlea2抗旱基因表达元件rd29A-Bhlea2和bar标记基因表达元件ubiquitin-bar.     

表达尾穗苋凝集素基因的转基因烟草对桃蚜(Myzus persicae)繁殖的影响

为研究尾穗苋凝集素(ACA)在植物中可能的抗虫作用,通过RH-PCR克隆了ACA cDNA并通过RACE分析证实了cDNA序列的正确性.构建了ACA基因的韧皮部特异表达载体pBCACAc并通过根癌杜菌介导转化了烟草(Nicotiana tabacum L.).PCR和Southern blot分析结果证明,ACA基因已经整合到转化再生植物的基因组中,其插入插贝数1~4个不等.对转基因烟草叶片蛋白时行行免疫反应的结果表明,ACA基因已被转录和翻译.用桃蚜(Myzuspersicae Sulzer)对转基因烟草离体叶片进行了的接虫试验结果表明,测试过的78%的烟草对桃蚜口密度增长的平均抑制率在75%以上,在抗性植株上观察到有桃蚜若虫死亡的现象.以上结果表明,ACA基因是一个有效的抗蚜基因,在作物抗蚜分子育种具有应具应用价值.     

表达尾穗苋凝集素基因的转基因烟草对桃蚜(Myzus persicae)繁殖的影响(英文)

To investigate the possible function of the agglutinin from Amaranthus caudatus L. (ACA) in plant defending against insect pests, ACA cDNA was cloned by RT-PCR and the 5‘ and 3‘ sequences were confirmed by rapid amplification of cDNA ends (RACE). The phloem-specific expression vector of ACA gene, pBCACAc, was constructed based on the plant binary vector pBC438 and transfered into tobacco plants via Agrobacterium-mediated transformation method. Results from PCR and Southern blotting analysis showed that AOA gene was integrated into the genomes of transformed plants and the transgene integration varied from one to four estimated copies per genome. Western blotting analysis indicated that ACA gene was transcribed and translated in the transgenic plants. The bioassay of Myzus persicae Sulzer on detached leaves demonstrated that the 78% transgenic tobacco plants displayed an average aphid-resistant rate of more than 75%. Some apterous progeny of M. persicae were found dead on the resistant plants. These results indicate that ACA gene should be an effective aphid-resistant gene and could be valuable for application in crop breeding for aphid resistance.     

转双基因烟草对棉铃虫的杀虫活性评价

以含Ｂｔ杀虫蛋白基因（单基因）烟草和常规烟草为对照,系统测定了含Ｂｔ与豇豆胰蛋白酶抑制剂蛋白基因（双基因）的抗虫烟草对棉铃虫不同龄期幼虫的杀虫活性。结果表明：１￣３龄幼虫取食转双基因烟草３ｄ后死亡率为８０．５％￣９９．３％,取食６ｄ后死亡率达１００％,均显著高于转单基因烟草。２龄幼虫取食转基因烟草３ｄ后死亡率为８０．５％￣９９．３％,取食６ｄ后死亡率达１００％,均显著高于转单基因烟草。２龄幼虫取食     

菠菜甜菜碱醛脱氢酶基因在烟草中的表达

质粒pLS9含有1．5kb的编码菠菜甜菜碱醛脱氢酶(BADH)基因。经限制酶切后克隆到植物表达载体的35S启动子和PolyA终止子之间。经农杆菌介导转化烟草,获得90多株抗卡那霉素再生植株。经PCR检测证明60％以上再生植株含有BADH基因。转基因植株经Western blot,BADH酶活性测定,BADH酶活性特异性染色法检查和耐盐性分析,证明菠菜BADH基因在烟草正常表达。在叶绿体和胞液中均有BADH酶存在。转基因植株能耐较高浓度盐。     

抗烟青虫转基因烟草的培育

烟青虫属鳞翅且(Lpidoptera)昆虫。前人研究表明,苏云金杆菌(Bacillus thuringiensis)中的Bt毒蛋白对其具有很强的毒杀作用。设法将Bt基因导入到烟草中,是防治这类虫害的一种有效途径。80年代后期以来,为了提高Bt基因在植物中的表达水平,在编码区密码子的优化、编码顺序的改进和高效启动子的选配方面．都有了很快的发展。有些经过部分改进或人工全合成的Bt基因,还被先后导入到番茄〔1.2〕、烟草〔2-4〕、棉花〔5.6〕、玉米〔7〕、水稻〔8〕等重要作物中。迄1995年止．在美国获准可供商业用的Bt毒蛋白转基因作物已有槔花、玉米和马铃薯等〔9〕。在烟草中迄今未见有Bt毒蛋白转基因株达到生产实用水平的报道。本实验试图通过农杆菌介导法,将密码子经过优化的Bt基因cryIA(b)及cryIA?〔10〕叫导入离体培养的烟草叶片,然后使之再生,形成转基因植株,再从其后代中选育出既保留原品种优良农艺性状,又具有对烟青虫稳定抗性的转基因株,以期提高该品种的稳产性,降低杀虫农药成本。保护烟田的良好生态环境。     

转WMV-2外壳蛋白基因西瓜植株的病毒抗性

西瓜是夏季的重要水果,病毒病是影响其品质和产量的重要原因之一。植物基因工程的发展为抗病育种提供了新途径。利用外壳蛋白(coat protein)基因转化高等植物,赋予转基因植物以相应抗病性的成功例子已很多。本文报道WMV-2CP基因在自交子一代的分离符合孟德尔3：1的分离比。经过连续4代的选择鉴定,已从T7、T11和T323个独立转化子的后代中筛选获得8个转基因纯合株系,性状表现整齐一致。Western blot结果表明,R4T7-1、T4T11-3以及R4T32-73个不同来源的株系均能表达产生外壳蛋白。转基因纯合株系WMV-2感染后的病毒抗性实验表明,与未转基因对照相比,转基因株系可以推迟发病时间,减轻发病程度。实验筛选获得的转基因株系R4T32-7表现出对WMV-2的高度抗性,为利用植物转基因技术选育抗病新品种奠定了基础。     

昆虫特异性神经毒素基因tox34的合成及转基因烟草的获得

对来自虱状蒲螨（Pyemotestritici）的昆虫特异性神经毒素基因tox34编码成熟蛋白部分的762bp片段,依据植物的偏爱密码子进行了序列改造。将改造后的基因插入细菌表达载体pBV221,经热诱导处理后细菌总蛋白在SDS-PAGE上特异条带的表观分子量为32～33kD。将tox34基因插入植物高效表达载体后以根癌土壤杆菌（Agrobacteriumtumefaciens（SmithetTownsend）Conn）介导的方法导入烟草（NicotianatabacumL．cv．SR1）,并获得了转基因烟草植株。经PCRSouthem杂交结果证实,完整的毒素基因已整合入烟草基因组。转基因幼苗根部的组织化学检测GUS显示阳性。再生植株叶片的棉铃虫（HeliothisarmigevaHubner）饲喂实验显示对低龄幼虫有很好的毒杀作用。     

马铃薯天冬氨酸蛋白酶抑制剂基因转化烟草研究

将马铃薯天冬氨酸蛋白酶抑制剂基因由重组质粒pAPI189中亚克隆到双元表达载体pGA643的XbaI和KpnI位点之间,构建成高效表达重组质粒pGAPI3。三亲融合法将其转移至农杆菌LB4404。通过叶盘法利用此融合后的含有目的基因的农杆菌转化烟草叶圆片,在含有Km的培养基上筛选抗性芽。将抗性芽接种到生根培养基至长成完整植株后再移栽到土质中以获得转基因植株。通过目的基因的特异引物进行PCR检测以及目的基因的片段为探针进行Southern杂交检测,证实已获得马铃薯天冬氨酸蛋白酶抑制剂基因的转基因烟草植株。 Abstract：An aspartic proteinese inhibitor gene contained in the recombinant plasmid pAPI189 was inserted into the XbaI and KpnI site of binary vector pGA643 for constructing subclone pGAPI.The pGAPI was introduced into Agrobacterium LB4404 by coculturing the mixture of DH5α(pGAPI)、HB101(pRK2013) and Agrobacterium LB4404.We transformed tobacco leaves by coculturing them with Agrobacterium LB4404 which contained aspartic proteinese inhibitor gene and then screening the shoots with T?medium containing kamnamycin (Km,100μg/ml).The shoots resistant to Km were transferred into the rooting medium.When roots were formed the whole plants were transferred into pots.PCR reaction using the primers complementing with potato aspartic proteinese inhibitor gene and Southern hybridization using the the fragments contained aspartic proteinese inhibitor gene as the probe were performed.The results of PCR and Southern hybridization showed that we obtained the transgenic tobacco plants.     

外源基因在转基因油菜染色体上的定位及分析

原位杂交技术 ( in situ hybridization,ISH)是基因定位的主要技术之一。近来 ,随着植物细胞染色体制片技术的发展 ,以及酶联放大检测系统的采用 ,在植物中已有低拷贝和单拷贝甚至小于 1 kb的 DNA序列定位的成功报道 [1 ,2 ]。染色体原位杂交技术不仅可以用于基因的物理作图 ,而且可以用来对转基因植物中的外源基因进行染色体定位 [3 5] 。研究表明 ,外源目的基因在转基因植物中的表达与整合位点有关 [6] 。因而 ,进行外源基因在转基因植物染色体上的定位以及研究外源基因的整合位点与表达之间的关系 ,对于开发和利用转基因植物具有重要…     

Expression of IAA-Lysine Synthetase Gene Caused Variations of Flower Develop ment in Transgenic Tobacco

The indoleacetic acid-lysine synthetase (iaaL) gene of Pseudornonas syringae subsp. savastanoi was fused to a rice Rchl0 promoter and introduced into tobacco plants. The expression pattern of this chimeric Rchl0-iaaL gene in the transgenic plants was studied, and the endogenous levels of indoleacetic acid (IAA) were assayed with the high-performance liquid chromatography (HPLC) method. The results showed that the Rchl0 promoter could direct the high level expression of the iaaL gene in young inflorescences, fully bloomed flowers, and immature fruits, resulted in a decrease of the IAA levels by 32%–82%. The transgenic plants displayed decreased apical dominance, enlongated stamens, abnormal inflorescences and fruits. The transgenie plant pYL614-T14, in which the endogenous IAA levels in inflorescences were reduced by 82 %, displayed extremely abnormal inflorescence with only two flowers and a much higher potential of vegetative growth than that of the controls after flowering. These Rchl 0-iaaL gene transgenic tobacco plants would pro- vide valuable materials for further studies of physiological functions of IAA in reproductive development.     

转LEA基因烟草的耐盐性分析

目的:验证柽柳LEA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LEA基因烟草当代(T0)和子一代(T1)分别进行不同浓度的NaCl胁迫处理,研究转基因烟草的耐盐性。结果:转基因烟草T0代组培苗耐受NaCl的临界浓度为230mmol/L,而对照耐受NaCl的临界浓度为130mmol/L以下;T1代幼苗耐受NaCl的临界浓度为150mmol/L,对照耐受NaCl的临界浓度为100mmol/L以下;在临界浓度转基因烟草的T0代、T1代根系发育良好,生长量明显高于非转基因对照烟草。结论:柽柳LEA基因的转化提高了烟草的耐盐性。     

转BmK IT4基因烟草的抗虫性

用酶切方法从「ｐＢＳ－ＢｍＫ ＩＴ４质粒中获得ＢｍＫ ＩＴ４基因片段,并构建ＣａＭＶ ３５Ｓ启动子下的ＢｍＫ ＩＴ４基因表达质粒ｐＥ３－ＢｍＫ ＩＴ４,以根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｒｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ）Ｃｏｎｎ）介导的叶盘法转化云烟（Ｎｉｃｏ－ｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）Ｋ３２６叶片,获得４５株抗卡那霉素的再生植株。用这些再生植株进行抗虫     

转BmK IT4基因烟草的抗虫性

The BmK IT4 gene was obtained from pBS-BmK IT4 by EcoRⅠ/KpnⅠdigestion and it was then cloned into pE3 intermediate vector. The resulting plasmid was named pE3-BmK IT4. The chimeric gene was transferred into the tobacco (Nicotiana tabacum L.) genome via Agrobacterium-mediated transformation. Forty-five regenerated kanamycin resistant plants were obtained, two individual lines showed strong toxicity to Manduca sexta (Linnaeus), Heliothis armigera (Hübner) and Leguminivora glycinivorella (Matsumura) by feeding experiments. Results from Southern blot indicated that BmK IT4 gene was transferred into tobacco genome. The mortality of M．sexta, H．armigera and L．glycinivorella larvae fed on transgenic plants was 95%-97%, 63%-70% and 65%-73%, respectively, and the growth of the surviving insects was remarkably retarded.     

转拟南芥ICE1基因增强烟草抗寒性的研究

ICE1是CBF冷响应通道的上游转录调控因子,通过与CBF启动子中MYC顺式作用元件的结合激活CBF3基因表达.采用RT-PCR方法,从拟南芥获得AtICE1基因,将AtICE1导入pCAMBIA1301构建35S:AtICE1植物表达载体.通过根癌农杆菌GV3101,将AtICE1基因导人烟草,T1代植株经潮霉素抗性筛选,PCR、RT-PCR检测,结果表明AtICE1基因已经整合到烟草基因组中,并在转录水平表达;在正常生长条件下,转基因烟草与对照烟草的生长未见明显区别,而在瞬时低温冻害下,转基因烟草存活率明显高于对照烟草植株,说明Atl-CEI基因可以提高低温敏感作物的耐寒性.