金属硫蛋白突变体的植物高效表达载体的构建及其在烟草中的表达

金属硫蛋白有α、β两个结构域（ｄｏｍ ａｉｎ）,其中α结构域优先结合Ｃｄ２＋ 和Ｈｇ２＋ ．小鼠αα突变体在大肠杆菌中已经构建并得到表达,其转基因植株已得到,可在Ｃｄ３００（３００ μｍ ｏｌ／Ｌ）中生长．为了进一步提高外源基因在烟草中的表达量,首先用ＰＣＲ 的方法设计引物,在基因翻译起始密码子ＡＴＧ 附近加入植物偏爱的碱基组合ＡＡＣＡＡＴＧ．另外,将该突变体基因插入具有双３５ Ｓ（ＣａＭＶ３５Ｓ）强启动子的植物双元表达载体ｐＧＰＴＶｄ３５Ｓ－ＢＡＲ中,获得了带有αα突变体的植物双元表达载体．通过农杆菌介导的叶盘转化法转化烟草ＮＣ８９,获得了抗除草剂的转基因植株．经ＰＣＲ－Ｓｏｕｔｈｅｒｎ 和蛋白Ｄｏｔ－ｂｌｏｔｔｉｎｇ 检测,证明了αα突变体在烟草中的嵌合与表达．抗重金属实验证明转基因烟草可以在Ｃｄ４００（４００ μｍ ｏｌ／Ｌ）中生长．     

尼虫特异性神经毒素基因tox34的合成及转基因烟草的获得中

对来自虱状蒲螨（Ｐｙｅｍｏｔｅｓ ｔｒｉｔｉｃｉ）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，依据植物的偏爱密友子进行了序列改造。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上特异条带的表现分子量为３２～３３ｋＤ。将ｔａｘ３４基因插入植物高效表达载体后以根吕土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅ     

转雪花莲外源凝集素基因烟草对桃蚜的抑制作用

将编码雪花莲外源凝集素成熟蛋白的cDNA GNA12和其前体蛋白cDNA GNA34插入到二元载体pBin438的双倍增强子CaMV 35S启动子或二元载体pBcop1的CoYMV启动子下游,分别构建成植物表达载体pBGna12、pBGna34\,pBCGna12和pBCGna34。土壤农杆菌介导的转化再生植株的PCR和Southern blot分析表明,GNA基因已整合到烟草DNA中。Western blot分析发现pBGna34和pBCGna34的转基因植株能有效地表达外源GNA,表达量约占可溶性总蛋白的0.08%～0.15%,并且前体蛋白基因编码的蛋白在植物体内进行了正确的加工;而pBGna12和pBCGna12的植株几乎检测不到外源GNA的表达。有效表达外源GNA的pBGna34和pBCGna34的转基因植株具有较强的抗蚜活性,平均能够抑制桃蚜(Myzus persicae)45%～６０％蚜口密度,有的高达90%以上。在转基因烟草中含双倍增强子的CaMV 35S启动子与韧皮部特异表达的CoYMV启动子介导GNA基因表达具有相似的强度,但它们的抗蚜活性存在差异。     

dsRNA介导的番木瓜环斑病毒（PRSV）的抗病性研究

以模式植物拟南芥（Arabidopsis thaliana）和烟草（Nicotiana tabacum）及PRSV寄主植物番木瓜（CaricapapayaL.）作为试验材料,开展了番木瓜环斑病毒外壳蛋白基因dsRNA介导的PRSV病原抗性的研究。利用农杆菌介导法将番木瓜环斑病毒外壳蛋白CP基因反向重复表达载体pHellsgate12-CPIR（简称PHG12-CPIR）分别转化到烟草和拟南芥中,获得阳性植株,并利用渗透法和农杆菌介导的瞬时表达体系将pHG12-CPIR载体导入到番木瓜中。对转基因植株进行攻毒试验并分析了其抗病性。在接种3~7d内,在拟南芥和番木瓜上转基因植株的发病情况较轻,而野生型植株叶片与转基因植株相比,均表现出不同程度的黄化、皱缩和枯斑等症状。在接种PRSV后,番木瓜和拟南芥转化植株表现症状的叶片的比例与对照相比,结果显著低于对照,而在烟草植株上症状表现的差异不明显。在3种植物上RT-PCR检测结果显示,在接种番木瓜环斑病毒PRSV后,野生型植株中有高浓度的病毒积累,而转pHG12-CPIR基因植株中几乎没有病毒积累,推测转pHG12-CPIR基因植株中瞬时表达系统已启动RNAi机制抑制了CP基因的表达。     

绿色荧光蛋白基因(GFP)在抗虫转基因植物研究中的应用(英文)

用合成的cry1Ac基因与绿色荧光蛋白基因 (GFP)构成融合蛋白基因 ,然后和改造的GNA基因构建双价抗虫基因植物表达载体pBGbfg ,经根癌农杆菌介导转化了烟草。在紫外灯照射下 ,观察到转基因植株叶片中有较强的绿色荧光 ;经抗虫试验、PCR、Southernblot和Westernblot等检测 ,表明该重组植物表达载体能够在转基因植物中有效表达外源基因 ,转基因植株绿色荧光的表型与其抗虫性密切相关。从而成功地建立了以绿色荧光蛋白基因与抗虫基因组成的融合基因转化系统 ,简化了抗虫转基因植物筛选程序 ,有助于快速获得双价抗虫转基因植株。     

绿色荧光蛋白基因（G卯）在抗虫转基因植物研究中的应用

用合成的crylAc基因与绿色荧光蛋白基因(GFP)构成融合蛋白基因,然后和改造的GNA基因构建双价抗虫基因植物表达载体pBGbfg,经根癌农杆菌介导转化了烟草。在紫外灯照射下,观察到转基因植株叶片中有较强的绿色荧光;经抗虫试验、PCR、Southern blot和Western blot等检测,表明该重组植物表达载体能够在转基因植物中有效表达外源基因,转基因植株绿色荧光的表型与其抗虫性密切相关。从而成功地建立了以绿色荧光蛋白基因与抗虫基因组成的融合基因转化系统,简化了抗虫转基因植物筛选程序,有助于快速获得双价抗虫转基因植株。     

霍乱毒素B亚单位基因分泌型表达载体的构建及转化烟草的研究

为了探索利用植物分泌特性来表达重组蛋白的可行性,先构建了含钙网蛋白信号肽的植物双元载体pBIcal,再向该载体中插入霍乱毒素B亚单位编码基因,最后得到表达载体pBIcal—ctb。通过根癌农杆菌介导,该表达载体转化烟草,在卡那霉素抗性培养基上筛选,得到30棵抗性植株。经PCR鉴定,霍乱毒素B亚单位基因已经整合到烟草基因组中。初步表达分析表明,转基因烟草中含有具生物活性的霍乱毒素B亚单位蛋白。     

含融合杀虫基因和VHb基因双价表达载体的构建

目的：探索分子育种的新途径,获得高产多抗虫谱的转基因植物。方法：将密码子优化后的透明颤菌血红蛋白（vgbM）基因与融合杀虫基因（GFMcryIA＋CPTI）构建成双价基因植物高效表达载体pGBI4ABCVHB,基因表达盒中含2个增强子、35S启动子、Ω前导序列、Kozak序列、多联终止密码子及Nos终止子以及正确切割、加工序列、Poly（A）信号序列。结果：通过根癌农杆菌介导转化烟草,获得了转基因植株,结论：vgbM基因在转基因烟草中有效表达;融合杀虫基因也表达出活性毒蛋白。     

华南象草PpCAD基因的亚细胞定位及其在转基因烟草中的异源表达

该研究根据已克隆的华南象草(Pennisetum purpureum cv.Huanan)肉桂醇脱氢酶(CAD)基因PpCAD的cDNA序列,构建亚细胞定位载体pAN580-PpCAD,用PEG介导法转化象草原生质体,以探究PpCAD蛋白在细胞内的定位;同时构建植物过表达载体pBA002-PpCAD,通过农杆菌介导法在烟草中异源表达,以研究PpCAD基因与植物木质素合成的关系。结果显示:(1)PpCAD定位在象草原生质体的细胞质内;(2)过表达载体pBA002-PpCAD转化烟草后获得27株转基因烟草,其中25株PCR鉴定为阳性;(3)半定量RT-PCR检测6株转基因烟草后发现,PpCAD基因在不同植株的表达量存在差异,通过Southern杂交检测后发现该差异与目的基因插入的拷贝数有关;(4)6株转基因烟草和野生型烟草表型上没有明显差异,除目的基因多拷贝插入的植株OEC6外,木质素含量有不同程度的提高,最高比野生型提高了56.50%。研究表明,PpCAD是一个细胞质蛋白,在烟草中过表达PpCAD能够提高植株木质素含量,表明PpCAD基因参与了植物的木质素合成,可用于象草的木质素调控研究。     

一种基于过敏性反应机制的抗植物病毒侵染策略

基于植物的过敏性反应机制,构建了PVY Nib基因和来自于细菌Bacillus amy—loliquefaciens的一类Rnase基因Barnase基因的融合基因的植物表达载体。在此表达载体内两基因的拼接处,保留了原来PVY蛋白酶识别PVYNIb和CP蛋白剪切位点的七肽保守序列。通过农杆菌介导获得此融台基因的转基因烟草植株。病毒侵染试验表明,转基因植物在病毒侵染后,发病症状被改变。少部分转融合基因的植株对病毒侵染表现局部抗性。     

The Synthesis of Insect specific Neurotoxin Gene tox 34 and Acquirement of Transgenic tobacco

A 762 bp mite (Pyemotes tritici) insect-specific neurotoxin tox34 gene fragment encoding mature protein was fully modified based on plant gene codon usage, tox34 was inserted into bacterial expression vector pBV221. Electrophoresis of protein mixture extracted from the bacteria treated with heat-induction showed that specific protein bands migrated with apparent molecular weight of 32 ~ 33 kD in denaturing gels. Tox34 was inserted into plant high-expression vector and then transferred into tobacco (Nicotiana tabacum L. cv. SRI ) using the Agrobacterium tumefaciens (Smith et Tomnsend) Conn binary vector system, followed by the obtainment of the transgenic tobacco plants. The PCR Southern blot assay indicated that the intact toxin gene was integrated into the tobacco genome. GUS assay of transgenic seedling roots showed positive results. The feeding trail of transgenic plants using bollworms (Heliothis armigeva Hubner) manifested strong toxity to lowstar bollworm larvae.     

Expression of human papillomavirus type 16 L1 protein in transgenic tobacco plants

To develop a plant expression system for the production of the human papillomavirus type 16 (HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV 16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV 16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV16 L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.     

转基因烟草中Bt毒蛋白基因的表达行为

Bt toxin genes were the insecticidal genes most widely used in genetic engineering of pest resistant plant, were of important significance to study their expression behavior in transgenic plants. In this work, a plant expression vector, pBinMoBc, was constructed. It contained the Cry IA(c) gene under control of chimeric OM promoter and the Ω factor. The vector was transferred into tobacco (Nicotiana tabacum L.) plant via Agrobacterium-mediated transformation. ELISA assay showed that the expression levels of the Cry IA(c) gene in transgenic tobacco plants were significantly higher than that in wild-type tobacco plants. The highest could be up to 0.255% of total soluble proteins; the expression level of CryIA(c) gene in transgenic tobacco plant was changeable during the development stages of tobacco plant. Bioassay showed that pBinMoBc transgenic tobacco plants had more notable insecticidal activity than the wild-type tobacco plants. The above results indicated that pBinMoBc was an effective pest-resistent plant expression vector. This study would be very helpful in screening transgenic cotton with high resistance to cotton bollworm (Heliothis armigeva Hubner).     

Transgenic Tobacco Plants with a Fully Synthesized GFM CryIA Gene Provide Effective Tobacco Bollworm (Heliothis armigera) Control

GFM CrylA gene is a fully modified synthetic gene derived from insecticidal crystal prorein gene of Bacillus thuringiensis Berliner (Bt). It was synthesized based on the codon usage of plant genes instead of changing the primary sequences of amino acids of insecticidal crystal protein (ICP) gene of Bacillus thuringiensis Htibner. To test the function of the synthetic GFM CrylA gene, we introduced the GFM CrylA gene into tobacco plant cells via an Agrobacterium tumefacieus (Smith et Townsedn) Conn binary vector system. As expected, the GFM CrylA gene is expressed under control of the cauliflower mosaic virus (CaMV) 35S promoter and allows efficient production of lepidopteran insectspecific toxic proteins in the transformed tobacco plants. Bioassays using transgenic tobacco plants with tobacco bollworm showed that the transgenic tobacco plants expressing proteins of GFM CrylA gene had effective control to tobacco bollworm. In this paper the authors firstly report the complete synthesis of GFM CryIA gene and the construction of plant expression vector pGBI4AB. The authors performed introduction of the synthetic GFM CrylA gene into the tobacco plants, and the integration of GFM CrylA gene into tobacco genome was confirmed by Southern blot analysis of the tobacco genomic DNA. The gene was efficiently expressed in the transgenic tobacco plants and effective tobacco bollworm control was verified by the insect-bioassays.     

含VHb基因和GFMcryIA基因的双价基因植物高效表达载体的构建

将人工合成的、密码子优化后的透明颤菌血红蛋白基因与人工合成的GFMcryIA基因构建成双价基因植物高效表达载体PGBI4ASVHBBt,vgbM基因表达盒中含2个增强子的35S启动子、Ω前导序列、Kozak序列、多联终止密码子及Nos终止子;Bt基因表达盒中,除含有以上提高转录和翻译的调控元件外,还包含有正确切割、加工序列、Poly(A)信号序列。利用根癌农杆菌介导转化烟草,获得了转基因植株;PCR及Southern blot检测,证实了双价基因在烟草基因组中的整合;Western blot检测证实了vgbM基因在转基因烟草中的表达;杀虫实验表明GFMcryIA基因也表达出活性毒蛋白。     

表达多肽抗生素apidaecin的转基因烟草及其抗病性的初步分析

将多肽抗生素apidaecin基因与病程相关蛋白的信号肽序列融合,构建了apidaecin的分泌型植物表达载体、apidaecin与另一多肽抗生素Shiva\|I的双价分泌型植物表达载体,以本实验室原来构建的Shiva-I分泌型植物表达载体做对照,转化了模式植物烟草。对3种转基因植物进行了分子检测,转化再生苗95%为PCR阳性,Southern杂交结果进一步证明外源基因已经整合到了烟草基因组中,RT-PCR检测表明外源基因可以在转基因烟草内正常转录。对T0代转基因烟草进行烟草野火病的抗病性实验,从3种转基因烟草中都得到了抗病植株,病情指数分析的初步结果显示,双价转基因烟草抗病性最好,apidaecin的次之,Shiva-I的最差。     

Production and secretion of human interleukin-18 in transgenic tobacco cell suspension culture

Interleukin-18 (IL-18), otherwise known as interferon-gamma-inducing factor (IGIF), is one of several well characterized and important cytokines that contribute to host defenses. The complementary DNA (cDNA) of mature human interleukin-18 gene (hIL-18) was fused with the signal peptide of the rice amylase 1A gene (Ramy1A) and introduced into the plant expression vector under the control of a duplicated CaMV 355 promoter. The recombinant plasmid was transformed into tobacco (Nicotiana tabacum L. cv Havana) using theAgrobacterium-mediated transformation method. The integration of the hIL-18 gene into the genome of transgenic tobacco plants was confirmed by polymerase chain reaction (PCR) amplification and its expression was observed in the suspension cells that were derived from the transgenic plant callus by using Northern blot analysis. The hIL-18 protein was detected in the extracts of the transgenic callus and in the medium of the transgenic tobacco suspension culture by using immunoblot analysis. Based upon enzyme-linked immunosorbant assay (ELISA) results, the expression level of the hIL-18 protein approximated 166 μg/L in the suspension culture medium. Bioassay results from the induction of interferon-γ from a KG-1 cell line indicated that the hIL-18 secreted into the suspension culture medium was bioactive.     

北非蝎昆虫毒素AaHIT1基因的原核表达和转基因分析

将北非蝎昆虫毒素 (AndroctonusaustralisHectorinsecttoxin ,AaHIT1)基因克隆入温度诱导表达载体pBV2 2 0 ,并在宿主菌E .coliDH5α中进行诱导表达 .表达定位分析发现 ,AaHIT1大部分存在于包涵体中 .蛋白质N 端测序证实了体外表达的AaHIT1蛋白的正确性 .将AaHIT1基因转化入烟草中并获得了转基因植株 ,基因组PCR、RT PCR及Northern印迹分别证实AaHIT1基因已正确地插入到烟草基因组中并已得到表达 .抗虫实验表明 ,该转基因烟草对白粉虱有明显的抗性 .该研究为蝎毒AaHIT1的生物学毒性实验以及获得抗虫转基因作物奠定了基础     

Expression of a modified Cry1Ie gene in E. coli and in transgenic tobacco confers resistance to corn borer

The wild-type Crylle gene from Bacillus thuringiensis was modified for its efficient expression in transgenic plants. Modified Crylle gene (designated as Cryllem) was cloned into prokaryotic expressionvector pET28b and its expression in E.coli was confirmed by SDS-PAGE analysis. Bioassays using crude expression products in E.coli revealed that CrylIem protein had a similar toxicity to corn borer as wild-type CrylIe. CrylIem gene was then inserted downstream of the maize ubiquitin-1 promoter in plant expression vector p3301. Transgenic tobacco plants carrying Cryllem showed insecticidal activity against corn borer.     

Expression of a Modified Cry1Ie Gene in E. coli and in Transgenic Tobacco Confers Resistance to Corn Borer

Gene transfer technology provides an alternativeapproach to breed insect-resistant crops. Insect-resistantgenes from plants or microbes could be introduced intoplants and the expressed insecticidal protein in plantcells could kill the target insects. Transgenic plantsexpressing a corresponding insecticidal crystal protein genefrom Bacillus thuringiensis (Bt) have been developed sincethe early 1980s [1,2]. Analysis of Bt gene sequencesrevealed that they contain numerous motifs seldom foundin p…