利用转基因标记NPTⅡ快速、规模化纯合转基因番茄

利用卡那霉素喷施方法对带有NPTⅡ标记基因和双价外源基因(烟草渗调蛋白基因AP24和菜豆几丁质酶基因Chi)的转基因番茄的3个世代在温室或田间环境进行规模化筛选,成功获得8个单拷贝转基因纯合植株。含有单个拷贝NPTⅡ标记基因的转基因株系后代,对卡那霉素的抗感分离符合3:1的孟德尔分离比例,T₂代中一些株系表现为对卡那霉素全抗,表明这些株系的外源基因已经纯合,这一结果在T₃代中进一步得到证实。但对于含有两个拷贝外源基因的转基因株系,外源基因的遗传则比较复杂。同时,结合Km喷施和多重PCR技术对外源基因的异常遗传进行了初步分析。用PCR分析进一步证实了该方法的准确性,该方法是对转基因番茄进行大规模、快速遗传分析的理想方法。     

利用转基因标记NPTⅡ快速、规模化纯合转基因番茄

利用卡那霉素喷施方法对带有NPTⅡ标记基因和双价外源基因(烟草渗调蛋白基因AP24和菜豆几丁质酶基因Chi)的转基因番茄的3个世代在温室或田间环境进行规模化筛选,成功获得8个单拷贝转基因纯合植株。含有单个拷贝NPTⅡ标记基因的转基因株系后代,对卡那霉素的抗感分离符合3∶1的孟德尔分离比例,T2代中一些株系表现为对卡那霉素全抗,表明这些株系的外源基因已经纯合,这一结果在T3代中进一步得到证实。但对于含有两个拷贝外源基因的转基因株系,外源基因的遗传则比较复杂。同时,结合Km喷施和多重PCR技术对外源基因的异常遗传进行了初步分析。用PCR分析进一步证实了该方法的准确性,该方法是对转基因番茄进行大规模、快速遗传分析的理想方法。     

抗逆调节转录因子DREB1B基因转化多年生黑麦草的研究

以逆境诱导型启动子rd29B为驱动,分别构建了含有抗逆调节转录因子DREB1B基因的表达载体pBAC123、pBAC128,选择标记为bar基因.用高压氦气基因枪PDS1000/He分别将表达载体和p35SIH3导入多年生黑麦草(Lolium perenne)品种Topgun成熟胚的愈伤组织.经除草剂Bialaphos抗性筛选和植株再生,获得了62株转基因植株.经PCR、Dot-blotting分子检测,DREB1B基因已整合到多年生黑麦草部分转基因株系的基因组中.用5种不同浓度的除草剂涂抹黑麦草叶片,非转基因植株表现为不抗,而转基因植株最高可以抗135~200 mg/L.脯氨酸含量测定表明,使用15%PEG8000处理后,转基因植株的叶片脯氨酸含量比非转基因植株提高1倍左右.经25 d人工温室干旱处理,有5棵转基因植株存活;复水后,有3棵植株恢复正常生长.结果表明,利用逆境诱导型启动子(rd29B)来增强外源DREB1B基因的表达,能显著改良黑麦草的抗旱能力.     

芥菜型油菜抗虫转基因植株及其后代株系的研究*

带有1～2mm子叶柄的芥菜型油菜子叶经农杆菌感染后,培养在附加10～20mg/L卡那霉素的MS选择培养基上筛选转化愈伤组织及不定芽。卡那霉素抗性苗相继在含30～50mg/L卡那霉素的选择培养基上继代培养,再转移到含20mg/L卡那霉素的生根培养基上诱导生根。以苏云金杆菌杀虫晶体蛋白基因为探针,进行Southern blot分子杂交,得到阳性结果。PCR分析也证明外源基因整合到油菜基因组并稳定传递到后代。转基因植株的抗虫性和卡那霉素抗性在自交后代中得到保持,筛选得到纯合的转基因植株后代株系。     

基因枪转化的bar基因表达盒在水稻中遗传行为复杂

基因枪介导基因表达盒(仅包括启动子、编码区和终止子)转化是基因枪转化植物的新趋势,它能消除质粒载体主干序列对转基因植物的不利影响。本文研究了基因枪转化的bar基因表达盒在转基因水稻T1～T3世代中的遗传行为。结果发现:作为筛选标记的bar基因表达盒在水稻基因组中多拷贝整合,遗传分离行为复杂,还出现了Basta抗感分离比在35:1～144:1之间的"假纯合体",但50%转基因株系中(5/10)bar基因可作为一个显性基因按孟德尔方式稳定遗传至自交T2代。虽然bar基因为多拷贝整合,30%的转基因株系(3/10)在自交低世代(T1)能获得纯合体。Southern杂交分析发现,多拷贝的bar基因表达盒倾向于连接成转基因串联子整合在水稻基因组内。我们发现在Basta抗性正常分离的株系后代中bar基因表达盒Southern杂交模式能稳定遗传,但异常分离的株系后代中bar基因表达盒的一些拷贝发生了丢失。我们推测,bar基因表达盒在水稻中遗传分离行为的复杂原因可能是bar基因表达盒多拷贝整合、基因丢失和基因表达互作。     

转金属硫蛋白及其突变体αα 基因烟草的Tl代性状分析

转金属硫蛋白MT及其突变体αα的烟草自交得到T1代,转基因植株的重金属抗性和除草剂抗性在后代中得到保持.不同的转基因株系在含有除草剂的培养基中萌发时表现出不同的抗性分离比,说明外源基因在植物染色体内可能有不同的整合拷贝数.两种转基因烟草在含有不同浓度Cd的培养基中萌发时表现出高的重金属抗性,其中转αα基因烟草对Cd的抗性优势在T1代种子萌发实验中更加明显.以MTcDNA为探针进行Southernblot杂交,在T0代和T1代同时检测到两种转基因烟草外源基因的存在,证明外源基因确实整合到烟草基因组内并稳定传递到后代.Westemblot结果证明了外源基因在烟草第二代的表达.纯合株系仍需要进一步的筛选.     

转金属硫蛋白及其突变体αα基因烟草的T1代性状分析

转金属硫蛋白MT及其突变体αα的烟草自交得到T1代,转基因植株的重金属抗性和除草剂抗性在后代中得到保持,不同的转基因株系在含有除草剂的培养基中萌发时表现出不同的抗性分离比,说明外源基因在植物染色体内可能有不同的整合拷贝数。两种转基因烟草在含有不同浓度Cd的培养基中萌发时表现出高的重金属抗性,其中转αα基因烟草对Cd的抗性优势在T1代种子萌发实验中更加明显。以MT cDNA为探针进行Southern blot杂交,在T0代和T1代同时检测到两种转基因烟草外源基因的存在,证明外源基因确实整合到烟草基因组内并稳定传递到后代。Western blot结果证明了外源基因在烟草第二代的表达,纯合株系仍需要进一步的筛选。     

RNA介导的病毒抗性在转基因烟草中的遗传分析

以含非翻译马铃薯Y病毒外壳蛋白(PVY—MCP)基因的抗病和感病T0代转基因烟草为材料,对转基因及RNA介导的病毒抗性在T1～T4代转基因植株中的遗传进行了研究。结果表明,在含低拷贝(1～2个)转基因的感病植株后代中,转基因是作为一个单显性遗传位点,遵循孟德尔遗传分离规律,各世代转基因植株仍表现感病。含多拷贝(4～6个)转基因的抗病植株,转基因在T1代的分离虽符合多位点插入的15：1和63：1遗传规律,但转基因发生了重排,RNA介导的病毒抗性表现为不稳定遗传。从含多拷贝转基因的抗病植株的T1代株系中,分离获得了两株含2个拷贝转基因的抗病植株;其后代中,转基因及抗性遗传遵循孟德尔遗传分离规律,可稳定遗传和表达,获得纯合的抗病转基因株系。对转基因在不同抗病类型植株中整合方式分析显示,抗病植株中多存在转基因的反向串联重复序列。     

转望江南核糖体失活蛋白基因cassin烟草及其对烟草花叶病毒的抗性

通过构建植物表达载体,由农杆菌介导,将望江南核糖体失活蛋白基因cassin转入烟草。PCR和Southern blot杂交结果证明：外源基因已经以单拷贝整合到烟草基因组内,并且在后代发生遗传分离。RT—PCR和Northern blot杂交结果显示：外源基因可以正常转录。用不同浓度的TMV机械摩擦接种转基因T1、T2代各3个自交株系,以非转基因烟草为阴性对照,实验结果表明转基因烟草对TMV表现出不同程度的抗性。     

转bar基因小麦和非转基因小麦抗除草剂鉴定方法比较

方便、快捷、准确地对转基因小麦中的bar基因进行检测,对于筛选纯合稳定转基因植株、获得无筛选标记转基因植株、鉴定常规小麦品种和商品小麦中的bar基因成分等具有一定价值。本试验对叶片涂抹、植株喷洒、培养基添加除草剂3种方法鉴定转bar基因小麦植株的效果进行了比较,表明3种方法都能很好鉴定转基因小麦中的bar基因,叶片涂抹200mg/LLiberty鉴别的准确性高于PCR检测,植株喷洒Basta的适宜浓度为100mg/L,喷洒Liberty的适宜浓度为150mg/L,培养基添加Bialaphos的适宜浓度为5～8mg/L。叶片涂抹和植株喷洒除草剂方法受环境条件影响较大,区别转基因植株和非转基因植株的标准不够明确。相比之下,成熟胚离体培养除草剂筛选不受外界环境条件的影响,具有鉴定效果直观明了、操作简单、试验周期短等优点,在检测小麦转入或飘入的bar基因方面具有潜在应用前景。     

带npt-Ⅱ基因转基因水稻快速检测技术的建立

以转溶菌酶基因水稻纯系材料中花9号(ZH9(R))及其受体品种中花9号(ZH9(CK))为材料, 以ZH9(R)中携带的npt-Ⅱ基因作为辅助筛选标记, 利用抗生素对其进行处理, 建立了一套快速检测带npt-Ⅱ基因转基因水稻的技术体系。使用不同浓度的卡那霉素(Kanamycin, Kan)和G418溶液将ZH9(R)和ZH9(CK)成株离体叶片于室内室温下置于培养皿中浸泡处理, 通过观察叶片变化, 确定G418为检测带npt-Ⅱ基因转基因水稻的最佳抗生素, 将G418(溶液)80 mg/L浓度(处理4天)作为检测该类转基因水稻成株离体叶片的临界浓度。进一步用G418对ZH9(R)和ZH9(CK)种子、幼胚和幼苗进行了不同处理。确定: (1) G418(溶液)300 mg/L浓度(处理7天)作为检测该类转基因水稻种子的临界浓度; (2) G418(1/2 MS+0.5 mg/L 6-BA+1.5%蔗糖培养基)200 mg/L浓度(处理10天)作为检测该类转基因水稻幼胚的临界浓度; (3) G418(1/2 MS+0.5 mg/L 6-BA+1.5%蔗糖培养基)150 mg/L浓度(处理12天)作为检测该类转基因水稻幼苗的临界浓度, 并且通过PCR方法证实了上述结论。将这些结论应用于ZH9(R)转育后代叶片、种子、幼胚和幼苗群体的检测, 检测效果都非常明显。这为带npt-Ⅱ基因转基因水稻建立了一套简便、直观且准确的检测方法。     

AtNHX1基因对荞麦的遗传转化及抗盐再生植株的获得

通过农杆菌介导法将拟南芥液泡膜Na /H 反向转运蛋白基因AtNHX1转入荞麦中,在2·0mg/L6-BA、0·1mg/LIAA、1mg/LKT、50mg/L卡那霉素和500mg/L头孢霉素的MS培养基上进行选择培养,从来源于864块外植体的36块抗性愈伤组织中共获得426棵再生植株(转化频率为4·17%)。经PCR、Southern印迹分析、RT-PCR和Northern检测,初步证实AtNHX1基因已整合至荞麦基因组中。用200mmol/L的盐水对转基因植株和对照植株进行胁迫处理6周,转基因植株能够生存,而对照植株死亡。用不同浓度的NaCl溶液处理转基因植株和对照植株,发现Na 及脯氨酸含量在转基因植株中的积累水平显著高于对照植株,而K 的含量在转基因植株中的积累水平低于对照植株。次生代谢产物黄酮类化合物芦丁在转基因植株根、茎和叶片中的含量也比对照植株明显要高。这些结果表明利用基因工程手段提高作物的耐盐性是可行的。     

AtNHX1基因对荞麦的遗传转化及抗盐再生植株的获得

通过农杆菌介导法将拟南芥液泡膜Na⁺/H⁺反向转运蛋白基因AtNHX1转入荞麦中,在2.0mg/L 6-BA、0.1mg/L IAA、1mg/L KT、50mg/L卡那霉素和500mg/L头孢霉素的MS培养基上进行选择培养,从来源于864块外植体的36块抗性愈伤组织中共获得426棵再生植株（转化频率为4.17%）。经PCR、Southern印迹分析、RT-PCR和Northern检测,初步证实AtNHX1基因已整合至荞麦基因组中。用200mmol/L的盐水对转基因植株和对照植株进行胁迫处理6周,转基因植株能够生存,而对照植株死亡。用不同浓度的NaCl溶液处理转基因植株和对照植株,发现Na⁺及脯氨酸含量在转基因植株中的积累水平显著高于对照植株,而K⁺的含量在转基因植株中的积累水平低于对照植株。次生代谢产物黄酮类化合物芦丁在转基因植株根、茎和叶片中的含量也比对照植株明显要高。这些结果表明利用基因工程手段提高作物的耐盐性是可行的。     

AtNHX1基因对荞麦的遗传转化及抗盐再生植株的获得

通过农杆菌介导法将拟南芥液泡膜Na⁺/H⁺反向转运蛋白基因AtNHX1转入荞麦中,在2.0mg/L 6-BA、0.1mg/L IAA、1mg/L KT、50mg/L卡那霉素和500mg/L头孢霉素的MS培养基上进行选择培养,从来源于864块外植体的36块抗性愈伤组织中共获得426棵再生植株（转化频率为4.17%）。经PCR、Southern印迹分析、RT-PCR和Northern检测,初步证实AtNHX1基因已整合至荞麦基因组中。用200mmol/L的盐水对转基因植株和对照植株进行胁迫处理6周,转基因植株能够生存,而对照植株死亡。用不同浓度的NaCl溶液处理转基因植株和对照植株,发现Na⁺及脯氨酸含量在转基因植株中的积累水平显著高于对照植株,而K⁺的含量在转基因植株中的积累水平低于对照植株。次生代谢产物黄酮类化合物芦丁在转基因植株根、茎和叶片中的含量也比对照植株明显要高。这些结果表明利用基因工程手段提高作物的耐盐性是可行的。     

Expression of the Bar Gene Confers Herbicide Resistance in Transgenic Lettuce

Resistance to bialaphos, a broad-spectrum herbicide, was introduced into Lactuca sativa cv. Evola by Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strains 0310 and 1310, both carrying the bialaphos resistance (bar) and neomycin phosphotransferase (nptII) genes, were used for transformation. Primary transformants were selected on kanamycin sulphate-supplemented shoot regeneration medium. Integration of both transgenes was confirmed by non-radioactive Southern hybridisation. The hypervirulent plasmid ToK47 in A. tumefaciens strain 1310 generated multiple insertions of T-DNA in some transgenic plants; the absence of pToK47 (strain 0310) resulted in single gene inserts in all plants tested. Resistance to glufosinate ammonium was observed in axenic seedlings grown on medium supplemented with the herbicide at 5 mg l–1 and in glasshouse-grown plants sprayed with the compound at 300 mg l–1. Stable expression of the bar gene was observed in R2 generation plants. The kanamycin resistance of R1 seedlings was observed by germinating seeds on medium supplemented with 200 mg l–1 kanamycin sulphate. The presence of NPTII protein and PAT enzyme activity were demonstrated by ELISA and PAT enzyme assay respectively. Transgenes segregated in a Mendelian fashion in some plant lines in the R1 generation; herbicide resistance also segregated in the expected ratio in the R2 generation in most transgenic lines. This study confirmed that an agronomically important transgene can be integrated and stably expressed over several generations in lettuce.     

Effects of kanamycin on tissue culture and somatic embryogenesis in cotton

The aminoglycoside antibiotic kanamycin was evaluated for its effects on callus initiation from hypocotyl and cotyledon explants, proliferation of non-embryogenic and embryogenic calli, initiation and development of somatic embryos in cotton (Gossypium hirsutum L.). On this basis, the potential use of kanamycin as a selective agent in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene was evaluated. Cotton cotyledon and hypocotyl explants, and embryogenic calluses were highly sensitive to kanamycin. Kanamycin at 10 mg/L or higher concentrations reduced callus formation, with complete inhibition at 60 mg/L. Kanamycin inhibited embryogenic callus growth and proliferation, as well as the initiation and development of cotton somatic embryos. The sensitivity of embryogenic callus and somatic embryos to kanamycin was different during the initiation and development stages. Kanamycin was considered as a suitable selective agent for transformed callus formation and growth of non-embryogenic callus. Forty to sixty mg/L was the optimal kanamycin concentration for the induction and proliferation of transformed callus. The concentration of kanamycin must be increased (from 50 to 200 mg/L) for the selection of transformation embryogenic callus and somatic embryos. A scheme for selection of transgenic cotton plants when kanamycin is used as the selection agent is discussed.     

Insect-resistant transgenic cabbage plants and their progenies

An insecticidal crystal protein gene of Bacillus thuringiensis was transferred into cabbage genome with the method of Agrobacterium infection. Cotyledons with petioles as explants were cocultivated with Agrobacterial suspension. Calli generated at the basis of petiole were subjected to selection on the MS medium containing 15-30 mg/L kanamycin (Km). About 5% explants produced calli growing continuously on the selective medium. Green shoots appeared on these calli when they were transplanted onto medium with Km and 6-BA for plant differentiation. The shoots were separated and cultivated on medium with kanamycin. About 80% shoots were rooted. Non-transformed control calli could not give normal shoots and roots and brownized and died gradually. Larvae of Pieris rapae showed poisonous symptoms: growth inhibition and mortality when fed with the leaf of the transgenic plants. About 80% of regenerated plants showed positive hybridization bands when their DNA were probed with crystal protein sequence of Bacillu     

Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea)

A rice chitinase cDNA (RCC2) driven by the CaMV 35S promoter was introduced into cucumber (Cucumis sativus L.) through Agrobacterium mediation. More than 200 putative transgenic shoots were regenerated and grown on MS medium supplemented with 100 mg/l kanamycin. Sixty elongated shoots were examined for the presence of the integrated RCC2 gene and subsequently confirmed to have it. Of these, 20 were tested for resistance against gray mold (Botrytis cinerea) by infection with the conidia: 15 strains out of the 20 independent shoots exhibited a higher resistance than the control (non-transgenic plants). Three transgenic cucumber strains (designated CR29, CR32 and CR33) showed the highest resistance against B. cinerea: the spread of disease was inhibited completely in these strains. Chitinase gene expression in highly resistant transgenic strains (CR32 and CR33) was compared to that of a susceptible transgenic strain (CR20) and a control. Different responses for disease resistance were observed among the highly resistant strains. CR33 inhibited appressoria formation and penetration of hyphae. Although CR32 permitted penetration of hyphae, invasion of the infection hyphae was restricted. Furthermore, progenies of CR32 showed a segregation ratio of 3:1 (resistant:susceptible). As the disease resistance against gray mold was confirmed to be inheritable, these highly resistant transgenic cucumber strains would serve as good breeding materials for disease resistance. Received: 31 March 1996 / Revision received: 2 July 1997 / Accepted: 18 July 1997     

不同浓度卡那霉素、潮霉素对楸树试管苗生长的影响

目的:将不同质量浓度的卡那霉素、潮霉素加入楸树培养基中,研究卡那霉素、潮霉素对楸树组培苗生长的影响,以确定抗生素对楸树茎段分化与生根的敏感质量浓度。方法:待楸树继代、生根培养基灭菌后温度降至30~50℃,将不同质量浓度的卡那霉素、潮霉素经抽滤式灭菌加入培养基中,在培养基中接入楸树组培无菌茎段培养,观测茎段继代(增殖芽数、芽长、叶数等)、生根(发根数、根长、芽长等)生长指标并统计分析。结果:楸树组培继代培养基添加卡那霉素质量浓度为100 mg/L时组培瓶苗生长缓慢,浓度为150 mg/L时叶片大部分发白并干枯,茎段基部无愈伤组织形成,瓶苗基本停止生长,楸树继代瓶苗对卡那霉素耐受性范围为100~150 mg/L;添加潮霉素质量浓度为5 mg/L时瓶苗生长较为缓慢,浓度为10 mg/L时叶片开始干枯,茎段基部愈伤组织较小,瓶苗基本停止生长,楸树继代瓶苗对潮霉素耐受性范围为10 mg/L左右。楸树组培生根培养基添加卡那霉素质量浓度为100 mg/L时大部分茎段干枯,少部分为绿但未分化芽与根,浓度为150 mg/L时大部分茎段干枯,极少上部为绿,基部干枯,但未分化芽与根,楸树组培瓶苗生根培养苗对卡那霉素耐受性范围为100~150 mg/L;添加潮霉素质量浓度为5 mg/L时少部分茎段干枯,浓度为10 mg/L时大部分茎段干枯,少部分为绿,茎段未出现芽的分化与根的萌发现象,楸树组培瓶苗生根培养苗对潮霉素耐受性范围为5~10 mg/L。结论:卡那霉素、潮霉素对楸树组培苗生长有明显的抑制作用且与抗生素浓度呈负相关,但低质量浓度(1 mg/L)的潮霉素对楸树继代分化芽数有促进作用;同一抗生素对楸树不同无性系间组培苗生长的影响无显著差异。