转BADH基因玉米植株的获得及其耐盐性分析

采用超声波辅助花粉介导植物转基因方法, 将甜菜碱醛脱氢酶(BADH)基因导入玉米自交系郑58, 获得了耐盐性强的转基因玉米植株。经卡那霉素抗性初筛、PCR扩增、Southern blot杂交分析, 证明BADH基因已导入转化植株并整合到其基因组中。用不同浓度的NaCl溶液对T₂代转基因玉米植株与对照进行盐胁迫处理, 结果表明, 转BADH基因玉米植株表现出一定的抗逆性, 生长状况明显优于对照; 根据非转化苗对NaCl的反应以及生长状况, 确定250 mmol L^-1 NaCl溶液为玉米幼苗耐盐性筛选的适宜浓度; 依据此临界浓度下形态指标和生理生化指标的测定结果, 与对照相比, 转基因植株的株高提高10.94%~25.7%, 鲜重增加8.62%~18.2%, 干重增加9%~18.18%, 相对电导率降低37.21%~58.14%, 叶绿素含量增加15.89%~90.65%, 超氧化物歧化酶(SOD)活性提高64.92%~148.29%, 丙二醛(MDA)含量减少26.97%~48.05%。综上所述, 转入甜菜碱醛脱氢酶(BADH)基因提高了玉米的耐盐性。这是首例将BADH基因导入优良玉米自交系郑58的报道。超声波辅助花粉介导法是一种经济、高效、实用和无基因型依赖性的植物基因转化方法。     

葫芦科植物的遗传转化研究进展

葫芦科植物中包括多种以采收果实为主的蔬菜作物，对其进行品质改良、延迟成熟和抗病虫害的遗传转化研究具有重要的理论和实践意义。目前，在葫芦科植物中已建立了子叶、茎尖、胚轴、茎段、原生质体和子叶节等的再生体系；用于遗传转化的方法有农杆菌介导法、基因枪法、花粉管通道法、DNA浸泡法等；用于转化的目的基因多为抗病毒病基因和延迟成熟的基因；NPTII基因为最常用的筛选标记基因。主要对以上几个方面进行综述，并讨论了葫芦科植物遗传转化过程中的影响因素及存在的问题。     

Ecological aspects of transgenic sugar beet: transfer and expression of herbicide resistance in hybrids with wild beets

Summary An increasing number of genetically engineered cultivars of several crops is being experimentally released into the environment. In future, crops with new transgenic traits will probably play an important role in agricultural practice. The long-term effect of transgenes on community ecology will depend on the distribution and establishment of transgenic plants in the wild, on the sexual transfer of their new genes to the environment and on the potential ecological impact of the transgenic trait. The starting point was the use of transgenic sugar beet lines, Beta vulgaris subspec. vulgaris var. altissima DÖLL (Helm 1957), with transgenes coding for rhizomania and herbicide (BASTA^®) resistance. The first two questions to answer were: Can the transgenes be transferred via pollen to wild beets, Beta vulgaris subspec. maritima (L.) ARCANG. or cultivated relatives such as red beet or spinach beet and are they expressed in the hybrids? Can transgenes be monitored in young Beta vulgaris-hybrids? The experimental transfer of transgenes was conducted in 1993 at a field location in northern Germany. The beets were hand-pollinated with transgenic pollen. In a non destructive biotest, the hybrid seedlings were tested for herbicide resistance. Transgenic plants showed no noxious phenotypic effects whereas control plants developed leaf necroses. All herbicide resistant hybrids within the biotest were assumed to be transgenic.     

花粉介导法将萝卜抗菌肽基因导入野罂粟的影响因素研究

以野罂粟开放花蕾为受体,载有萝卜抗菌肽AFP(antifungal protein)基因的质粒pBIAFP为供体,在不同浓度的蔗塘溶液中,加入新鲜花粉与含有目的基因的质粒DNA,应用SKH250LH型超声波震荡器进行震荡处理得到花粉处理液,在不同的时间用带有Coolsnap的OLYPUS光学显微镜下观察野罂粟花粉萌发情况;在不同授粉时间后,辅以人工授粉的方法将花粉处理液滴注野罂粟柱头上,套袋后获得种子,测定其结实率。实验证明：利用花粉介导法进行萝卜抗菌肽基因转化,以蔗糖浓度11%,处理后10-15h后的野罂粟花粉萌发最好;在蔗糖浓度8%的溶液中,以5h的结籽量较好。后代经卡那霉素筛选和PCR检测,初步证明外源AFP基因整合到野罂粟基因组中,并获得转基因T1代。为特殊中药材育种提供一个新方法。     

Environmental risks of genetic engineering

Summary Before release into commerce, genetically engineered organisms are first assessed for possible risks, including risks to the environment. The present paper first identifies the environmental risks recognized by regulators, and reviews the parameters considered predictive of risk. Recent field-scale studies suggest opportunities for improvement of the environmental risk assessment process. Risks unique to genetically engineered crops – if any – could pertain to the specific traits chosen for commercialization and to unintended trait expression caused by the process of transgene insertion itself. Both the standard against which to compare genetically engineered traits and the scale of exposure need to be considered when assessing environmental impact. Evidence of environmental risk in the recognized areas of weediness on agricultural land, invasiveness of unmanaged systems, and non-target impacts from Bacillus thuringiensis (Bt) maize is presented. Targeted, statistically sound, rigorously conducted, multi-trophic studies analogous to the Field Scale Evaluation trials recently completed in the UK are needed to clarify the many questions which remain unanswered.     

转基因小麦外源品质基因1Dx5表达的遗传研究

转基因小麦B72-8-11b中外源品质基因1Dx5表达量是内源相应基因表达量的6倍。利用小麦转基因品系为父本，常规品种鄂麦12、鄂麦18和日喀则8号分别为母本，配置3个杂交组合。采用SDS—PAGE技术，检测各组合亲本、F1、F2代的HMW-GS组成，研究转基因小麦B72-8-11b中外源品质基因1Dx5表达的遗传。结果表明：外源1Dx5基因有功能拷贝整合在1个位点，如同内源品质基因，遵从孟德尔遗传模式。这对育种选择策略的制订具有指导意义，也表明了基因枪转化法能够使得外源基因有功能拷贝整合在1个位点并能稳定传递。     

Transmission of a Bacillus thuringiensis cry3Aa transgene from diploid to tetraploid potato using 4x-2x hybridization: effect of ploidy increase on transgene expression and implications for TPS hybrid production

The study examined the effect of ploidy elevation through unreduced gametes on transgene expression in potato. Tetraploid transgenic progenies were obtained from one tetraploid potato cultivar crossed with 2n pollen producing diploid clones harbouring an exogenous transgene (cry3Aa). Both single‐ and multiple‐insert diploid transgenic lines that were regenerated by Agrobacterium tumefaciens leaf disc inoculation were used in crosses. A DAS‐ELISA system and no‐choice feeding bioassay enabled characterization of the parental lines as either ‘high’ or low’ expressers of the Cry3Aa protein. High Cry3Aa expression was observed for both single‐insert transgenic diploids and their 4x‐2x progeny. On the contrary, 68% of 4x‐2x progeny derived from a multiple‐insert, diploid transgenic had significantly reduced Cry3Aa expression compared with the parent, with 32% demonstrating nearly complete silencing of the transgene. Multiple copies of a transgene, like homologous native genes, may be susceptible to transgene silencing following polyploidization. Therefore, incorporation of exogenous transgenes into a true potato seed (TPS) production system is feasible if a single‐insert diploid parent is used. Gene‐centromere mapping of the cry3Aa transgene demonstrated that a non‐transgenic refuge might be naturally created in a TPS hybrid system through genetic recombination.     

转双价抗虫基因BmkIT-Chitinase玉米株系的获得

通过超声波辅助花粉介导法,将双价抗虫基因BmkIT-Chitinase分别导入以玉米自交系昌7-2及郑58的花粉为受体的不同基因型的自交系中。本研究共处理玉米雌穗1072穗,获得T0代种子1563粒,经卡那霉素初筛,T1代～T4代PCR及SouthernBlot杂交分子跟踪检测共获得20个转化株系,田间抗虫性鉴定表明共有16个转化株系与对照在抗虫性方面有显著差异,且此抗性随着各代稳定遗传。农艺性状调查结果表明,所获得的转基因玉米株系中大部分材料的农艺性状与对照无显著差异,除了N55材料及N20-1材料。N55材料的穗位高度与对照相比略低6±0.5cm,而穗粒数增加75±5粒。而N20-1材料百粒重增加5±0.5g。因此,转入此双价抗虫基因对玉米农艺性状影响不是很大。经过分子检测、田间抗虫性鉴定及农艺性状调查我们最终选育了9个转双价抗虫基因昌7-2自交系优良株系,6个郑58转双价抗虫基因自交系优良株系。     

风、蜜蜂因素对转Cry1Ac基因棉花花粉介导的基因漂移的影响

在温室内人工创造风力和释放传粉昆虫蜜蜂的条件下,应用PCR和蛋白试纸条结合的方法检测外源Cry1Ac基因通过花粉漂移至非转基因棉的频率和距离.结果表明:风力处理和蜜蜂处理的基因漂移频率均显著高于空白对照.漂移至非转基因亲本棉石远321的频率显著高于陆地棉中棉所35和海岛棉吉扎1号.漂移至石远321的频率随距离远近差异显著,而漂移至中棉所35和吉扎1号的频率在不同距离上差异不显著.风力处理共检测到阳性样本72个,在检测范围内,漂移至石远321的最远距离为25.6 m,漂移至中棉所35和吉扎1号的最远距离均为19.2 m.蜜蜂处理中共检测到阳性样本75个,在检测范围内,漂移到常规棉的最远漂移距离均达到设置最远处36 m,并在此处达到峰值.本研究可为转基因棉花基因漂移生态风险性评估提供参考.     

Occasional loss of expression of phosphinothricin tolerance in sexual offspring of transgenic oilseed rape (Brassica napus L.)

The commercial and economic value of genetically modified crops is determined by a predictable, consistent and stable transmission and expression of the transgenes in successive generations. No gene inactivation is expected after selfings or crosses with non-transformed plants of homozygous transgenic oilseed rape plants if the expression of the transgene in homozygous or hemizygous nature in such plants is stable. The segregation ratios of phosphinothricin (PPT) tolerance in successive generations of selfings and mutual crosses of a few independent transgenic PPT-tolerant oilseed rape plants indicated a dominant, monogenic inheritance. In within-variety and between-variety crosses no transgene inactivation was observed. However, after selfings and backcrosses with non-transgenic oilseed rape infrequent loss of the expression of the PPT tolerance transgene was observed independent from its homozygous or hemizygous nature. Molecular analysis of PPT-susceptible plants showed that the loss of expression was due to gene inactivation and not to the absence of the transgene. Methylation and co-suppression are mechanisms that might cause reduced or even loss of expression of the transgene in later generations. The implications of this observation for seed multiplication of varieties and breeding activities with transgenic oilseed rape are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Overwintering of genetically modified sugar beet, Beta vulgaris L. subsp. vulgaris, as a source for dispersal of transgenic pollen

The potential impact of transgenic crops on community ecology will depend on the distribution and establishment of the new transgenic traits, on the sexual transfer of their new genes to the environment (Bartsch &; Pohl-Orf, 1996) and on the potential ecological impact of the transgenic trait. Flowering and pollen dispersal is important for outcrossing of the genetically engineered trait. For a biennial plant, like the cultivars of Beta vulgaris L., overwintering is normally necessary to become generative and to produce pollen and seeds (Abe et al., 1997), which usually does not happen with sugar beet as a field crop harvested in autumn (Longden 1989). The starting point for the project was a transgenic sugar beet, Beta vulgaris L. subsp. vulgaris (Lange et al., 1998), with rhizomania and herbicide ( Basta®, Liberty®) resistance. Cold tolerance is one of the most important factors for survival of sugar beet in Central- and North-Europe. Among other ways, spreading of transgenic traits into weed beet (Boudry et al., 1993) or wild beet can occur if genetically engineered – biennial – plants survive the winter, flower in spring and spread their pollen. Field experiments were performed with transgenic breeding lines and their hybrids, transgenic and non-transgenic hybrids with Swiss chard and three conventional beet cultivars to evaluate winter survival rates at seven different field sites. We could show that survival of sugar beet – transgenic as well as conventional ones – in Germany and at the Dutch border is possible. Survival rates were well correlated with temperature data and were unexpectedly high. Differences between sugar beet hybrids and breeding lines could be detected but not within different breeding lines or hybrids. There were no differences detectable between transgenic and non-transgenic plants. The data are crucial for the risk assessment of the release of transgenic sugar beet and are the basis for further experiments towards outcrossing and establishment.     

纳米磁珠介导牡丹花粉转基因技术的初步分析

磁性纳米颗粒(magnetic nanoparticles,MNPs)可以吸附DNA,作为一种新型基因载体应用于转基因育种。本研究旨在探索MNPs介导外源基因进入牡丹花粉,为利用携有外源基因的花粉进行授粉,获得转基因种子提供依据,本研究制备了MNPs与DNA的复合物;利用扫描电子显微镜与透射电子显微镜观察了牡丹花粉的形态结构;用花粉离体萌发法测定了供试牡丹花粉磁转染前后的生活力。结果表明:MNPs与DNA的质量比为1:1~1:10时,MNPs均可以与DNA稳定结合,并可以保护DNA免受酶的消化作用;牡丹花粉为长球形,萌发孔为三孔沟类型,位于萌发沟内;‘凤丹’牡丹、‘京红’牡丹及紫斑牡丹的花粉均具有较高的萌发力,且经磁转染后的三种牡丹花粉萌发力保持在50%以上,能够用于正常杂交授粉。本研究首次采用四氧化三铁纳米磁转化技术介导牡丹花粉转基因,弥补了花粉介导牡丹转基因方法研究的空白,从而有助于牡丹分子育种进程的推进。     

花粉致死基因ZmAA1对玉米遗传转化的研究

为了研究花粉致死基因ZmAA1的功能,进而创制转ZmAA1基因玉米不育新材料。在Gen Bank中找到花粉致死基因ZmAA1及启动子Pg47,并在目的片段的上下游设计増加酶切位点,基因合成构建到克隆载体puc57上,采用传统酶切连接的方法构建了植物表达载体pCAMBIA3300-Pg47-ZmAA1-35S-bar,并利用农杆菌介导法转化优良玉米自交系郑58萌动胚。成功构建植物表达载体pCAMBIA3300-Pg47-ZmAA1-35S-bar;共获得94株除草剂抗性植株,其中47株目的片段PCR呈阳性反应,对温室中表现为花粉败育的PCR阳性植株进行目的片段及筛选标记bar基因RT-PCR与蛋白免疫学试纸条检测,结果表明,花粉致死基因ZmAA1及启动子Pg47已经整合到玉米基因组并表达。     

转水稻OsSIK1基因玉米植株的获得及抗旱性分析

类受体激酶基因Os SIK1具有通过激活抗氧化系统,增强水稻对于干旱和盐胁迫抗性的作用。为了丰富可利用的作物抗旱基因,获得具有较高抗旱水平的玉米新种质,通过超声波辅助花粉介导法,将水稻类受体激酶基因Os SIK1导入玉米自交系郑58中,并对转化株进行卡那霉素筛选及T1、T2、T3的PCR及Southern Blotting杂交等分子检测,获得转化植株并在T3获得转基因纯合株系。对T3转基因玉米和非转基因玉米对照以16.1%的PEG模拟水分胁迫进行抗旱性分析。结果表明,与对照相比,在水分胁迫处理下,转基因玉米株系叶片相对含水量提高了7.4%~19.8%,叶绿素含量提高了11.3%~106.9%,SOD活性上升45.8%~93.4%,而转基因玉米叶片的相对电导率下降了35.4%~58.1%,MDA含量下降了25.7%~50.4%,说明转Os SIK1基因玉米植株抗旱性得到提高,其中,5个转化株系与对照在抗旱性方面有显著差异,且生长状况明显优于对照。综上所述,研究最终获得5个转Os SIK1基因玉米株系,并证明导入水稻Os SIK1基因可以提高玉米植株的抗旱性。     

The phenotypic characterisation of R2 generation transgenic rice plants under field and glasshouse conditions

Summary The phenotypes of seed progeny (R₂ generation) of Oryza sativa L. cv. Taipei 309, which carried the neomycin phosphotransferase II (npt II) gene, were compared with those of non-transformed, protoplast-derived plants of the same generation and non-transformed, seed-derived plants under field and glasshouse conditions. Under both conditions the transgenic plants were generally smaller, took longer to flower and had reduced fertility. Significant differences were observed between individuals within the group of transgenic plants. The npt II gene was present in most of the transgenic plants, but NPT II activity was only detected in a minority of individuals.     

棉花转基因材料的获得及主要农艺性状变异分析

 利用不同棉花受体材料进行花粉管通道法遗传转化研究。通过卡那霉素鉴定、抗虫性鉴定和PCR分子生物学鉴定,证明已成功将Bt基因转化到泗棉3号、辽棉15、中棉所19、中棉所29母本、中棉所35、中棉所36等棉花材料中。通过对转基因材料后代与非转基因材料的铃重、衣分和纤维品质等差异分析,发现花粉管通道转基因后代材料存在广泛的变异。     

利用新的外植体建立棉花高效转化系统的研究

利用陆地棉 (Gossypium hirsutum cv.Cok-er3 1 2和 G.hirsutum.晋棉 7号 ,冀合 3 2 1 ) 8～1 2 d无菌苗侧根 1 0 mm切段作为新的外植体与农杆菌 L BA440 4 p BK9(3 5 s∶∶ LUC∶∶ Nos)共培养将外源基因导入棉花 ,成功的获得转基因工程植株 ,提高了愈伤组织和转基因工程植株的转化效率。 p BK9质粒上携带的萤光素酶基因 [Lucif-erous(lux) ]为报告基因 (reporter gene) ,新霉素磷酸转移酶基因 (npt II)为选择标记基因 (markergene) ,经选择获得的转基因工程植株通过 VedioImage System进行整株活体萤光素酶基因 (lux)活性表达检测 ,获得 To 代和 T1代萤光素酶基因(lux)阳性表达植株。进行 DNA分子杂交 (South-ern blot)分析 ,证明外源基因已经整合到棉花基因组中。转基因工程植株萤光素酶基因 (lux)和新霉素磷酸转移酶基因 (npt II)在自交后代中得到保持 ,外源基因呈核基因单一位点显性遗传。     

转基因高油酸油菜T-DNA插入拷贝数及整合位点分析

为获得转基因高油酸油菜T-DNA插入拷贝数及整合位点相关信息,本研究应用地高辛标记的NPTII基因片段为探针,与经BamHI酶切的转基因甘蓝型油菜高油酸株系W-4的T2代单株的基因组DNA进行Southern杂交。结果显示:W-4的T2代单株的基因组含有一个T-DNA拷贝。用3到4个根据载体pCNFIRnos序列设计的嵌套特异性引物分别与简并引物组合进行TAIL-PCR反应,扩增得到转基因油菜T-DNA插入位点的左、右边界旁侧序列。经分析右边界旁侧序列长度为470bp,其中180bp为载体序列,290bp为W-4的基因组序列;左边界旁侧序列长度为641bp,其中365bp为W-4的基因组序列,276bp为载体序列。序列比对结果发现该转基因事件中,T-DNA左边界序列完全整合到油菜基因组中,仅有1个碱基由G转换成了A。而右边界则缺失了包括RBborder在内的62个碱基。结果表明:转基因高油酸油菜T-DNA的整合是一次无其他额外载体序列的整合。blast分析获得的与左右边界相连的油菜基因组序列,未检索到与之高度同源的序列,推测T-DNA插入位点可能位于油菜基因组非编码区。综上所述,本研究分析了转基因油菜W-4基因中T-DNA拷贝数、整合特点和旁侧序列,研究结果为转基因油菜的生物安全性评价以及转基因高油酸油菜的检测提供重要的基础信息。     

水稻育性调控相关基因RNAi载体的构建及其表型鉴定

利用RNAi干扰技术研究不同基因对花粉发育、卵细胞发育和合子胚发育的影响是一种重要的手段。本研究通过筛选水稻在生殖发育过程中的9个重要调控基因,构建基因的RNAi表达载体,分析转基因植株育性及相关性状表型,以期探明RNAi表达载体对靶标性状的干扰效应。其中,AT61~AT63、AT64~AT66、AT67~AT69表达载体分别靶标花粉育性、卵细胞发育以及合子胚发育的调控。结果表明,除RNAi表达载体AT64没有获得转基因植株外,其余8个RNAi表达载体均获得了转基因植株;对T0代转基因植株的花粉育性、结实率以及潮霉素筛选(40 mg/L)发芽率检测的统计结果显示,RNAi表达载体AT62(花粉发育调控)、AT65(卵细胞发育调控)和AT67(合子胚发育调控)的干扰效应较强。本研究结果将为创制新型水稻基因工程不育系提供策略和选择。     

Agronomic traits and gene containment capability of cleistogamous rice lines with the superwoman1-cleistogamy mutation

Pollen-mediated transgene flow is a major concern for the production of genetically modified (GM) rice. Cleistogamy is a useful tool for preventing this form of gene flow. We previously identified the cleistogamous rice mutant superwoman1-cleistogamy (spw1-cls) and determined its molecular genetic mechanism. In the present study, we cultivated spw1-cls over five years to examine effects of cleistogamy on agronomic traits. Simultaneously, we cultivated cleistogamous backcross lines created by continuous backcrossing with “Yumeaoba” (a japonica cultivar) as the recurrent parent and by application of a DNA marker. In these experimental cultivations, spw1-cls and its backcross lines showed almost equal or slightly lower, but acceptable, agronomic traits compared with each control line. We also conducted natural crossing tests in paddy fields to assess the gene containment capability of spw1-cls. In a series of field experiments, there was no natural crossing between spw1-cls (pollen donor) and pollen recipient lines, but the wild-type donor and recipient lines were crossed. Thus, the cleistogamy of the spw1-cls mutation is able to inhibit natural crossing effectively, without significant loss of commercial benefits, such as yield. We conclude that spw1-cls cleistogamy is a practical tool for gene containment in GM rice cultivation.