几种转基因植物体细胞克隆变异的多样性研究

分析了杨树、水稻和甘蔗转基因植株体细胞克隆的表型变异和基因组DNA多态性。探讨了以下问题:转基因植株体细胞克隆的1)表型多样性, 2)基因组DNA多样性, 3)二者的相关性, 4)表型变异和DNA变异的可遗传特性, 5)产生的可能原因,以及6)在农业生产上的应用。     

转基因白桦的遗传变异分析

应用细胞学方法分析了由农杆菌介导法获得的转基因白桦的细胞学变异情况,结果表明转基因白桦的染色体变异频率为78.5％,远远高于非转基因白桦的变异频率(15.3％),且变异以非整倍体占多数。同时用RAPD标记方法研究了转基因白桦在DNA水平的变异情况,结果显示DNA多态性指数为31.67,并与其它转基因植物的变异情况作了比较研究。最后分析、讨论了产生变异的原因:(1)组织培养过程中产生突变;(2)外源基因的整合及重排时宿主基因组的插入位点及相邻基因转录表达的干扰;(3)应用抗生素和除草剂等筛选转基因植株时促进了转基因植株的变异程度。并提出减少转基因植物体细胞克隆变异的建议。     

高表达水稻WRKY72基因影响拟南芥生长素信号传导

植物转录调控因子WRKY基因家族是一个拥有众多成员的超家族,功能涵盖了植物生长发育的控制与抗病耐逆的调节。我们主要分析了OsWRKY72基因在外源植物拟南芥中的生物学功能。通过转基因拟南芥(Arabidopsis thaliana)的遗传学研究发现外源高表达该基因不单明显地抑制转基因植株的顶端优势,增强植株侧枝的生长,还改变了转基因植株叶片和角果的发育。进一步分析证实,高表达OsWRKY72基因所导致转基因拟南芥植株的表型和其它生理现象都与生长素信号通路改变所导致的表型和生理变化极其相近。这些结果说明OsWRKY72基因在外源植物拟南芥体内高表达后很可能改变了其正常的生长素信号通路。     

一种快速鉴定转基因植物纯合体的新方法

植物转化中鉴定转基因植物的整合性是一个很重要的步骤,常规方法是对独立分离的转基因T1代植株产生的T2代进行转基因分离比率研究,以检测T1代的转基因整合状态,不仅费时费力,而且浪费了T1代资源。本介绍一种应用双重定量实时PCR技术鉴定转基因植物纯合子的新方法：以T1代植物DNA为模板,根据转基因后代的Ct表型值鉴定其转基因整合状态,Ct值接近2的为转基因纯合型,Ct值接近1的为转基因杂合型。用这种方法,可以同时对数十个T1代转基因幼苗的整合状态进行快速鉴定,准确率为100％。     

植物基因功能鉴定新工具--病毒诱导基因沉默技术的研究进展

病毒诱导基因沉默（Virus—induced gene silencing,VIGS）技术是指带一段靶基因序列的VIGS重组病毒侵染植物、引起植物同源基因沉默与表型变异,进而通过表型变异进行基因功能分析的方法,是近年发展起来的从反向遗传学方向快速鉴定植物基因功能的技术,是转录后基因沉默机制的一种表现。①VIGS的分子机制,涉厦基因沉默的起始、维持和信号放大、传播共3个阶段;②VIGS技术、栽体与方法学的发展;③VIGS作为基因功能研究的优越性,如不必进行转基因、操作方法简便、获得结果快速等;④应用VIGS对植物抗病途径、代谢与发育调控中参与基因功能进行研究的概况。同时。展望了VIGS技术作为植物功能基因组学功能分析的高通量技术平台的美好前景。     

滇池流域富磷山地优势植物种群生态分化

为了解植物在富磷山地条件下的生态分化情况,本试验以滇池流域柴河子流域山地富磷区4种常见优势植物(紫茎泽兰Eupatorium adenophorum、戟叶酸模Rumex hastatus、蔗茅Saccharum rufipilum、马桑Coriaria sinica)为研究对象,分别对采自不同土壤全磷条件下4种植物各6个种群进行了同质园栽培试验,通过测量其表型性状,并采用单因素方差分析、变异系数分析和相关性分析等统计方法,分析了4种植物各种群的表型变异情况。结果表明:4种植物不同种群间在株高、叶面积、比叶面积和叶干物质量上存在显著差异,同种植物不同种群同一表型性状上变异系数差别较大,不同表型性状变异幅度也不相同。蔗茅、戟叶酸模、紫茎泽兰和马桑各种群表型性状的变异平均有24.01%、4.58%、16.80%和9.42%来源于种群间,59.86%、66.17%、63.15%和68.51%的变异存在于各种群内部。这说明,富磷区山地优势植物对土壤异质环境的适应不仅仅只是表型上的响应,已出现一定的遗传分化。     

青藏高原特有属——固沙草属表型变异及其对环境因子的响应

该研究通过测量固沙草属(Orinus)3个物种(青海固沙草、固沙草和居间固沙草)的40个天然居群145个个体的22个表型性状,依据VIF膨胀系数筛选得到年平均降雨(MAP)等9个气候因子和海拔等因子,并采用冗余分析(db-RDA)考察不同物种、不同居群植物表型多样性与多元地理气候环境的关系,探讨该属植物表型多样性驱动机理。结果显示:(1)基于22个植物表型性状,固沙草属天然居群在组内最小方差(Ward)55时可归为2类,居群间表型性状不存在显著的空间自相关性(P 0.05),植物表型多样性高(Shannon-Wiener index,H:1.045～2.734),17个定量的表型性状变异系数(CV)平均值47.84%,而居群间小穗颜色多样性最低(H:0.170),证明固沙草属植物表型性状具有独特的环境响应模式。(2)巢式方差分析显示,固沙草属植物表型在居群间和居群内均存在丰富的变异(F 10,P0.01),旗叶、颖片和外稃分别是主成分分析中表型总变异的重要组成部分,表型分化系数居群间变异(71.10%)大于居群内(28.90%),固沙草属植物表型变异来源于居群间。(3)冗余分析结果表明,海拔(ALT)和年平均风速(Wind)是决定青海固沙草(O.kokonoricus)和固沙草(O.thoroldii)居群间表型差异性的主要影响因子;极端气候条件[最冷季平均温度(Bio9)和最干季降雨量(Bio14)]对固沙草居群间表型相似性起促进作用,相反干燥度指数(AI)与水气压(Vapr)对其差异性起促进作用;居间固沙草(O.intermedius)居群间表型差异受年均降雨(MAP)、潜在蒸散(PET)和水气压(Vapr)影响。研究认为,固沙草属植物天然居群之间表型变异反映出明显的地理气候变化趋势,这些表型变异是高山植物环境适应性的重要标志。     

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。     

中国转基因植物安全管理走上法制化轨道转基因抗虫棉进入商品化生产阶段

自１９８３年世界上第一例转基因植物问世,１９８６年美国第一例转基因植物进入田间实验,１９９４年世界第一种转基因植物获准商品化生产以来,世界生物技术发展十分迅速,中国也不例外。由于目前的科学技术水平不能精确地预测转基因可能产生的所有表型效应,故需要对基...     

植物遗传转化中存在的问题及对策

自 1 98 4年转基因植物问世以来 ,经过 1 0多年的发展 ,转基因技术已在近 2 0 0种植物中获得成功[1] 。转基因植物在提高植物的农业和园艺价值 ,作为某些重要蛋白质和次生代谢产物的廉价生物反应器 ,以及研究基因在发育和其它生理生化过程与代谢途径中的作用等方面 ,均充当了核心角色。目前 ,科学家们研究的兴趣已不再停留在简单地将基因导入植物中。遗传转化研究更多地转向转化的高效性、可预测性、稳定性和安全性等方面。本文就目前植物遗传转化中存在的一些主要问题及对策作一些介绍并提出我们的看法。1　组织培养中的体细胞变异　　目前…     

Phenotypic and genotypic characterization of antagonistic bacteria associated with roots of transgenic and non-transgenic potato plants

Rhizobacteria obtained during a risk assessment study from parental and transgenic T4 lysozyme-expressing potato plants were investigated to determine whether or not the strains could be grouped based on the source of isolation, transgenic or non-transgenic plants, respectively. A total of 68 representative bacterial strains of the group of enterics and pseudomonads were investigated by phenotypic profiling (the antagonistic activity towards bacterial and fungal plant pathogens, the production of the plant growth hormone indole-3-acetic acid [auxin], and the sensitivity to T4 lysozyme in vitro) and genotypic profiling by PCR fingerprints using BOX primers. All isolates were identified by fatty acid methyl ester (FAME) analysis. Computer-based analysis of the phenotypic characteristics showed that both, enterics and Pseudomonas strains clustered into six to seven groups at an Euclidian distance of 10. According to their BOX-PCR-generated fingerprints the Pseudomonas strains clustered into seven groups and the enterobacteria into two groups at the same genetic distance level of 10. The majority of groups were heterogeneous and contained isolates from all plant lines. In conclusion, cluster analysis of the phenotypic and genotypic features did not reveal correlations between bacterial isolates and transgenic character of plants.     

Expression of MsLEC1- and MsLEC2-antisense genes in alfalfa plant lines causes severe embryogenic, developmental and reproductive abnormalities

Although it has been proposed that plant lectins play a number of roles, the function of these proteins in normal plant growth and development has been unclear. To analyze the functions of putative alfalfa lectin genes, lines of transgenic alfalfa plants expressing approximately half of the open reading frame of MsLEC1 or MsLEC2, in the antisense or sense orientation, were established and analyzed. The antisense plants displayed severe abnormalities in embryogenesis, and both vegetative and reproductive development were perturbed. Some differences were observed between MsLEC1- and MsLEC2-antisense plants, and abnormalities were especially severe during the early stages of development in both the primary and secondary transgenic generations. In contrast, vector-control and sense-transgene plants exhibited normal growth and development. MsLEC1 and MsLEC2 mRNA accumulation levels were reduced in cognate antisense plants, especially during the later stages of embryogenesis, but also tended to be low in MsLEC1 sense-transgene plants. However, correlated with the phenotypic abnormalities observed in the MsLEC1-antisense plants was the specific reduction in the accumulation of a candidate MsLEC1 protein. Our results suggest that the MsLEC1 and MsLEC2 gene products, in addition to being important for embryogenesis, are required throughout alfalfa development.     

Transgenic tobacco plants expressing the geminivirus BL1 protein exhibit symptoms of viral disease.

Bipartite geminiviruses, such as squash leaf curl virus (SqLCV), encode two movement proteins (MPs), BR1 and BL1, that are essential for viral movement in and subsequent infection of the host plant. To elucidate the biochemical functions of these MPs and define their respective contributions to viral infection, we have generated transgenic Nicotiana benthamiana plants expressing SqLCV BR1 and BL1. Transgenic plants expressing BR1 or a truncated BL1 were phenotypically indistinguishable from wild-type N. benthamiana. In contrast, transgenic plants expressing full-length BL1, alone or in combination with BR1, were strikingly abnormal both in their growth properties and phenotypic appearance, with leaves that were mosaic and curled under, thus mimicking typical SqLCV disease symptoms in this host. BL1 was localized to the cell wall and plasma membrane fractions, whereas BR1 was predominantly in the microsomal membrane fraction. These findings demonstrate that expression of BL1 in transgenic plants is sufficient to produce viral disease symptoms, and they further suggest that BL1 and BR1 carry out distinct and independent functions in viral movement.     

Wheat LEA genes, PMA80 and PMA1959, enhance dehydration tolerance of transgenic rice (Oryza sativa L.)

Drought and salt stresses are two major factors that lower plant productivity. Transgenic approaches offer powerful means to better understand and then minimize loss of yield due to these abiotic stresses. In this study, we have generated transgenic rice plants expressing a wheat LEA group 2 protein (PMA80) gene, and separately the wheat LEA group 1 protein (PMA1959) gene. Molecular analysis of the transgenic plants revealed the stable integration of the transgenes. Immunoblot analysis showed the presence of the LEA group 2 protein (39 kDa) and the LEA group 1 protein (25 kDa) in most of the plant lines. Second-generation transgenic plants were subjected to dehydration or salt stress. The results showed that accumulation of either PMA80 or PMA1959 correlates with increased tolerance of transgenic rice plants to these stresses.     

Excision of the maize transposable element Ac in periclinal chimeric leaves of 35S-Ac-rolC transgenic aspen-Populus

The transposable element Ac from maize, in combination with the phenotypic selectable marker rolC, was employed in transformation experiments of a hybrid aspen clone. A number of transgenic clones exhibited light-green sectors on green leaves. In vitro regeneration from leaves showing a high number of light-green spots resulted in R2 plants, which also showed light-green sectored leaves. However, only one out of 385 regenerated plants obtained showed green leaves. Both PCR and northern analysis indicated Ac excision and restoration of rolC expression. In Southern blot analysis of this green plant additional bands were observed as compared to the original R1 plant. The occurrence of these bands and a suggested Ac excision in the non-green L1-epidermal layer leading to periclinal chimerism of this plant is discussed.     

Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth

Rice (Oryza sativa), a monocotyledonous plant that does not cold acclimate, has evolved differently from Arabidopsis (Arabidopsis thaliana), which cold acclimates. To understand the stress response of rice in comparison with that of Arabidopsis, we developed transgenic rice plants that constitutively expressed CBF3/DREB1A (CBF3) and ABF3, Arabidopsis genes that function in abscisic acid-independent and abscisic acid-dependent stress-response pathways, respectively. CBF3 in transgenic rice elevated tolerance to drought and high salinity, and produced relatively low levels of tolerance to low-temperature exposure. These data were in direct contrast to CBF3 in Arabidopsis, which is known to function primarily to enhance freezing tolerance. ABF3 in transgenic rice increased tolerance to drought stress alone. By using the 60 K Rice Whole Genome Microarray and RNA gel-blot analyses, we identified 12 and 7 target genes that were activated in transgenic rice plants by CBF3 and ABF3, respectively, which appear to render the corresponding plants acclimated for stress conditions. The target genes together with 13 and 27 additional genes are induced further upon exposure to drought stress, consequently making the transgenic plants more tolerant to stress conditions. Interestingly, our transgenic plants exhibited neither growth inhibition nor visible phenotypic alterations despite constitutive expression of the CBF3 or ABF3, unlike the results previously obtained from Arabidopsis where transgenic plants were stunted.