Cre/loxP定位重组系统在植物雄不育和杂种优势中的利用研究

Cre—loxP是来源于噬菌体P1的一种位点特异性重组系统。目前，它已广泛应用于基因功能鉴定，动植物及微生物基因组的修饰以及基因的表达和调控。雄性不育系在杂种优势的利用中具有重要意义。本实验是以Cre—loxP系统调控细胞毒素基因barnase在植物雄性器官发育的特异时期在特异组织表达，从而达到控制植物育性的目的。1．在本实验中，通过PCR方法克隆了解淀粉芽孢杆菌Bacillus amyloliquefaciens的barnase基因，及其抑制因子barstar的DNA序列，同时，构建成功双元表达载体及基因枪转化载体，并已获得经分子险测呈阳性的小麦植株。2．osg6B是来源于水稻花药绒毡层的特异性的启动子，它在小孢子发育的四分体时期至单核花粉期在花药绒毡层中特异启动基因的表达，它具有较为严紧的时空特异性。以osg6B为启动子，构建了一组受控于Cre／loxP位点特异性重组系统的适用于基因枪法转化小麦的载体系统。其中，转不育基因(og6B—barnase)小麦植株生长正常，但开花后，花粉量明显减少，自交不能得到正常种子。进行花粉活力检测发现，转基因小麦大部分花粉表现为败育。花粉离体萌发试验表明，转不育基因小麦的花粉离体萌发率极低，几近败育。说明以osg6B驱动barnase在小麦花药中的表达可以导致雄性不育。3．以osg6B为启动子，构建了一组受控于Cre／loxP的用于控制双子叶植物育性的双元表达载体系统。以转cre基因的烟草作为受体，以含阻断序列的质粒农杆菌进行二次转化。二次转化株营养生长正常，但开花期花器官表现异常。具体表现为花瓣异常开裂或不开裂，花丝变短或粘连，花药提早萎蔫或异常开裂。推测这种现象的出现与启动子的组织特异性或barnase的渗露表达有关。4．二次转化株花粉染色因不同株系出现全部败育、部分不育和多数可育的情况。花粉离体萌发实验也得到了相似的结果。这说明通过筛选，利用Cre／loxP得到完全不育的植物株系是完全可行的。5．获得了9个barnase的突变体，初步推测第十四位氨基酸及第八十位氨基酸与核糖核酸酶活性有关。同时，发现barnase的第316位核苷酸序列为易突变位点，自然发生的突变多插入一个C，从而产生终止位点的改变。这或许会为今后barnase的克隆与利用提供一些参考。     

The Application of TDZ in Enhancing Regeneration of Transgenic Plants

At present, transgenic plants are globally grown. Availability of a reliable regeneration system predominantly from a single transformed cell is the prerequisites for gene transfer, but regeneration is still a key problem （Wenzel, 2006）. The application of genetic modification technology in plants is closely related to the efficiency of the regeneration protocol. Shoot formation is oilen enhanced by the combination of auxins and cytokinins. TDZ （Thidiazuron, N-phenyl-N＇-1,2,3-thiodiazol-5-ylurea）, a non-purine, has been shown to promote differentiation of organized centers of growth in cultured tissues at much lower concentrations, and shoot regeneration occurs with an efficiency comparable to or greater than that of other cytokinins （Fiola et al., 1990）. By addition of TDZ, a series of plants which were difficult to culture in vitro or less sensitive to plants growth regulators obtained somatic embryos and regenerated plants, some of them have become the eminent transformation system for genetic engineering. TDZ has been reported to be very efficient in stimulating adventitious shoot production in several recalcitrant plants. TDZ is considered as a potential growth regulator for in vitro shoot regeneration and somatic embryogenesis of several crops. This review summarized how the new growth regulator TDZ to affect the regeneration of transgenic plants.     

The Experimental and Commercial Release of Transgenic Crop Plants

With advances in recombinant DNA methods and transformation procedures, it is possible to transfer genes into crop plants from unrelated plants, microbes and animals. Many of the modifications being carried out, or envisaged, are for disease and pest resistance, product quality and tolerance to environmental stress, but there are additional opportunities to modify crops to give specialized products for industrial or pharmaceutical use. Some of the characteristics of transgenic plants are considered, including: transgene copy number, position, expression, stability, pleiotropy, selectable marker genes and somaclonal variation. There have been several hundreds of field trials with transgenic plants, and the first transgenic varieties are likely to be approved for commercial production in 1993. Before releasing transgenic plants, it is necessary to carry out a risk assessment to determine whether the transgenic variety will behave differently from a conventionally bred variety. Assessment procedures are being harmonized internationally by various organizations. There is a growing commitment to apply these genetic modification methods to crops in developing countries, as genes relevant to their crops and environments become available.     

转基因植物对农业、农民和生态发展的意义

转基因植物商业化生产10年以来,尽管遇到各种各样的阻力,仍以前所未有的速度发展。长期积累的数据表明,在经济利益上,采用转基因植物的农民获得了显著的经济效益。由于采用转基因植物,农药特别是高毒农药使用量减少,减少了农田环境和水环境的化学毒素毒物的残留。由于转基因抗虫植物虫害减少,在粮食产品中有害微生物及其毒素毒物残留大大降低。这表明,在现有技术基础上,转基因植物商业化有利于农民生活质量的提高和环境的改良。     

转基因抗虫棉Bt基因的遗传连锁分析

对中美两国所获得的转基因抗虫棉品种GK— 1 2、新棉 33B分别进行了 Bt基因的连锁测验。分析表明两个品种的试验结果相似 ,Bt基因未整合在棉花第 I、II、III、IV、V、VI、VII、IX、X、XI、XII、XIII、XVII连锁群及 cu、pg、gl1等基因所涉及的染色体上。阐明了 Bt基因染色体图谱定位的复杂性 ,并提出以原位杂交技术实现 Bt基因在棉花染色体组上的物理标图     

利用卡那霉素间接鉴定转基因抗虫棉的研究

研究了利用卡那霉素间接鉴定转基因抗虫棉的方法。结果表明,在棉花苗期使用8000mg/kg卡那霉素溶液处理子叶,或者在蕾期使用6000mg/kg卡那霉素溶液处理棉株顶部第2片展开叶,7～9d后非转基因棉株的叶片点液处出现黄色斑块,而转基因抗虫棉的叶片仍为正常绿色,从而简便有效地鉴别出转基因抗虫棉。经网室接虫测试和室内饲养生物学测试方法验证,该方法结果准确。     

密度与施钾量对转基因抗虫棉产量的影响

在田间试验条件下,研究了密度与施钾量对转基因抗虫棉DPH37B产量的影响。结果表明:在一定范围内,随着密度和施钾量的增加,DPH37B皮棉产量呈上升趋势;但当密度超过37 500株/hm2、施钾量超过450 kg/hm2时,皮棉产量反而下降。试验还发现,适当的密度和施钾量能增加单株果枝数和果节数;密度和施钾量对纤维长度、衣分的影响较小,无明显规律,但对单铃重有影响。密度和施钾肥有互作现象,在密度30 000~37 500株/hm2和施钾量450 kg/hm2的水平下,DPH37B产量最高。     

转基因玉米研究进展

植物转基因技术开辟了作物遗传改良的新途径。转基因玉米是植物基因工程领域研究的热点之一。近年来,应用转基因技术已经获得抗虫、抗除草剂和其他有用性状的转基因玉米。在此,就玉米转基因在受体系统、目的基因及转化方法方面的研究成果,尤其是近年来玉米转基因研究方面存在的问题、解决途径以及其他类型玉米的转基因研究进展做了综述。     

抗除草剂转基因水稻及其生物安全性的探讨

抗除草剂转基因水稻在转基因水稻的研究和利用中占主导地位,其安全性问题也成为人们关注的焦点。文章简述了稻田中主要使用的几种除草剂及其作用机理,水稻抗除草剂基因的转化方法及抗性机理,讨论了抗除草剂转基因水稻的应用及生物安全性问题,并对今后的研究与应用进行了展望。     

Traditional Mexican Agricultural Systems and the Potential Impacts of Transgenic Varieties on Maize Diversity

The discovery of transgenes in maize landraces in Mexico, a center of diversity for this crop, raises questions about the potential impact of transgene diffusion on maize diversity. The concept of diversity and farmers’ role in maintaining diversity is quite complex. Farmers’ behavior is expected to have a significant influence on causing transgenes to diffuse, to be expressed differently, and to accumulate within landraces. Farmers’ or consumers’ perceptions that transgenes are “contaminants” and that landraces containing transgenes are “contaminated” could cause these landraces to be rejected and trigger a direct loss of diversity. Mauricio R. Bellon is a human ecologist working in the Economics Program of the International Maize and Wheat Improvement Center (CIMMYT) in Texcoco, Mexico. He received his MSc and PhD in ecology at the University of California, Davis. His current research includes projects that deal with on-farm conservation of maize, gene flow in traditional farming systems, and the impact of improved germplasm in the livelihoods of poor farmers. Julien Berthaud is a population geneticist working for the Institut de Recherche pour le Développement (IRD). He received his PhD in plant science at the University of Paris 11. His current research includes projects related with the dynamics of genetic diversity, especially in traditional maize farming systems.