植物安全转基因技术研究现状与展望

转基因技术是进行基因功能研究和作物遗传改良的重要工具。根癌农杆菌介导转化法和基因枪轰击法是两种主要的遗传转化方法。从1996年首例转基因作物商业化种植以来，2012年全球有28个国家和地区种植转基因作物，种植面积已达1.7亿公顷。随着全球转基因作物的大面积商业化种植，转基因植物的安全性越来越多地受到公众关注。安全转基因技术的研发对于转基因植物的商业化生产具有重要意义。文章详细介绍了目前已报道的安全转基因技术原理及其应用现状。按其作用原理和目的将其分为4类：安全选择标记、标记基因删除及基因叠加、基因漂移防控法和基因定点编辑整合技术。其中，安全选择标记按其筛选原理分为糖代谢相关标记、氨基酸代谢相关标记、激素相关标记和抗逆相关标记。与抗生素和除草剂抗性标记相比，这类标记基因及其表达产物对人和其他生物更安全。标记基因删除及基因叠加技术包括共转化法、位点特异性重组法、转座子法、同源重组法以及基于位点特异性重组的基因叠加技术，其中共转化法又包括农杆菌介导的共转化法和基因枪介导的表达盒共转化法。基因叠加技术为复合性状转基因植物研发奠定了基础，有望成为未来多性状转基因植物研发的重要技术之一。基因漂流防控法包括叶绿体转化法和基因拆分法。基因定点编辑和整合技术包括锌指核酸酶、TALEN和CRISPR/Cas9系统介导的基因定点编辑和整合技术。其中，TALEN和CRISPR/Cas9技术，设计简单，成本低，易操作，靶点分布广泛，有望成为安全转基因研发和应用的重要技术。文章对这些技术原理及其应用现状进行了综述，讨论了其优缺点，并展望了安全转基因技术研发重点和发展趋势。

Current Status of Transgenic Technologies for Safety Consideration in Plants and Future Perspectives

Genetic modification (GM) facilitates research into fundamental questions of plant functional genomics and provides a route for developing novel commercial varieties. Agrobacterium-mediated transformation and microparticle bombardment are two widely used methods for plants genetic transformation. Since the first commercial planting of GM crop in 1996, the planting area of GM crops has reached 170 million hectares in 28 countries and regions in 2012. Along with the increased commercialization and planting of a range of GM crops globally, the biosafety of GM crops has become a hot topic and major public concern. Development of the precise and marker-free transgenic technologies would be essential for the commercialization of transgenic crops. In this article, current development status of biosafe and precise transgenic technologies such as the use of selective markers avoidable of biosafety concerns, marker gene deletion and gene pyramiding, the method of control transgene flow as well as target genome editing and integration technologies are reviewed thoroughly. According to the selection principle, four classifications of biosafe selective marker genes have been explored so far such as marker genes related to carbohydrates metabolism, amino acids metabolism, auxins metabolism and abiotic stresses. Compared with antibiotic and herbicide resistant marker genes, the use of these marker genes and their products may not raise any biosafety concerns. Marker gene deletion and gene pyramiding includes co-transformation, site-specific recombination, transposon, intrachromosomal homologous recombination and gene stacking-based site-specific recombination. Among them, co-transformation includes Agrobacterium-mediated co-transformation and microparticle bombardment mediated co-transformation of gene expression cassettes. Gene deletion and stacking technology is essential for production of GM crops with improved complex traits, and gene stacking technology based on site-specific recombination system is expected to become an important technology for producing GM crops with multiple transgenes. The methods for control of transgene flow include chloroplast transformation and transgene split system. Target genome editing and integration technologies include ZFNs, TALEN and CRISPR/Cas9 mediated technologies. Among these three technologies, TALEN and CRISPR/Cas9 technologies are expected to become a powerful tool in future because of their advantages of simple design and operation, low cost and wide range of targets existing in plant genome. At last, future perspectives and applications of these transgenic technologies for safety consideration in agricultural practice are proposed.