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随着拟南芥、水稻等模式植物基因组测序计划的全面完成,数据库中大量的DNA序列需要进行功能注释,而用传统的正向遗传学进行基因克隆和近年来发展的反向遗传学(如插入突变、反义RNA、RNAi等技术)方法已不能适应基因组学的发展需求,因此,研发大规模、高通量的基因功能分析方法成为当务之急。TILLING技术(Targetinginducedlocallesionsingenomes)就是在基因组生物学大背景下出现的一种全新的反向遗传学技术。TILLING技术的基本步骤是通过化学诱变方法产生一系列点突变,经过PCR扩增放大和变性复性过程产生异源双链DNA分子,再通过特异性酶切和双色电泳分析识别异源双链中错配碱基,从而检测出突变发生的准确位置。由于具有高通量、大规模、高灵敏度和自动化等特点,能够适应植物功能基因组学研究的要求,TILLING技术已经和即将在功能基因组领域发挥越来越重要的作用。TILLING技术应用于已测序完成的拟南芥和水稻中的突变位点检测并取得了巨大成功;TILLING技术应用于农作物的品种改良,可以帮助实现快速、定向改良作物的品种,同时由于TILLING采用的化学诱变技术与传统诱变育种并无二致,因此在作物改良中采用TILLING技术不存在外源基因转入引发的转基因作物(GMO)争论;由TILLING技术发展来的EcoTILLING技术,具有通量高、成本低、定位准确等优点,可以很好地进行多态性检测和研究基因的功能,已成为开展物种DNA多态性检测和不同物种演替进化研究的有力工具。本文简要介绍了TILLING的原理及操作步骤,讨论了TILLING技术的特点和优点及TILLING技术的应用前景。
With the complete sequencing of plant genomes such as Arabidopsis and rice, a large number of DNA sequences in the database need to be functionally annotated. However, the use of traditional forward genetics for gene cloning and reverse genetics developed in recent years Insert mutation, antisense RNA, RNAi and other technologies) methods can no longer meet the needs of the development of genomics, therefore, the development of large-scale, high-throughput methods of gene function analysis has become a top priority. TILLING technology (Targetinginducedlocallesionsingenomes) is a brand new reverse genetics technology in the background of genomics. The basic steps of TILLING technology is to generate a series of point mutations through chemical mutagenesis. After amplification and denaturation by PCR amplification, heteroduplexes generate double-stranded DNA molecules, and then specific heteroduplexes In the mismatch base, in order to detect the exact location of the mutation occurred. Due to its high throughput, large-scale, high sensitivity and automation, TILLING technology has been and will soon play an increasingly important role in the field of functional genomics. TILLING technology has been used successfully in the detection of the mutated sites in Arabidopsis and rice that have been sequenced. TILLING technology has been applied to the improvement of crop varieties, which can help to improve crop varieties quickly and directionally. At the same time, Chemical mutagenesis is the same as conventional mutagenesis. Therefore, there is no controversy over the use of TILLING in crop improvement without the introduction of GMO. The EcoTILLING technology developed by TILLING has the following advantages: High, low cost, accurate positioning, etc., which can well perform the function of polymorphism detection and gene research. It has become a powerful tool for the study of species DNA polymorphism and evolutionary evolution of different species. This article briefly introduces the principle and operation steps of TILLING, discusses the features and advantages of TILLING technology and the application prospect of TILLING technology.