Chen Kunling, Shan Qiwei, Gao Caixia
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
Methods. 2014 Aug 15;69(1):2-8. doi: 10.1016/j.ymeth.2014.02.013. Epub 2014 Feb 17.
Targeted gene mutagenesis is a powerful tool for elucidating gene function and facilitating genetic improvement in rice. TALENs (transcription activator-like effector nucleases), consisting of a custom TALE DNA binding domain fused to a nonspecific FokI cleavage domain, are one of the most efficient genome engineering methods developed to date. The technology of TALENs allows DNA double-strand breaks (DSBs) to be introduced into predetermined chromosomal loci. DSBs trigger DNA repair mechanisms and can result in loss of gene function by error-prone non-homologous end joining (NHEJ), or they can be exploited to modify gene function or activity by precise homologous recombination (HR). In this paper, we describe a detailed protocol for constructing TALEN expression vectors, assessing nuclease activities in vivo using rice protoplast-based assays, generating and introducing TALEN DNAs into embryogenic calluses of rice and identifying TALEN-generated mutations at targeted genomic sites. Using these methods, T0 rice plants resulting from TALEN mutagenesis can be produced within 4-5 months.
靶向基因诱变是阐明水稻基因功能和促进其遗传改良的有力工具。转录激活样效应物核酸酶（TALENs）由一个定制的TALE DNA结合结构域与一个非特异性FokI切割结构域融合而成，是迄今为止开发的最有效的基因组工程方法之一。TALEN技术可将DNA双链断裂（DSB）引入预定的染色体位点。DSB触发DNA修复机制，可能通过易出错的非同源末端连接（NHEJ）导致基因功能丧失，或者可以通过精确的同源重组（HR）来利用它们修饰基因功能或活性。在本文中，我们描述了构建TALEN表达载体、使用基于水稻原生质体的分析方法在体内评估核酸酶活性、将TALEN DNA生成并导入水稻胚性愈伤组织以及在靶向基因组位点鉴定TALEN诱导的突变的详细方案。使用这些方法，可在4至5个月内产生由TALEN诱变产生的T0代水稻植株。
