Srivastava Vibha
Department of Crop, Soil & Environmental Sciences, University of Arkansas, Fayetteville, AR, USA.
Department of Horticulture, University of Arkansas, Fayetteville, AR, USA.
Methods Mol Biol. 2019;1864:267-277. doi: 10.1007/978-1-4939-8778-8_18.
Biotechnology methods for inserting genes one by one or as a block of fragment into plant genomes are needed to introduce valuable traits into crop varieties. Insertion of multiple genes into a single site, called as molecular stacking, is important to allow co-inheritance of the genes into the progeny. Generally, two approaches are available for creating gene stacks: nuclease-induced targeted gene integration into native sites and recombinase-mediated gene integration into the engineered sites. The recombinase application is attractive as several recombinases show high efficiency and precision in plant genomes. This chapter describes a gene stacking method based on the use of Cre-lox site-specific recombination system to integrate genes into the engineered sites and nucleases to delete selection genes leading to stacking of traits into a single genomic site. High efficiency and precision, and undetectable off-target effects of Cre-lox in a number of plant species, make it an attractive tool for complex applications such as gene stacking.
为了将有价值的性状引入作物品种，需要采用生物技术方法将基因逐个或作为片段块插入植物基因组。将多个基因插入单个位点，即所谓的分子堆叠，对于使这些基因共同遗传给后代很重要。一般来说，创建基因堆叠有两种方法：核酸酶诱导的靶向基因整合到天然位点以及重组酶介导的基因整合到工程化位点。重组酶的应用很有吸引力，因为几种重组酶在植物基因组中显示出高效率和精确性。本章描述了一种基于使用Cre-lox位点特异性重组系统将基因整合到工程化位点以及使用核酸酶删除选择基因从而将性状堆叠到单个基因组位点的基因堆叠方法。Cre-lox在许多植物物种中具有高效率、精确性以及不可检测的脱靶效应，使其成为基因堆叠等复杂应用的有吸引力的工具。
