中文摘要：植物的稳定转化是假设检验的有力工具。在分析这些转化时，转基因拷贝数和纯合性的快速可靠评价方法十分重要。本文比较了Southern杂交分析、热不对称交错PCR（TAIL PCR）、定量PCR（qPCR）和数字微滴PCR（ddPCR）四种分析方法对评估转基因烟草的T-DNA拷贝数、轨迹的复杂性和纯合性中的适用性。研究发现，在Southern杂交分析和ddPCR分析中，转基因后代的纯合性和拷贝数是完全一致的，而TAIL PCR往往低估拷贝数；qPCR偏离了Southern杂交分析的结果，且相对于ddPCR来说，有较低的精度和较高的变异性。研究结果表明，最新开发的ddPCR是最合适的方法，因为其与Southern杂交分析的可靠性一致，但速度更快。另外，本文还提供了ddPCR的应用协议以证明其功效。
外文摘要："Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL-)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T-DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL-PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T-1 progeny from 26 T-0 plants showed that at least 19% of the lines carried multiple T-DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T-DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided.Genetic transformation is being used increasingly in the public domain to test a range of hypotheses concerning gene action, not only in Arabidopsis, but now in a broad range of plants. A major challenge though, particularly with species with relatively long life cycles, is in identifying individuals that are homozygous for the insert or DNA modification at T2, which is necessary to provide homozygous lines. Southern blotting has been the traditional approach, but it is slow and requires considerable skill. Various PCR methods have been used to accelerate testing, but have not been as reliable. However, we show here that the recently developed digital droplet PCR is as effective as Southern blotting, yet faster and capable of high-throughput. A protocol for application of ddPCR is provided together with evidence of its efficacy."
外文关键词：ddPCR；digital droplet PCR；qPCR；segregation analysis；selectable marker；Southern blot；TAIL-PCR；transformation
作者：Glowacka, Katarzyna；Kromdijk, Johannes；Leonelli, Lauriebeth；等.
作者单位：伊利诺伊大学
期刊名称：PLANT CELL AND ENVIRONMENT
期刊影响因子：6.169
出版年份：2016
出版刊次：4
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