博文

Nature Plants：作物中长末端重复序列LTR反转录转座子鉴定方法

已有 7268 次阅读 2018-12-12 09:10 |个人分类:每日摘要|系统分类:论文交流

Sensitive detection of pre-integration intermediates of long terminal repeat retrotransposons in crop plants

First author: Jungnam Cho; Affiliations: University of Cambridge (剑桥大学): Cambridge, UK

Corresponding author: Jerzy Paszkowski

Retrotransposons have played an important role in the evolution of host genomes. Their impact is mainly deduced from the composition of DNA sequences that have been fixed over evolutionary time. Such studies provide important ‘snapshots’ reflecting the historical activities of transposons but do not predict current transposition potential. We previously reported sequence-independent retrotransposon trapping (SIRT) as a method that, by identification of extrachromosomal linear DNA (eclDNA), revealed the presence of active long terminal repeat (LTR) retrotransposons in Arabidopsis. However, SIRT cannot be applied to large and transposon-rich genomes, as found in crop plants. We have developed an alternative approach named ALE-seq (amplification of LTR of eclDNAs followed by sequencing) for such situations. ALE-seq reveals sequences of 5′ LTRs of eclDNAs after two-step amplification: in vitro transcription and subsequent reverse transcription. Using ALE-seq in rice, we detected eclDNAs for a novel Copia family LTR retrotransposon, Go-on, which is activated by heat stress. Sequencing of rice accessions revealed that Go-on has preferentially accumulated in Oryza sativa ssp. indica rice grown at higher temperatures. Furthermore, ALE-seq applied to tomato fruits identified a developmentally regulated Gypsy family of retrotransposons. A bioinformatic pipeline adapted for ALE-seq data analyses is used for the direct and reference-free annotation of new, active retroelements. This pipeline allows assessment of LTR retrotransposon activities in organisms for which genomic sequences and/or reference genomes are either unavailable or of low quality.

反转录转座子在寄主基因组的演化过程中扮演了十分重要的作用。我们一般只能通过现有的DNA序列组成推测反转录转座子对于基因组演化的影响，但这种影响可能在历史长流中已经被修复了。这些研究会鉴定出一些反应转座子历史上发挥作用的事件，但并不能预测现在的潜在转座活性。作者之前报道了一种不依赖于序列的反转录转座子鉴定方法SIRT，即通过鉴定染色体外线性DNA（eclDNA）来揭示拟南芥中现存的有活性的长末端重复序列LTR反转录转座子。然而，SIRT并不适合较大、富含转座子的基因组，比如说某些作物的基因组。本文中，作者又开发了一套适合这种情况的新方法，叫做ALE-seq。ALE-seq通过两步扩增法揭示eclDNA的5′ LTR：体外转录和反转录。作者在水稻中借助于ALE-seq检测到了一个新的Copia家族LTR反转录转座子Go-on，这一类LTR反转录转座子会被高温胁迫所激活。通过对水稻不同品种的测序显示Go-on在相对生长在温度更高的籼稻中优先积累。此外，作者在番茄果实中应用ALE-seq鉴定了一个发育调控的Gypsy家族LTR反转录转座子。作者构建了一套适用于ALE-seq数据分析的生物信息操作流程，可用于注释无参考信息的新的活性反转录元件。该流程无论有无参考基因组或参考基因组质量好坏都可以对LTR反转录转座子活性进行评估。