基因表达通常通过调节转录物产生的顺式调节元件(CREs)来控制。对于多基因基因工程和合成生物学,精确控制转录至关重要,既可以使转基因与不需要的天然调控隔离,又可以防止多基因盒内转基因的连读或交叉调控。为了防止这种活动,类似绝缘体的元件,更恰当地称为转录阻断剂,可以插入以分离转基因,以便它们被独立调节。然而,只有少数经过验证的类似绝缘体的元素可用于植物,它们往往比理想的要大。
■为了识别额外的潜在的类似绝缘子的序列,我们对GibbaUtricularia(驼峰叶草)进行了全基因组分析,已知最小的植物基因组之一,与自然紧密相连的基因。在体内评估了10种最佳的类似绝缘体的候选物的类似绝缘体的活性。
■我们确定了总共4,656个基因间区域,其表达谱表明具有类似绝缘子的活性。这些地区与其他45种植物物种的比较(代表单子叶植物,星号,和Rosids)显示这些区域的同伦保守水平较低。对未甲基化区域(UMR)的全基因组分析表明,约87%的靶向区域未甲基化;然而,对此的解释很复杂,因为U.gibba在整个基因组中的甲基化水平非常低,因此,大型UMR经常在多个基因和基因间空间上延伸。我们也无法在选定的基因间区域中找到任何保守的基序,也无法与现有的植物类绝缘体元素共享。尽管缺乏保护,然而,对10个选定的基因间区域进行绝缘体样活动的测试发现,有两个元素与先前发布的元素(EXOB)相当,但要小得多。
■鉴于目前可用于植物的类似绝缘子的元件数量很少,我们的结果是对现有工具的重要补充.高命中率(10个中的2个)还意味着在我们选择的基因间区域中可能存在更有用的序列;将需要额外的验证工作来鉴定哪些对植物基因工程最有用。
UNASSIGNED: Gene expression is often controlled via cis-regulatory elements (CREs) that modulate the production of transcripts. For multi-gene genetic engineering and synthetic biology, precise control of transcription is crucial, both to insulate the transgenes from unwanted native regulation and to prevent readthrough or cross-regulation of transgenes within a multi-gene cassette. To prevent this activity,
insulator-like elements, more properly referred to as transcriptional blockers, could be inserted to separate the transgenes so that they are independently regulated. However, only a few validated
insulator-like elements are available for plants, and they tend to be larger than ideal.
UNASSIGNED: To identify additional potential insulator-like sequences, we conducted a genome-wide analysis of Utricularia gibba (humped bladderwort), one of the smallest known plant genomes, with genes that are naturally close together. The 10 best
insulator-like candidates were evaluated in vivo for
insulator-like activity.
UNASSIGNED: We identified a total of 4,656 intergenic regions with expression profiles suggesting insulator-like activity. Comparisons of these regions across 45 other plant species (representing Monocots, Asterids, and Rosids) show low levels of syntenic conservation of these regions. Genome-wide analysis of unmethylated regions (UMRs) indicates ~87% of the targeted regions are unmethylated; however, interpretation of this is complicated because U. gibba has remarkably low levels of methylation across the genome, so that large UMRs frequently extend over multiple genes and intergenic spaces. We also could not identify any conserved motifs among our selected intergenic regions or shared with existing insulator-like elements for plants. Despite this lack of conservation, however, testing of 10 selected intergenic regions for
insulator-like activity found two elements on par with a previously published element (EXOB) while being significantly smaller.
UNASSIGNED: Given the small number of
insulator-like elements currently available for plants, our results make a significant addition to available tools. The high hit rate (2 out of 10) also implies that more useful sequences are likely present in our selected intergenic regions; additional validation work will be required to identify which will be most useful for plant genetic engineering.