Artificial optimization of bamboo Ppmar2 transposase and host factors effects on Ppmar2 transposition in yeast.
Artificial optimization of bamboo Ppmar2 transposase and host factors effects on Ppmar2 transposition in yeast.
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竹子Ppmar2转座酶的人工优化及宿主因子对酵母Ppmar2转座的影响
DOI：
10.3389/fpls.2022.1004732
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发表时间：
2022
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
--
作者：
水手样元件(MLEs)是基因克隆、基因表达和基因标记的重要工具。我们从毛竹中鉴定了两个MLE转座子，Ppmar1和Ppmar2。Ppmar2，体积较小，具有较高的自然活性，因此与Ppmar1相比，它是一种更有潜力的基因组工具。利用在酵母中共转化的转座酶表达框和非自主转座子组成的双组分系统，研究了Ppmar2的转座活性，产生了高活性的转座酶。在Ppmar2的19个氨基酸突变中，有5个的催化活性高于野生型，特别是S347R突变的转座活性是野生型的6.7倍。此外，还选择了36株单基因缺失的酵母突变株，对宿主因素对Ppmar2NA转座的影响进行了筛选。与对照菌株(His3Δ)相比，9个突变体的迁移率显著增加，7个突变体的迁移率显著降低。Efm1Δ突变体的转座能力是对照的15倍，而rtt10Δ突变体的转座能力降低到1/66。转录分析表明，EFM1缺陷导致DDR2、HSP70的表达显著降低，JEN1的表达显著增强，而RTT10缺陷导致UTP20、RPA190和Rrp5的表达显著抑制。蛋白质甲基化、染色质和RNA转录可能影响Ppmar2NA在酵母中的转座效率。总体而言，这些发现为转座调控提供了证据，并为毛竹和其他植物提供了一种替代的基因组工具。
Mariner-like elements (MLEs) are promising tools for gene cloning, gene expression, and gene tagging. We have characterized two MLE transposons from moso bamboo, Ppmar1 and Ppmar2. Ppmar2, is smaller in size and has higher natural activities, thus making it a more potential genomic tool compared to Ppmar1. Using a two-component system consisting of a transposase expression cassette and a non-autonomous transposon cotransformed in yeast, we investigated the transposition activity of Ppmar2 and created hyperactive transposases. Five out of 19 amino acid mutations in Ppmar2 outperformed the wild-type in terms of catalytic activities, especially with the S347R mutant having 6.7-fold higher transposition activity. Moreover, 36 yeast mutants with single-gene deletion were chosen to screen the effects of the host factors on Ppmar2NA transposition. Compared to the control strain (his3Δ), the mobility of Ppmar2 was greatly increased in 9 mutants and dramatically decreased in 7 mutants. The transposition ability in the efm1Δ mutant was 15-fold higher than in the control, while it was lowered to 1/66 in the rtt10Δ mutant. Transcriptomic analysis exhibited that EFM1 defection led to the significantly impaired DDR2, HSP70 expression and dramatically boosted JEN1 expression, whereas RTT10 defection resulted in significantly suppressed expression of UTP20, RPA190 and RRP5. Protein methylation, chromatin and RNA transcription may affect the Ppmar2NA transposition efficiency in yeast. Overall, the findings provided evidence for transposition regulation and offered an alternative genomic tool for moso bamboo and other plants.
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影响因子：
14.9
作者：
Liu D;Chalmers R
通讯作者：
Chalmers R
影响因子：
16
作者：
Dawson, A;Finnegan, DJ
通讯作者：
Finnegan, DJ
影响因子：
3.3
作者：
Lazarow, Katina;Du, My-Linh;Kunze, Reinhard
通讯作者：
Kunze, Reinhard
DOI：
10.1073/pnas.94.4.1293
发表时间：
1997-02-18
影响因子：
11.1
作者：
Lohe, AR;DeAguiar, D;Hartl, DL
通讯作者：
Hartl, DL
影响因子：
64.8
作者：
Dragon, F;Gallagher, JEG;Baserga, SJ
通讯作者：
Baserga, SJ