Mapping subgenomic promoter of coat protein gene of Cucumber green mottle mosaic virus
Mapping subgenomic promoter of coat protein gene of Cucumber green mottle mosaic virus
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黄瓜绿斑驳花叶病毒外壳蛋白基因亚基因组启动子定位
DOI：
10.1016/s2095-3119(19)62647-x
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发表时间：
2020-01-01
影响因子：
4.8
通讯作者：
Gu Qin-sheng
中科院分区：
文献类型：
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作者：
Liu Mei;Liu Li-ming;Gu Qin-sheng
许多植物病毒利用亚基因组RNA作为基因表达策略，因此定位亚基因组启动子（subgenomic promoter， SGP）对于构建病毒载体至关重要。黄瓜绿斑驳花叶病毒（CGMMV）载体虽已构建，但其外壳蛋白（CP）的SGP尚未定位。为此，我们首先假设起始密码子上游的13个核苷酸为转录起始位点（TSS），这是先前研究通过cDNA末端随机扩增（RACE）确定的。其次，从核苷酸-110到+175的区域是假定的CP SGP，正如预测的那样，这是一个由RNA覆盖运动蛋白（MP）和CP的二级结构组成的长茎环结构。为了绘制CGMMV CP SGP，我们进一步根据RNA二级结构预测构建了一系列缺失突变体。上游TSS的缺失显著增强了CP转录，CPTSS前保留了105个核苷酸。在CPTSS下游，我们分析了增强绿色荧光蛋白（enhanced green fluorescent protein， EGFP）在CGMMV CP部分缺失的一系列载体中的表达情况，发现+71 ~ +91核苷酸在转录水平上对EGFP的表达起关键作用，而在CPTSS下游保留160个核苷酸时，EGFP的表达水平最高。为了证实这些结果，我们应用在线软件MEME预测了466个核苷酸的基序和顺式作用元件，这些核苷酸覆盖了缺失分析的序列。保守基序和相关作用元件位于转录水平最高或增强的区域。据我们所知，这是CGMMV SGP的首次映射。
Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter (SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus (CGMMV)-based virus vectors have been constructed, SGP of the coat protein (CP) has not yet mapped. To this end, we firstly presumed 13 nucleotides upstream of the start codon as the transcription starting site (TSS) as previous study identified by random amplification of cDNA ends (RACE). Secondly, the region from nucleotides -110 to +175 is the putative CP SGP, as predicted, a long stem loop structure by the secondary structure of RNA covering movement protein (MP) and CP. To map the CGMMV CP SGP, we further constructed a series of deletion mutants according to RNA secondary structure prediction. The deletion of TSS upstream significantly enhanced CP transcription when 105 nucleotides were retained before the CPTSS. For the downstream of CPTSS, we analyzed the expression of enhanced green fluorescent protein (EGFP) in a series of vectors with partial deletion of the CGMMV CP and found that the nucleotides from +71 to +91 played a key role in the EGFP expression at the transcription level, while EGFP showed the highest expression level when 160 nucleotides were retained downstream of the CP TSS. To confirm these results, we applied online software MEME to predict the motifs and cis-acting elements in the 466 nucleotides covering the sequences of deletion analysis. Conserved motifs and relative acting elements were in regions in which transcription levels were the highest or enhanced. To our best knowledge, this is the first mapping of CGMMV SGP.