Suppression of cotton leaf curl disease symptoms in Gossypium hirsutum through over expression of host-encoded miRNAs
Suppression of cotton leaf curl disease symptoms in Gossypium hirsutum through over expression of host-encoded miRNAs
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DOI：
10.1016/j.jbiotec.2017.10.003
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发表时间：
2017-12-10
影响因子：
4.1
通讯作者：
Khan, Jawaid A.
中科院分区：
文献类型：
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作者：
Akmal, Mohd.;Baig, Mirza S.;Khan, Jawaid A.
棉花曲叶病（Cotton Leaf Curl Disease，CLCuD）是由与棉花曲叶病（Cotton Leaf Curl木尔坦Betasatellite，CLCuMB）相关的一种独特的单分型菜豆花叶病毒（Monoparticebegomovirus）引起的，是造成棉花产量损失的主要原因。已知微RNA（mi）调节真核生物中的基因表达，包括植物中的抗病毒防御。在之前的研究中，我们已经通过计算鉴定了一组棉花miRNA，这些miRNA在棉花曲叶木尔坦病毒（CLCuMuV）和CLCuMB基因组的多个位点上具有潜在的靶点。在本研究中，亚洲棉编码的miRNAs在植物中对CLCuMuV和CLCuMB基因组的影响进行了研究。选择了两种计算预测的棉花编码的miRNA（miR 398和miR 2950），其显示出结合CLCuMuV的多个开放阅读框架（ORF; C1、C4、V1和非编码基因间区）和CLCuMB的（β C1）的潜力。miR 398和miR 2950在G.多毛变种HS6。从独立事件产生总共十株体外棉花植物，并进行生物学和分子分析。通过PCR和Southern印迹法确认各前体（pre）-miRNA的存在，并通过半定量RT-PCR、真实的时间定量PCR和PCR阳性细胞系中的北方杂交评估它们的表达水平。Southern杂交结果显示，转化株系的基因组中整合了2-4个拷贝的T-DNA。值得注意的是，如通过真实的时间PCR所揭示的，在转基因（T-0）系中显示premiRNA的表达高达5.8倍。用含病毒的烟粉虱（Bemisia tabaci）昆虫载体接种转基因棉花品系后，监测病毒抗性。接种后，4个转基因株系保持明显无症状。虽然通过滚环扩增可以检测到非常低滴度的病毒DNA，但在任何看起来健康的转基因品系中都不能检测到负责症状诱导的β卫星。在本研究中，首次对宿主（G。通过在G. arboreum）中过表达miR 398和miR 2950，实验证明了针对CLCuD症状的miRNAs。多毛变种HS 6植物。计算预测靶向病毒基因组的miRNAs及其随后通过过表达在基于翻译抑制或切割的病毒mRNA抑制中的意义可以帮助产生抗病毒植物。
Cotton leaf curl disease (CLCuD), a major factor resulting in the enormous yield losses in cotton crop, is caused by a distinct monopartite begomovirus in association with Cotton leaf curl Multan betasatellite (CLCuMB). Micro (mi) RNAs are known to regulate gene expression in eukaryotes, including antiviral defense in plants. In a previous study, we had computationally identified a set of cotton miRNAs, which were shown to have potential targets in the genomes of Cotton leaf curl Multan virus (CLCuMuV) and CLCuMB at multiple loci. In the current study, effect of Gossypium arboreum-encoded miRNAs on the genome of CLCuMuV and CLCuMB was investigated in planta. Two computationally predicted cotton-encoded miRNAs (miR398 and miR2950) that showed potential to bind multiple Open Reading Frames (ORFs; C1, C4, V1, and non-coding intergenic region) of CLCuMuV, and (beta C1) of CLCuMB were selected. Functional validation of miR398 and miR2950 was done by overexpression approach in G. hirsutum var. HS6. A total of ten in vitro cotton plants were generated from independent events and subjected to biological and molecular analyses. Presence of the respective Precursor (pre)-miRNA was confirmed through PCR and Southern blotting, and their expression level was assessed by semi quantitative RT-PCR, Real Time quantitative PCR and northern hybridization in the PCR-positive lines. Southern hybridization revealed 2-4 copy integration of T-DNA in the genome of the transformed lines. Remarkably, expression of premiRNAs was shown up to 5.8-fold higher in the transgenic (T-0) lines as revealed by Real Time PCR. The virus resistance was monitored following inoculation of the transgenic cotton lines with viruliferous whitefly (Bemisia tabaci) insect vector. After inoculation, four of the transgenic lines remained apparently symptom free. While a very low titre of viral DNA could be detected by Rolling circle amplification, betasatellite responsible for symptom induction could not be detected in any of the healthy looking transgenic lines. In this study for the first time, efficacy of the host (G. arboreum)-encoded miRNAs against CLCuD symptoms was experimentally demonstrated through overexpression of miR398 and miR2950 in G. hirsutum var. HS6 plants. Computational prediction of miRNAs targeting virus genome and their subsequent implication in translational inhibition or cleavage based suppression of viral mRNA via overexpression could help in generating virus resistant plants.