A versatile system for functional analysis of genes and microRNAs in cotton
A versatile system for functional analysis of genes and microRNAs in cotton
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DOI：
10.1111/pbi.12169
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发表时间：
2014-06-01
影响因子：
13.8
通讯作者：
Zhou, Xueping
中科院分区：
文献类型：
--
作者：
Gu, Zhouhang;Huang, Changjun;Zhou, Xueping
棉花是世界范围内重要的经济作物。由于该作物生长周期长、基因组大小大、不易稳定转化，传统的基因功能分析方法难度大、劳动强度大。在这里，我们报告了一个基于棉花皱叶病毒（CLCrV）的载体及其在基因功能分析中的应用，通过病毒诱导的基因沉默（VIGS）和过表达的microRNA（miRNA），小串联靶模拟物（STTM）和人工miRNA（amiRNA）在棉花通过农杆菌介导的渗透方法。使用这个系统，我们能够有效地沉默两个内源基因，镁螯合酶亚基I（CHLI）和延伸因子-1 α（EF-1 α），在棉属物种和苏云金芽孢杆菌cry 1A基因在转基因棉花。此外，我们的研究结果表明，该载体可用于异位表达内源性miR 156在陆地棉，导致减少miR 156靶向的RNA转录，导致异常叶表型的发展。用该载体异位表达miR 165/166 STTM导致向下卷曲和起皱的叶片，以及miR 165/166靶mRNA的显著增加。这种通用的系统易于使用，并且可以在棉花中提供更均匀和持久的基因沉默，从而为棉花基因发现提供了强有力的方法。
Cotton is an important economic crop worldwide. Due to its long growth cycle, large genome size and recalcitrance to stable transformation, traditional methods for the analysis of gene function in this crop are difficult and labour intensive. Here, we report a cotton leaf crumple virus (CLCrV)-based vector and its application in gene function analysis through virus-induced gene silencing (VIGS) and overexpression of microRNAs (miRNAs), small tandem target mimic (STTM) and artificial miRNA (amiRNA) in cotton via an Agrobacterium-mediated infiltration approach. Using this system, we were able to efficiently silence two endogenous genes, magnesium chelatase subunit I (CHLI) and elongation factor-1 alpha (EF-1 alpha), in Gossypium species and the Bacillus thuringiensis cry1A gene in transgenic cotton. Furthermore, our results show that this vector can be used to ectopically express endogenous miR156 in G.hirsutum, causing a reduction in miR156-targeted RNA transcripts resulting in the development of abnormal leaf phenotypes. Ectopic expression of miR165/166 STTM with this vector led to downward curling and crumpled leaves, and a significant increase in the miR165/166 target mRNAs. This versatile system is easy to use and can provide more uniform and persistent gene silencing in cotton, thereby providing a powerful approach for gene discovery in cotton.