title: A versatile system for functional analysis of genes and microRNAs in cotton.
abstract: 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-1α (EF-1α), 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.
authors: State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, China.
authors: Zhouhang
authors: Z
authors: Gu
authors: Changjun
authors: C
authors: Huang
authors: Fangfang
authors: F
authors: Li
authors: Xueping
authors: X
authors: Zhou
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism; D004731:Endotoxins / Q000378:metabolism; D020868:Gene Silencing; D017343:Genes, Plant; D005821:Genetic Techniques; D005822:Genetic Vectors; D003368:Gossypium / Q000235:genetics* / Q000254:growth & development / Q000821:virology; D006460:Hemolysin Proteins / Q000378:metabolism; D007814:Larva / Q000254:growth & development; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D009036:Moths / Q000254:growth & development; D005810:Multigene Family; D018515:Plant Leaves / Q000235:genetics / Q000821:virology; D010942:Plant Viruses / Q000502:physiology; D013045:Species Specificity; D019076:Transgenes
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D035683:MicroRNAs; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: Begomovirus; cotton; microRNA; small tandem target mimic; virus-induced gene silencing