An increasing number of microRNAs （miRNAs） have been shown to play crucial regulatory roles in the process of plant development. Here, we used high-throughput sequencing combined with computational analysis to chara...An increasing number of microRNAs （miRNAs） have been shown to play crucial regulatory roles in the process of plant development. Here, we used high-throughput sequencing combined with computational analysis to characterize miRNAomes from the ovules of wild-type upland cotton and a fiberless mutant during fiber initiation. Comparative miR- NAome analysis combined with northern blotting and RACE-PCR revealed seven fiber initiation-related miRNAs expressed in cotton ovules and experimentally validated targets of these miRNAs are involved in different cellular responses and metabolic processes, including transcriptional regulation, auxin and gibberellin signal transduction, actin bundles, and lignin biosynthesis. This paper describes a complex regulatory network consisting of these miRNAs expressed in cotton ovules to coordinate fiber initiation responses. In addition, 36 novel miRNAs and two conserved miRNAs were newly iden- tified, nearly doubling the number of known cotton miRNA families to a total of 78. Furthermore, a chromatin remodeling complex subunit and a pre-mRNA splicing factor are shown for the first time to be miRNA targets. To our knowledge, this study is the first systematic investigation of fiber initiation-related miRNAs and their targets in the developing cotton ovule, deepening our understanding of the important regulatory functions of miRNAs in cotton fiber initiation.展开更多
Ethylene-responsive factors （ERFs） are important regulators of plant gene expression. In this study, three novel ERF genes, GhERF2, GhERF3 and GhERF6, were isolated from cotton （Gossypium hirstum） using rapid ampl...Ethylene-responsive factors （ERFs） are important regulators of plant gene expression. In this study, three novel ERF genes, GhERF2, GhERF3 and GhERF6, were isolated from cotton （Gossypium hirstum） using rapid amplification of cDNA ends-polymerase chain reaction. Transient expression analysis using GhERF-green fluorescent protein fusions showed that these three proteins were targeted to the nucleus. Fusion proteins consisting of GhERF2, GhERF3 or GhERF6 coupled to the GAL4 DNA binding domain strongly activated transcription in yeast. Furthermore, GhERF6 was shown to be able to bind specifically to GCC boxes using a particle bombardment assay in tobacco cells. Semi-quantitative reverse transcription-polymerase chain reaction revealed that GhERF2 and GhERF3 are constitutively expressed in all organs, while GhERF6 is only constitutively expressed in vegetative organs. When plants were treated with ethylene, abscisic acid, salt, cold and drought, the transcripts of GhERF2, GhERF3 and GhERF6 were rapidly induced to high levels. Promoter analysis also indicated that the 5＇ upstream regions of the three genes possess elements induced by these physiological and environmental factors. Collectively, our data suggest that GhERF2, GhERF3 and GhERF6 might function as positive trans-acting factors in the plant responses to ethylene, abscisic acid and other stresses and provide useful clues for further research into the mechanism of them in regulating cotton multiple stress responses.展开更多
基金This work was supported by grants from the State Key Basic Re- search and Development Plan （2010CB126003）, the National Natural Science Foundation of China （90608016）, the Hi-Tech Re- search and Development Program of China （2008AA02Z116）, and the National Transgenic Animals and Plants Research Project （2009ZX08009-069B, 2009ZX08005-026B, and 2008ZX08009- 003）.
文摘An increasing number of microRNAs （miRNAs） have been shown to play crucial regulatory roles in the process of plant development. Here, we used high-throughput sequencing combined with computational analysis to characterize miRNAomes from the ovules of wild-type upland cotton and a fiberless mutant during fiber initiation. Comparative miR- NAome analysis combined with northern blotting and RACE-PCR revealed seven fiber initiation-related miRNAs expressed in cotton ovules and experimentally validated targets of these miRNAs are involved in different cellular responses and metabolic processes, including transcriptional regulation, auxin and gibberellin signal transduction, actin bundles, and lignin biosynthesis. This paper describes a complex regulatory network consisting of these miRNAs expressed in cotton ovules to coordinate fiber initiation responses. In addition, 36 novel miRNAs and two conserved miRNAs were newly iden- tified, nearly doubling the number of known cotton miRNA families to a total of 78. Furthermore, a chromatin remodeling complex subunit and a pre-mRNA splicing factor are shown for the first time to be miRNA targets. To our knowledge, this study is the first systematic investigation of fiber initiation-related miRNAs and their targets in the developing cotton ovule, deepening our understanding of the important regulatory functions of miRNAs in cotton fiber initiation.
基金supported by grants from the National Natural Science Foundation of China (90608016)the Hi-Tech Research and Development Program of China (Grant No. 2007AA02Z100)the National Transgenic Animals & Plants Research Project (2008ZX08009-003 and 2008ZX08005-003)
文摘Ethylene-responsive factors （ERFs） are important regulators of plant gene expression. In this study, three novel ERF genes, GhERF2, GhERF3 and GhERF6, were isolated from cotton （Gossypium hirstum） using rapid amplification of cDNA ends-polymerase chain reaction. Transient expression analysis using GhERF-green fluorescent protein fusions showed that these three proteins were targeted to the nucleus. Fusion proteins consisting of GhERF2, GhERF3 or GhERF6 coupled to the GAL4 DNA binding domain strongly activated transcription in yeast. Furthermore, GhERF6 was shown to be able to bind specifically to GCC boxes using a particle bombardment assay in tobacco cells. Semi-quantitative reverse transcription-polymerase chain reaction revealed that GhERF2 and GhERF3 are constitutively expressed in all organs, while GhERF6 is only constitutively expressed in vegetative organs. When plants were treated with ethylene, abscisic acid, salt, cold and drought, the transcripts of GhERF2, GhERF3 and GhERF6 were rapidly induced to high levels. Promoter analysis also indicated that the 5＇ upstream regions of the three genes possess elements induced by these physiological and environmental factors. Collectively, our data suggest that GhERF2, GhERF3 and GhERF6 might function as positive trans-acting factors in the plant responses to ethylene, abscisic acid and other stresses and provide useful clues for further research into the mechanism of them in regulating cotton multiple stress responses.
