龙眼miR396分子进化特性及响应蓝光的表达分析

关于蓝光对龙眼胚性愈伤组织miRNA组学研究中发现大量响应蓝光的miRNAs,挑选关键且差异表达的miR396a-3p_2和miR396b-5p进行分子进化特性分析,以进一步明确它们在龙眼响应蓝光过程中的作用。该研究对前体和成熟体进化树、前体二级结构、启动子顺式作用元件、靶基因预测及其响应蓝光表达模式等进行分析。结果表明:(1)由5p臂上形成的miR396成熟体序列保守性较高,由3p臂上形成的miR396成熟体序列特异性较大;茎序列的保守性高于环序列,5p臂上保守性高于3p臂。(2)miR396a-3p_2和miR396b-5p的启动子主要包括光、激素、生物和非生物胁迫响应元件,可能通过这些顺式元件在龙眼响应蓝光中起调控作用;其响应蓝光的靶基因或蛋白主要包括生长素调控因子(GRF2)、LRR类受体丝氨酸(FLS2)、乙烯不敏感蛋白(EIN3)和DNA定向RNA聚合酶α亚基(rpoA)等。(3)实时荧光定量PCR结果表明,miR396a-3p_2和miR396b-5p在不同光质的表达模式存在差异;在不同光强的蓝光条件下,miR396b-5p与其靶基因的表达趋势基本呈负相关,而miR396a-3p_2与rpoA的表达趋势未呈负相关,可能存在其他miR396家族成员或miRNAs同时调控,从而实现靶基因转录水平的调控。     

A common miRNA160‐based mechanism regulates ovary patterning,floral organ abscission and lamina outgrowth in tomato

Plant microRNAs play vital roles in auxin signaling via the negative regulation of auxin response factors (ARFs). Studies have shown that targeting of ARF10/16/17 by miR160 is indispensable for various aspects of development, but its functions in the model crop tomato (Solanum lycopersicum) are unknown. Here we knocked down miR160 (sly–miR160) using a short tandem target mimic (STTM160), and investigated its roles in tomato development. Northern blot analysis showed that miR160 is abundant in developing ovaries. In line with this, its down‐regulation perturbed ovary patterning as indicated by the excessive elongation of the proximal ends of mutant ovaries and thinning of the placenta. Following fertilization, these morphological changes led to formation of elongated, pear‐shaped fruits reminiscent of those of the tomato ovate mutant. In addition, STTM160‐expressing plants displayed abnormal floral organ abscission, and produced leaves, sepals and petals with diminished blades, indicating a requirement for sly–miR160 for these auxin‐mediated processes. We found that sly–miR160 depletion was always associated with the up‐regulation of SlARF10A, SlARF10B and SlARF17, of which the expression of SlARF10A increased the most. Despite the sly–miR160 legitimate site of SlARF16A, its mRNA levels did not change in response to sly–miR160 down‐regulation, suggesting that it may be regulated by a mechanism other than mRNA cleavage. SlARF10A and SlARF17 were previously suggested to function as inhibiting ARFs. We propose that by adjusting the expression of a group of ARF repressors, of which SlARF10A is a primary target, sly–miR160 regulates auxin‐mediated ovary patterning as well as floral organ abscission and lateral organ lamina outgrowth.     

Dwarf and deformed flower 1, encoding an F‐box protein,is critical for vegetative and floral development in rice (Oryza sativa L.)

Recent studies have shown that F‐box proteins constitute a large family in eukaryotes, and play pivotal roles in regulating various developmental processes in plants. However, their functions in monocots are still obscure. In this study, we characterized a recessive mutant dwarf and deformed flower 1‐1 (ddf1‐1) in Oryza sativa (rice). The mutant is abnormal in both vegetative and reproductive development, with significant size reduction in all organs except the spikelet. DDF1 controls organ size by regulating both cell division and cell expansion. In the ddf1‐1 spikelet, the specification of floral organs in whorls 2 and 3 is altered, with most lodicules and stamens being transformed into glume‐like organs and pistil‐like organs, respectively, but the specification of lemma/palea and pistil in whorls 1 and 4 is not affected. DDF1 encodes an F‐box protein anchored in the nucleolus, and is expressed in almost all vegetative and reproductive tissues. Consistent with the mutant floral phenotype, DDF1 positively regulates B‐class genes OsMADS4 and OsMADS16, and negatively regulates pistil specification gene DL. In addition, DDF1 also negatively regulates the Arabidopsis LFY ortholog APO2, implying a functional connection between DDF1 and APO2. Collectively, these results revealed that DDF1, as a newly identified F‐box gene, is a crucial genetic factor with pleiotropic functions for both vegetative growth and floral organ specification in rice. These findings provide additional insights into the molecular mechanism controlling monocot vegetative and reproductive development.     

miR‐148a inhibits early relapsed colorectal cancers and the secretion of VEGF by indirectly targeting HIF‐1α under non‐hypoxia/hypoxia conditions

Vascular endothelial growth factor (VEGF) is correlated with angiogenesis and early relapse of colorectal cancer (CRC). This study investigated the role of miR‐148a in the regulation of VEGF/angiogenesis and early relapse of CRC. We established a stable clone with miR‐148a expression in HCT116 and HT29 cell lines and created a hypoxic condition by using CoCl₂ to determine the underlying mechanism of miR‐148a. The effects of miR‐148a on the phosphoryl‐ERK (pERK)/hypoxia‐inducible factor‐1α (HIF‐1α)/VEGF pathway were evaluated through Western blotting and the inhibitory effect of miR‐148a on angiogenesis was demonstrated through a tube formation assay. Sixty‐three CRC tissues (28 early relapse and 35 non‐early relapse) were analysed to assess the relationship between miR‐148a and HIF‐1α/VEGF. The protein expression of pERK/HIF‐1α/VEGF in HCT116 and HT29 cells was significantly decreased by miR‐148a (all P < 0.05). The protein expression of VEGF/HIF‐1α was strongly inversely associated with the expression of miR‐148a in the 63 CRC tissue samples (all P < 0.05). Tube formation assay demonstrated that miR‐148a significantly obliterated angiogenesis. miR‐148a suppresses VEGF through down‐regulation of the pERK/HIF‐1α/VEGF pathway and might lead to the inhibition of angiogenesis; miR‐148a down‐regulation increased the early relapse rate of CRC. This demonstrates that miR‐148a is a potential diagnostic and therapeutic target.     

Phylogeny and Molecular Evolution of miR820 and miR396 microRNA Families in <Emphasis Type="Italic">Oryza</Emphasis> AA Genomes

The phylogeny and evolution of the microRNA families, miR820 and miR396, was analysed across the AA genomes of the Oryza species, the close relatives of domesticated rice. A highly dynamic evolution of the miR820 family was revealed. The number of copies of MIR820 genes, their chromosomal location and the mature microRNA sequence varied greatly with a total of 16 novel miR820 variants being identified. The phylogeny of pre-MIR820 sequences revealed that MIR820 genes of recently evolved Oryza AA genomes may have derived from sequence divergence of one or a few ancestral genes found in wild Australian perennial rice populations, Taxon B (jpn2)-MIR820 genes. Genomic scale duplication played an important role in the evolution of some of the miR396 family genes in AA genome Oryza species. miR396 family contained a MIR396 gene cluster (MIR396a and MIR396c) which was conserved across the cereal genomes. Nucleotide diversity analysis at these two MIR396 loci revealed that domesticated rice has retained less than 10% of the total diversity present in wild species. In contrast, the nucleotide sequence of four MIR396 loci remained almost conserved across domesticated and wild rices, indicating that they were under extreme functional constraint and may be involved in regulating some fundamental processes which are important both for wild and domesticated rices. Expression analysis demonstrated that miR820 variants were expressed in O. glaberrima O. barthi and O. longistaminata genome. These findings pose new challenges to explain the possible role of miR820 variants identified.