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转BpmiR156基因白桦株系的耐盐性分析
叶查龙, 颜斌, 申婷婷, 宁坤, 李慧玉
南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (6) : 147-151.
PDF(1923 KB)
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转BpmiR156基因白桦株系的耐盐性分析
Analysis of salt tolerance in BpmiR156 overexpression Betula platyphylla
【目的】探究植物年龄响应因子miR156的耐盐功能。【方法】以BpmiR156过表达白桦株系和非转基因对照株系为材料,通过PCR及qPCR分析各转基因株系中外源BpmiR156的稳定性及表达情况;同时对转基因及对照株系进行NaCl胁迫处理,调查胁迫后盐害指数、生理指标及生长速度。【结果】各转基因株系中外源BpmiR156已整合到白桦基因组中,且表达量显著高于非转基因对照株系;NaCl胁迫后,转基因株系的H2O2、丙二醛(MDA)含量及盐害指数高于对照株系,生长速度变慢。【结论】BpmiR156基因在白桦中稳定表达,BpmiR156基因过表达白桦株系与对照株系相比,在盐胁迫后盐害指数增加,生长速度变慢,膜系统损伤更严重,说明转入miR156基因在一定程度上降低了白桦的耐盐性。
【Objective】This study aimed to explore the role of miR156, an age-response factor in plants, in Betula platy-phylla (birch) salt tolerance.【Method】Transgenic BpmiR156 overexpression lines and non-transgenic line were generated. The polymerase chain reaction (PCR) and quantitative PCR were used to analyze the stability and expression of the exogenous BpmiR156 in each transgenic line. Under NaCl stress, the salt injury index, physiological index and growth rate of transgenic and non-transgenic lines were analyzed. 【Result】Results indicated that the BpmiR156 was integrated into the genome of the transgenic lines and more highly expressed than those in the non-transgenic line. Following NaCl treatment, the contents of H2O2, MDA and the salt injury index were higher in the transgenic lines than those in the non-transgenic line. Moreover, the transgenic lines exhibited a slower growth rate than the non-transgenic line. 【Conclusion】BpmiR156 gene was stably expressed in transgenic birch. Compared with the non-transgenic line, the index of salt injury increased, the growth rate slowed down and the membrane system damage was more serious. This indicates that over-expression of miR156 reduces the salt tolerance of birch.
白桦 / miR156 / 转基因 / 盐胁迫 / 丙二醛 / H2O2
Betula platyphylla (birch) / miR156 / transgenic lines / salt stress / malondialdehyde (MDA) / H2O2
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The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events. Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated. Here we show that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3' untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly controlled by let-7. A reporter gene bearing the lin-41 3' untranslated region is temporally regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates lin-14 and lin-28 through RNA-RNA interactions with their 3' untranslated regions. We propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing.
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MicroRNAs (miRNAs) are an extensive class of ~22-nucleotide noncoding RNAs thought to regulate gene expression in metazoans. We find that miRNAs are also present in plants, indicating that this class of noncoding RNA arose early in eukaryotic evolution. In this paper 16 Arabidopsis miRNAs are described, many of which have differential expression patterns in development. Eight are absolutely conserved in the rice genome. The plant miRNA loci potentially encode stem-loop precursors similar to those processed by Dicer (a ribonuclease III) in animals. Mutation of an Arabidopsis Dicer homolog, CARPEL FACTORY, prevents the accumulation of miRNAs, showing that similar mechanisms direct miRNA processing in plants and animals. The previously described roles of CARPEL FACTORY in the development of Arabidopsis embryos, leaves, and floral meristems suggest that the miRNAs could play regulatory roles in the development of plants as well as animals.
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CHUCKG,
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Alfalfa (Medicago sativa) is an important forage crop that is often grown in areas that frequently experience drought and water shortage. MicroRNA156 (miR156) is an emerging tool for improving various traits in plants. We tested the role of miR156d in drought response of alfalfa, and observed a significant improvement in drought tolerance of miR156 overexpression (miR156OE) alfalfa genotypes compared to the wild type control (WT). In addition to higher survival and reduced water loss, miR156OE genotypes also maintained higher stomatal conductance compared to WT during drought stress. Furthermore, we observed an enhanced accumulation of compatible solute (proline) and increased levels of abscisic acid (ABA) and antioxidants in miR156OE genotypes. Similarly, alfalfa plants with reduced expression of miR156-targeted SPL13 showed reduced water loss and enhanced stomatal conductance, chlorophyll content and photosynthetic assimilation. Several genes known to be involved in drought tolerance were differentially expressed in leaf and root of miR156 overexpression plants. Taken together, our findings reveal that miR156 improves drought tolerance in alfalfa at least partially by silencing SPL13.
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BACKGROUND: Developing Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop's sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. RESULTS: To understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13, and DIHYDROFLAVONOL-4-REDUCTASE (DFR) regulating WD40-1. Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40-1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40-1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression. CONCLUSIONS: Taken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40-1 expression, whereas higher miR156 overexpression results in drought susceptibility.
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