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张婉露, 丁勇. RNA结合蛋白U2BL调控拟南芥下胚轴伸长的研究[J]. 植物科学学报. doi: 10.11913/PSJ.2095-0837.23065
引用本文: 张婉露, 丁勇. RNA结合蛋白U2BL调控拟南芥下胚轴伸长的研究[J]. 植物科学学报. doi: 10.11913/PSJ.2095-0837.23065
WanLu ZHANG, Yong DING. Regulation of hypocotyl elongation in Arabidopsis thaliana by RNA binding protein U2BL[J]. Plant Science Journal. doi: 10.11913/PSJ.2095-0837.23065
Citation: WanLu ZHANG, Yong DING. Regulation of hypocotyl elongation in Arabidopsis thaliana by RNA binding protein U2BL[J]. Plant Science Journal. doi: 10.11913/PSJ.2095-0837.23065

RNA结合蛋白U2BL调控拟南芥下胚轴伸长的研究

Regulation of hypocotyl elongation in Arabidopsis thaliana by RNA binding protein U2BL

  • 摘要: 下胚轴是子叶着生处以下生出的最初茎部分,连接着子叶与胚根,是高等植物的胚性器官。在种子萌发过程中,下胚轴在植物地上和地下部分的营养成分及信号分子的运输和传递中发挥着重要作用。因此,下胚轴伸长过程对于植物的早期存活和后期的生长发育都十分重要。本研究对拟南芥(Arabidopsis thaliana)突变体进行表型观察和筛选,发现了一个具有短下胚轴表型的突变体u2bl,并对其调控拟南芥下胚轴伸长的机制进行了初步探究。基因型鉴定以及半定量实验证明,u2bl-1和u2bl-2是T-DNA插入且功能缺失的突变体。表型观察显示,突变体u2bl在长日照(16 h光照/8 h黑暗)、短日照(8 h光照/16 h黑暗)及黑暗条件下均表现出较短的下胚轴表型。细胞学显微观察实验表明,突变体u2bl下胚轴细胞数目与Col-0相比没有明显差异,但其下胚轴细胞长度明显短于Col-0。以上结果说明,突变体u2bl的短下胚轴表型是其下胚轴细胞长度变短所致。植物激素赤霉素(GA)是促进下胚轴伸长的主效应因子。为了验证U2BL是否通过参与GA途径调控下胚轴伸长,使用不同浓度的GA及内源GA合成抑制剂多效唑(PAC)处理突变体u2bl及Col-0。结果显示,不同浓度的GA处理后,u2bl突变体下胚轴长度增长幅度明显小于Col-0,表现为对外源GA处理不敏感的表型;不同浓度PAC处理的结果显示PAC对突变体u2bl下胚轴伸长的抑制作用相对于Col-0亦不明显。这表明,U2BL影响GA的信号转导。将U2BL与mCherry荧光标签融合后稳转至Col-0获得转基因植株,将生长7天的幼苗置于激光共聚焦显微镜下观察其根尖亚细胞定位。结果表明U2BL在细胞核中富集。RT-qPCR实验显示,在u2bl突变体中,PRE1、SAUR16、YUC2、YUC8和PIF4基因的转录水平均有明显下调。这表明U2BL通过调控上述基因来间接调控下胚轴伸长。综上,U2BL参与GA信号传导途径,且通过促进下胚轴伸长相关基因的表达来调控拟南芥下胚轴伸长。本研究初步阐述了U2BL调控植物下胚轴伸长的分子机制,拓展了U2BL在拟南芥生长发育过程中发挥的作用,为进一步研究U2BL与GA互作调控植物生长发育提供参考。

     

    Abstract: Hypocotyl is the part between the origin of radicle and cotyledon node of seedling, and it is the embryonic organ of higher plants. During seed germination, hypocotyls play an important role in the transport and transmission of nutrients and signal molecules between the aboveground and underground parts of plants. Therefore, the hypocotyl elongation process is important for plant survival and growth.In this study, a large number of Arabidopsis mutants were observed and screened, and a mutant u2bl with short hypocotyl phenotype was identified, and its mechanism of regulating the elongation of Arabidopsis hypocotyl was identified explored. Genotype identification and semi- quantitative experiments proved that u2bl-1 and u2bl-2 were mutants with T-DNA insertion and functional deletion. Secondly, phenotypic observation identified that mutant u2bl showed shorter hypocotyls under long-day (16 h light /8 h darkness), short-day (8 h light /16 h darkness) and dark conditions. Cytological microscopic observation showed that the number of hypocotyl cells of mutant u2bl was not significantly different from that of Col-0, and the length of hypocotyl cells was significantly shorter than that Col-0.The above experimental results show that the short hypocotyl phenotype of mutant u2bl was caused by the reduced hypocotyl cell length. Gibberellin (GA), a plant hormone, is the main effector that promotes hypocotyl elongation. To verify whether U2BL regulated hypocotyl elongation by involving in the GA pathway, u2bl mutants and Col-0 were treated with different concentrations of GA and paclobutrazol (PAC), an endogenous GA synthesis inhibitor. The results showed that after treatment with different concentrations of GA, the growth amplitude of hypocotyl length of u2bl mutant was significantly smaller than Col-0, which presented as a phenotype insensitive to exogenous GA. The results of treatment with different concentrations of PAC also showed that the inhibition of PAC on the hypocotyl elongation of mutant u2bl was not significant relative to Col-0.The above results indicated that U2BL affected the signal transduction of GA. U2BL was fused with the mCherry fluorescent tag and stably transferred to Col-0 to obtain transgenic plants. The seedlings, which grew for 7 days, were placed under a laser confocal microscope to observe the subcellular localization in the root tips.The results showed that U2BL was enriched in the nucleus . The RT-qPCR assay showed that in the u2bl mutants, the transcription levels of PRE1, SAUR16, YUC2, YUC8 and PIF4 genes were all significantly down-regulated. This indicated that U2BL indirectly regulated hypocotyl elongation by regulating the above genes.In summary, U2BL is involved in the GA signal transduction pathway and regulates hypocotyl elongation in Arabidopsis thaliana by promoting the expression of genes related to hypocotyl elongation.This study preliminarily expounded the molecular mechanism of U2BL regulating plant hypocotyl elongation, expanded the role of U2BL in the growth and development of Arabidopsis thaliana, and provided reference for further study on the interaction between U2BL and GA regulating plant growth and development.

     

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