大豆microRNA基因GmMIR160A负调控植物叶片衰老进程

GmMIR160A, a class of soybean microRNA gene, negatively regulates progress of leaf senescence

关键词:  大豆  MicroRNAs  生长素响应因子  过表达

Key words:  Glycine max  MicroRNAs  auxin response factors  over-expression

摘要:

叶片衰老是受内外多种因子影响的遗传发育进程。生长素、细胞分裂素和乙烯等多种植物激素是调控叶片衰老的重要内部因子,它们通过长或短距离运输形成叶片组织内特定的区域分布和浓度梯度,从而直接或间接参与植物叶片衰老过程。分子遗传学表明,细胞分裂素和乙烯分别是叶片衰老的抑制子和正调节子,而生长素如何参与叶片衰老的分子机制目前还不清晰。植物体内成熟小分子RNA由小RNA基因转录并通过特定酶加工形成的21～23 bp的双链RNA分子。这些小分子通过不完全配对方式抑制其靶基因转录和/或表达,参与植物生长发育多个过程,然而这类小RNA分子如何调控植物叶片衰老发育过程目前则还鲜有报告。大豆是重要的油料作物,具有典型的单次结实性衰老特征。研究大豆叶片衰老具有重要的科学意义和深远的应用价值。该文采用实时荧光定量PCR(qPCR)技术分析大豆(Glycine max)micro RNA基因GmMIR160A的表达模式,发现大豆第一复叶中GmMIR160A表达受外源生长素和黑暗处理的诱导,暗示该基因是生长素快速响应的叶片衰老相关基因。为进一步探究GmMIR160A在大豆叶片发育中的功能,构建了肾上腺皮质激素(Glucocorticoid,GR)类似物地塞米松(Dexamethasone,DEX)诱导表达GmMIR160A双元表达载体并通过农杆菌介导的子叶节方法转化野生型大豆。通过抗性筛选和基因组PCR鉴定并结合表型分析,共获得了4株诱导表达的稳定遗传转基因植株(株系OX-3、OX-5、OX-7和OX-8)。GmMIR160A过表达植株根、茎、叶、花和果实在形态学上与野生型相比无显著差异,但叶片的叶绿素含量增加、最大光量子效率(Fv/Fm)增强。进一步分子分析发现,转基因大豆叶片中GmARFs和衰老标记基因(GmCYSP1)表达明显下降,表明大豆Gma-miR160通过抑制靶基因GmARFs的表达来负调控植物叶片的衰老进程。该文揭示了生长素通过小分子RNA调控叶片发育一条新途径,为研究植物激素调控植物叶片衰老提供了新的思路。

Abstract:

Leaf senescence is known to be a genetic programmed process regulated by the interior and/or exterior factors. Plant hormones including cytokinins,ethylene and auxin directly or indirectly regulate leaf senescence as pivotal interior factors through their redistribution and local concentration gradient by long and/or short distance transport. The molecular genetics study has shown that the cytokinins work as a repressor and the ethylene as a positive regulator respectively during the processes of leaf senescence. However,the molecular mechanism of auxin involved in regulating leaf senescence is largely unknown. The mature microRNAs are a kind of small and short double-strand RNA molecules with 21-23 bp length,which are first transcripted from mircroRNA(miRNA)genes and then processed by the special enzymes. These small RNAs are proved to be involved in many plant growth and development processes by matching their specific targets in an inaccuracy manner. However,there is no evidence that the specific microRNA could regulate plant leaf senescence in soybean so far. Soybean is a both oil crop and the classical seed-setting dependent aging species. To uncover the molecular mechanism of soybean leaf senescence should have both the important scientific significance and the profound application value in the future. In this report,we used real-time fluorescence quantitative PCR(qPCR)approach to explore the expression pattern of GmMIR160A gene during the development of the first trifoliage soybean leaves. We found that the messenger RNA of GmMIR160A was rapidly induced by both exogenous applied auxin and darkness treatment,suggesting that this gene could be both an immediate auxin-responsive gene and leaf senescence-associated gene. To further investigate the role of GmMIR160A gene during the soybean leaf development,the binary vector over-expressing GmMIR160A induced by glucocorticoid-like chemical,dexamethasone( DEX),was constructed and then transformed into wild-type soybean through Agrobacterium-mediated method with cotyledon node as the explants. Using the time order screening approaches including the antibiotic screen,the genome PCR identification and the phenotypic analysis,we finally generated four transgenic lines(Line OX-3,5,7 and 8)with stable integrated insertion T-DNA. Compared with wild types control,these transgenic plants,successful expressing the transgene showed normal morphological characteristics in respect to roots,stem,leaves,flowers,fruits and seeds but exhibited the increased chlorophyll content and higher maximum quantum efficiency(Fv/Fm)for the first trifoliage leaves during the mature stage. Moreover,GmARFs and GmCYSP1,in which the former are targets of GmMIR160 and the latter is thought as a soybean senescence marker,were down-regulated dramatically in the transgenic trifoliage leaves. Taking together,these data indicated that Gma-miR160 might negatively regulate leaf senescence by repression of its targets in soybean. This report uncovered a novel pathway that the plant hormone auxin could modify the processes of leaf senescence by regulating the transcriptional expression of microRNA gene Gma-miR160 and then repressing the messenger RNA level of auxin responsive factors GmARFs and also provided the new clues for investigating how the plant hormones control the progress of leaf senescence.