红花miR397a基因表达及对靶基因LAC2的调控作用

Expression of safflower miR397a gene and its role in LAC2 regulation

【目的】研究红花miR397a前体基因及成熟基因在红花不同组织中的表达水平,并对miR397a基因序列、靶基因及基因功能进行分析,为深入研究红花抗逆机制提供参考。【方法】提取红花籽粒、花瓣、茎、根、叶片等5个组织材料的总RNA,使用荧光定量PCR鉴定miR397a在不同组织中的表达水平;利用生物信息学方法分析红花miR397a基因序列,用原生质体转化方法验证红花miR397a调控的靶基因LAC2,并利用转基因拟南芥表型研究miR397a基因的功能。【结果】miR397a前体基因和成熟基因均能在红花不同组织中表达,且均以叶片组织中的表达量最高,分别是籽粒组织的2.5与1.9倍,差异达极显著水平;miR397a序列在不同物种间高度保守,序列中仅存在1-2个碱基的错配或缺失;miR397a对LAC2基因有调控作用,miR397a的过表达可引起LAC2表达水平降低,红花miR397a表达引起植物对NaCl的敏感性增加。【结论】红花miR397a在叶片组织中的表达量最高,基因序列保守,且红花miR397a表达可增强植物对NaCl的敏感性。

[Objective] This study analyzed the expression levels of precursor of miR397a and mature miR397a gene in different safflower tissues,identified the sequence of miR397a, predicted its target genes and explored its function. [Method] Total RNA was extracted from 5 issues including seed, petal,leaf, stem and root of safflower and qRT-PCR was employed to analyze the gene expression levels. Sequence analysis and target genes prediction were conducted by bioinformatics methods. Protoplast transformation method was employed to validate target gene regulated by safflower miR397a. At last,transgenic Arabidopsis was used to discuss function of miR397a gene. [Result] The expressions of miR397a precursor and mature miR397a were higher in safflower leaf than in other tissues. Their expressions in leaf were 2. 5 and 1. 9 times higher than in seed with extremely significant difference. The miR397a was highly conservative in different plants with only one or two bases mismatching or missing. Gene miR397a played role in LAC2 regulation. Over expression of miR397a could decrease LAC2 expression level and miR397a expression could increase plant sensibility to NaCl. [Conclusion] Conserved miR397a expresses the highest in leaf,andit could increase plant sensibility to NaCl.