外源脱落酸和茉莉酸甲酯调控低温胁迫下冬小麦miR444a及其靶基因TaMADS57表达

Exogenous abscisic acid and methyl jasmonate regulate the expression of miR444a and target gene TaMADS57 in winter wheat under cold stress

为探究小麦(Triticum aestivum)中mi RNA响应低温胁迫的机制,本研究以强抗寒冬小麦品种‘东农冬麦1号’(Dn1)为材料,依据实验室前期建立的miRNA数据库,筛选出与低温变化相关差异表达的tae-miR444a前体(pre-tae-miR444a),并预测出其靶基因为TaMADS57。构建pre-tae-miR444a和TaMADS57的pBI121-GUS表达载体,并利用根癌农杆菌(Agrobacterium tumefaciens)瞬时转化本氏烟草(Nicotiana benthamiana)。结果显示,共转叶片中β-葡萄糖苷酸酶(GUS)活性明显降低。利用实时荧光定量PCR对大田自然降温下Dn1中A、B、D三条同源染色体上的tae-miR444a初始体(pri-tae-miR444a)和TaMADS57进行表达模式分析,结果表明:分蘖节和叶片中pri-tae-miR444a-D和TaMADS57-D表达水平差异极显著。低温下外源施加脱落酸(ABA)促进麦苗内pri-tae-miR444a-D表达并抑制TaMADS57-D表达;茉莉酸甲酯(MeJA)处理抑制pri-tae-miR444a-D表达,但促进TaMADS57-D表达。以上结果表明,低温胁迫促进miR444a表达,从而抑制下游靶基因TaMADS57表达,其中D染色体上pri-tae-miR444a和TaMADS57作为主效基因,调控Dn1响应低温胁迫,并且外施ABA能够增强低温下小麦miR444a对TaMADS57的抑制作用,使Dn1的抗寒能力增强。

In order to explore the mechanisms of microRNA(miRNA)in winter wheat(Triticum aestivum)under cold stress,a winter wheat cultivar’Dongnong Dongmai 1’(Dn1)was used as material.The pre-taemiR444a with higher expression level was screened from previous miRNA database,and the potential target gene of TaMADS57 was predicted by psRNATarget program.The expression vectors containingβ-glucuronidase(GUS)gene labeled of pre-tae-miR444a and TaMADS57 were constructed and introduced into tobacco(Nicotiana benthamiana)leaves by Agrobacterium tumefaciens–mediated transformation system.The results of GUS staining showed that GUS activity of co-transformation leaves were significantly decreased.Real-time fluorescent quantitative analysis of Dn1 showed that there was a negative regulation relationship between miR444a and TaMADS57.The results of expression of primary miR444a(pri-tae-miR444a)and TaMADS57 on chromosome A,B and D of Dn1 under cold stress showed that the expression of pri-miR444a-D and TaMADS57-D were significantly in tillering nodes and leaves.Exogenous application of abscisic acid(ABA)under cold stress promoted pri-tae-miR444a-D expression and inhibited TaMADS57-D expression in wheat seedlings,and exogenous application of methyl jasmonate(MeJA)inhibited pri-tae-miR444a-D expression and promoted TaMADS57-D expression.Based on these data,we conclude that cold stress may promote the expression of miR444a to inhibit the expression of its target gene TaMADS57.The pri-tae-miR444a-D and TaMADS57-D may be the key genes to regulate Dn1 in response to cold stress.Moreover,exogenous application of ABA can promote the inhibitory effect of miR444a on TaMADS57 to enhance the cold tolerance of Dn1.