FmJAZ1基因瞬时侵染水曲柳对 JA通路相关基因表达的影响

Effects of transient infection of FmJAZ1 gene on JA pathway related gene expression in Fraxinus mandshurica

关键词:  水曲柳,FmJAZ1基因,瞬时侵染,JA通路,基因表达

Key words:  Fraxinus mandshurica, FmJAZ1 gene, transient infection, JA pathway, gene expression

摘要:

该文对水曲柳JAZ蛋白家族的一员FmJAZ1的功能及对其上下游基因影响进行了初步分析。首先,利用无缝克隆的方法构建FmJAZ1-pROK2-GUS过表达载体,利用三亲杂交的方法将载体转入农杆菌;然后,使用农杆菌对水曲柳组培苗进行瞬时侵染,得到FmJAZ1过表达的侵染苗;最后,在侵染后36h使用茉莉酸合成途径抑制剂(DIECA)对侵染苗进行处理,分别取0、1、3、6、18、21、24h七个时间点的样品,通过荧光定量PCR对FmJAZ1、FmJAZ2、GL1、EIN3、MYC25个基因的表达进行了分析。结果表明:农杆菌瞬时侵染水曲柳幼苗后,FmJAZ1表达显著升高,为空载侵染的3.2倍,说明FmJAZ1瞬时转入水曲柳并完成基因表达,侵染有效。经过DIECA处理的侵染苗FmJAZ1的相对表达量初期下降并出现明显波动,18h后恢复平稳,证明它是JA通路的作用基因。同时检测了4个JA通路相关基因的表达,在1h时JAZ2、GL1表达下调,其余均有轻微上调,随后各基因表达均有上调。FmJAZ1瞬时转化水曲柳后FmJAZ1过表达,说明瞬时侵染有效;DIECA处理后FmJAZ1表达显著下调说明FmJAZ1的合成受JA调控。在水曲柳中,FmJAZ1抑制转录因子GL1、EIN3、MYC2、FmJAZ2的表达,且FmJAZ2的合成也受JA调控。综上结果表明,JAZs不仅调节JA通路的关键蛋白,还参与其他信号通路的调节,最终通过体内JA的表达与其他相关信号分子的协同表达来调节植物的生长发育及其对应激的反应。

Abstract:

In this paper, the function of FmJAZ1, a member of the Jasmonates(JAZs), and the influence of its upstream and downstream genes were preliminarily analyzed. Firstly, the FmJAZ1-pROK2-GUS overexpression vector was constructed by using the method of seamless cloning, and then the vector was transferred into Agrobacterium by triparental hybridization. Then, Agrobacterium was used to conduct instantaneous infection on the tissue culture seedlings of Fraxinus mandshurica, and the infected seedlings with FmJAZ1 overexpression were obtained. Finally, the infected seedlings were treated with jasmonic acid synthetic pathway inhibitor(DIECA)at 36 h after infection. Samples were taken at 0, 1, 3, 6, 18, 21, 24 h, respectively. The expressions of FmJAZ1, FmJAZ2, GL1, EIN3 and MYC2 were analyzed by fluorescence quantitative PCR. After Agrobacterium instantaneously infected the tissue culture seedlings of Fraxinus mandshurica, FmJAZ1 expression increased significantly, which was 3.2 times as much as that of no-load infection. After treatment with DIECA, the relative expression level of FmJAZ1 fluctuated significantly at the initial, but was stable after 18 h, which proved that FmJAZ1 was acting on JA pathway. The expressions of JAZ2 and GL1 are down-regulated at 1 h, while the others were slightly up-regulated, and later the expressions of all the genes were up-regulated. Overexpression of FmJAZ1 after instantaneous infection of F. mandshurica indicated that transient infection was effective. After DIECA treatment, FmJAZ1 expression was significantly down-regulated, indicating that the synthesis of FmJAZ1 was regulated by JA. In Fraxinus mandshurica, FmJAZ1 inhibited the expression of transcription factors GL1, EIN3, MYC2, and FmJAZ2, and the synthesis of FmJAZ2 was also regulated by JA. JAZs not only regulates the key proteins of JA pathway, but also participates in the regulation of other signaling pathways, and finally regulates the growth and development of plants and their response to stress through the expression of JA in vivo and the synergistic expression of other related signaling molecules.