Xu Juan, Zhang Shuqun
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, China,
Methods Mol Biol. 2014;1171:91-103. doi: 10.1007/978-1-4939-0922-3_8.
Arabidopsis genome contains 20 genes encoding mitogen-activated protein kinases (MAPKs, or MPKs), and ten genes encoding MAPK kinases (MAPKKs, or MKKs), the upstream kinases that activate MAPKs in the signaling cascades. They play critical roles in many different biological processes ranging from growth/development to response to environmental stimuli and pathogen invasion. T-DNA knockout lines are not currently available for all these genes. There is also functional redundancy at both MAPK and MAPKK levels. In addition, embryo lethality is associated with some double mutant combinations, which makes it difficult to investigate their specific functions in plants. In such situation, the use of RNA interference technology by which mRNA of interested gene is targeted by double-stranded RNA (dsRNA) for degradation and gene silencing provides a powerful tool for loss-of-function analyses. In this chapter, we describe the hairpin-RNA interference (hpRNAi) method we employed to silence MPK3/MPK6 and their upstream MKK4/MKK5 in the model plant Arabidopsis, with particular emphasis on the generation of hpRNAi constructs for single gene RNAi, tandem RNAi of two MAPKK genes, and tissue-specific RNAi.
拟南芥基因组包含20个编码促分裂原活化蛋白激酶（MAPKs，或MPKs）的基因，以及10个编码MAPK激酶（MAPKKs，或MKKs）的基因，后者是在信号级联反应中激活MAPKs的上游激酶。它们在从生长/发育到对环境刺激和病原体入侵的反应等许多不同的生物学过程中发挥着关键作用。目前并非所有这些基因都有T-DNA敲除株系。在MAPK和MAPKK水平上也存在功能冗余。此外，胚胎致死与一些双突变组合有关，这使得研究它们在植物中的特定功能变得困难。在这种情况下，利用RNA干扰技术，即通过双链RNA（dsRNA）靶向感兴趣基因的mRNA进行降解和基因沉默，为功能缺失分析提供了一个强大的工具。在本章中，我们描述了我们用于在模式植物拟南芥中沉默MPK3/MPK6及其上游MKK4/MKK5的发夹RNA干扰（hpRNAi）方法，特别强调了用于单基因RNAi、两个MAPKK基因的串联RNAi和组织特异性RNAi的hpRNAi构建体的生成。
