RNA沉默在植物逆境生物中的作用

RNA沉默在植物逆境生物中的作用

Abstract :Antimicrobial peptides, including defensins, are components of the innate immune system in ticks that have been shown to provide protection against both gram-negative and gram-positive bacteria. Varisin, one of the defensins identified in Dermacentor variabilis, was shown to be produced primarily in hemocytes but transcript levels were also expressed in midguts and other tick cells. In this research, we studied the role of varisin in the immunity of ticks to the gram-negative cattle pathogen,Anaplasma marginale. Expression of the varisin gene was silenced by RNA interference (RNAi) in which male ticks were injected with varisin dsRNA and then allowed to feed and acquire A. marginale infection on an experimentally-infected calf. Silencing expression of varisin in hemocytes, midguts and salivary glands was confirmed by real time RT-PCR. We expected that silencing of varisin would increase A. marginale infections in ticks, but the results demonstrated that bacterial numbers, as determined by an A. marginale msp4 quantitative PCR, were significantly reduced in the varisin-silenced ticks. Furthermore,colonies of A. marginale in ticks used for RNAi were morphologically abnormal from those seen in elution buffer injected control ticks. The colony shape was irregular and in some cases the A. marginale appeared to be free in the cytoplasm of midgut cells. Some ticks were found to be systemically infected with a microbe that may have been related to the silencing of varisin. This appears to be the first report of the silencing of expression of a defensin in ticks by RNAi that resulted in reduced A. marginale infections.

引自: Katherine M. Kocan Æ Jose´ de la Fuente Æ Rau´ l Manzano-Roman ÆVictoria Naranjo ÆWayne L. Hynes ÆDaniel E. Sonenshine Silencing expression of the defensin, varisin, in male Dermacentor variabilis by RNA interference results in reduced Anaplasma marginale infections Exp Appl Acarol (2008) 46:17–28 DOI 10.1007/s10493-008-9159-5

RNA 沉默在植物的逆境生理中具有重要作用,植物中已经发现了多种小RNAs, 如miRNAs 和siRNAs。其中siRNAs 可以由外源性核酸产生, 如侵入的病毒, 这种外源性siRNAs 是植物、真菌和一部分动物重要的抗病毒机制。siRNAs 也可以由植物自身基因组转录物产生, 如天然反义转录siRNAs (natsiRNAs)和长小干扰RNAs(long short interfering RNAs,lsiRNAs), 这些内源性siRNAs 和miRNAs 一样, 不仅可以调节植物基因表达, 而且在植物防御细菌和食草动物过程中也具有重要作用。病毒或细菌则分别进化出了病毒沉默抑制子(Viral suppressors of RNA silencing, VSRs)或细菌沉默抑制子(Bacterial suppressors of RNA silencing, BSRs)在不同阶段抑制植物的RNA 沉默反应。此外, miRNAs 或内源siRNAs 还可以和转录因子家族WRKY(Tryptophanarginine-lysine-tyrosine)相互作用, 参与植物的

生物逆境胁迫反应, 本文就RNA 沉默在植物生物逆境反应中的作用机制、VSRs 和BSRs 的研究新进展做一概述。

1 植物参与RNA 沉默途径的主要蛋白质因子

1.1 RNA 沉默的引发因子: Dicer-like 蛋白(Dicer-like proteins, DCLs)DCLs 可以认为是RNA 沉默的引发因子, 拟南芥(Arabidopsis thaliana L.)中有4 种DCLs: DCL1、DCL2、DCL3 和DCL4, 分别产生21 nt、22 nt、24 nt和21 nt-siRNAs。DCLs 具有以下特点: (1) 功能冗余和互补, 如dcl3 发生突变时, siRNAs 可由DCL1、DCL2 和DCL4 剪切产生; (2) 作用过程中具有顺序性, 往往在dcl4 发生突变时, DCL2 才发挥功能[1]; (3)相互配合与协作, 在沉默花椰菜花叶病毒(CaMV)时,

有多种DCLs 参与[2]。在上述4 种DCLs 中, DCL1主要参与miRNAs 途径, 对(+)RNA 病毒siRNAs 积累贡献不大, 但可以利用发夹结构RNAs(hairpinRNAs, hpRNAs)为前体形成siRNAs[3]。另外DCL1还参与nat-siRNAs[4]和lsiRNAs[5]的合成, 揭示DCL1 在防御DNA 病毒和细菌中具有重要作用。但芜菁萎缩病毒感染拟南芥时, DCL1 可以负调节DCL4 和DCL3 表达, 影响植物抗性[6]。DCL2 除了作为其他DCLs 的后备之外, 也涉及RNA 沉默转移性[7]及nat-siRNAs 合成[8]。DCL3 多在转录水平发挥作用, 可使植物转座成分、重复序列或病毒DNA 发生甲基化修饰[9, 10], 也可在DCL4 上游发挥作用增强siRNAs 的积累[11]。虽然现在还不知道长距离转运的RNA 沉默信号的本质, 但已经发现DCL3 参与这种沉默信号的接收过程[12]。DCL4 是拟南芥防御病毒的主要DCLs, 因为病毒感染的细胞中往往21 nt-siRNAs 最多[10]。此外, DCL4 还参与细胞对细胞的沉默信号的产生和接受[13], 长距离沉默信号的接受和次级siRNAs 的引物合成。

1.2 RNA 沉默的增强因子: 依赖RNA 的RNA 聚合酶(RNA dependent RNA polymerases, RDRs)

RDRs 不仅能够产生次级siRNAs, 使RNA 沉默效应放大, 还可在RNA 沉默

上游识别病毒, 也许病毒RNA 无帽子或无poly(A)的特点被RDRs 识别并引发RNA 沉默效应。而生物自身有多种酶, 可防止自身正常mRNAs 进入RNA 沉默途径造成伤害[14]。拟南芥中有6 种RDRs, RDR1 和RDR6 是防御病毒的主要RDRs, 其中RDR1 的表达可被水杨酸强烈诱导[11]。烟草(Nicotiana rustica

L.)RDR1 则参与烟草防御食草动物的反应[15, 16]。RDR6 不仅涉及转基因沉默、病毒防御、nat-siRNAs 合成、反式作用siRNAs (trans-acting siRNAs, ta-siRNAs)