Pocket K No.34:用于作物改良的RNAi

介绍

RNA interference (RNAi) is a method of blocking gene function by inserting short sequences of ribonucleic acid (RNA) that match part of the target gene's sequence,thus no proteins are produced.Since Science named it as "Breakthrough of the Year" and Fortune magazine hailed it as "Biotech's Billion Dollar Breakthrough" in 2003,RNAi作为研究人员研究重要基因的结构和功能的首选方法,已经得到了显著的重视。

RNAi有潜力成为针对性和个性化药物的强大治疗方法。它可能解决的疾病和紊乱的范围是前所未有的;从癌症到心血管疾病,神经退行性疾病甚至艾滋病。更令人兴奋的是RNAi在农业领域的潜力。RNAi提供了一种防治病虫害的方法,引进新的植物性状,提高作物产量。Using RNAi,scientists have developed novel crops such as nicotine-free tobacco,non-allergenic peanuts,无咖啡因咖啡,以及营养强化玉米。

Discovery of RNAi

完整的植物基因组序列为改善植物特性在基因改造中带来了新的维度。Scientists believed that they could produce any gene products by just introducing foreign genes in plants,which was not always the case.Plant biologists found out that introducing multiple copies of a gene that codes for purple petunia flowers led,not as expected to a deeper purple hue,but rather to plants with white or variegated flowers.通过一种未知的机制,引进的转基因和植物的“紫色花”基因都被沉默了。2,3.在另一项研究中,当植物感染含有植物基因片段的RNA病毒时,也观察到基因沉默。

直到美国科学家安德鲁费尔(AndrewFire)和克雷格·梅洛(CraigMello)发现向蠕虫体内注入双链核糖核酸(DSRNA)后,才知道造成这些影响的机制。Caenorhabditis eleganstriggered the silencing of genes with sequences identical to that of the dsRNA.他们称这种现象为RNA干扰。Re-examining the co-suppression pathway in petunia and virus-induced gene silencing revealed that all these processes led to the accumulation of dsRNAs,因此RNAi通路。Fire and Mello were awarded the 2006 Nobel price for Physiology or Medicine for their discovery.

In addition to its roles in regulating gene expression,RNAi被用作对感染的免疫应答。5and as a natural defense mechanism against molecular parasites such as jumping genes and viral genetic elements that affect genome stability6.特定类型的细菌也被证明能触发植物的RNAi通路。

RNAi是如何工作的

  1. 长双链RNA的进入,例如导入的转基因,a rogue genetic element or a viral intruder,触发细胞的RNAi通路。这导致了酶切器的招募。
  2. Dicer将dsrna剪短,20-25 basepairs long,碎片,称为小干扰RNA(sirna)。
  3. An RNA-induced silencing complex (RISC) then distinguishes between the two siRNA strands as either sense or antisense.感觉链(与目标基因序列完全相同)被降解。
  4. The antisense strands on the other hand are incorporated to the RISC.These are used as guide to target messenger RNAs (mRNA) in a sequence-specific manner.
  5. 信使RNA(mRNA)哪些是氨基酸的代码,被RISC割裂。激活的RISC可反复参与mRNA降解,inhibiting protein synthesis.


Figure 1.RNAi机制

用于抗病和病原抵抗的RNAi

Gene silencing was first used to develop plant varieties resistant to viruses.植物中的工程抗病毒策略模拟天然RNA沉默机制。This was first demonstrated when scientists developed Potato virus Y- resistant plants expressing RNA transcripts of a viral proteinase gene7、8.从那时起,对黄瓜和烟草花叶病毒等其他病毒的免疫力就得到了证实。Tomato Spotted Wilt Virus,黄豆花叶病毒,香蕉苞叶花叶病毒,还有水稻通格罗杆菌型病毒等。
In addition,植物也可以被修饰产生抑制害虫和寄生线虫基本基因的dsrna。This approach was used to develop root-knot nematode9,corn rootwormand cotton bollworm11  resistant varieties.

男性不育RNAi

RNAi has also been used to generate male sterility,which is valuable in the hybrid seed industry.Genes that are expressed solely in tissues involved in pollen production can be targeted through RNAi.例如,科学家通过抑制TA29的表达,培育出雄性不育烟草系。花粉发育所必需的基因12.RNAi也被用来破坏烟草和番茄中msh1的表达,导致与自然发生的细胞质雄性不育相关的线粒体DNA的重排。十三.

RNAi and Plant Functional Genomics

A major challenge in the post-genomic era of plant biology is to determine the functions of all genes in the plant genome十六.Compared to other techniques,RNAi在沉默一个基因或多个基因家族成员方面提供了特异性和有效性。In addition,the expression of dsRNAs with inducible promoters can control the extent and timing of gene silencing,such that essential genes are only silenced at chosen growth stages or plant organs14,15.

There are several ways of activating the RNAi pathway in plants.各种RNAi技术的优缺点在于其效果的持久性和可应用的植物范围。这些包括使用发夹RNA表达载体,particle bombardment,农杆菌介导的转化及病毒诱导基因沉默(VIGS)十六.

通过RNAi工程化植物代谢途径

RNAi has been used to modify plant metabolic pathways to enhance nutrient content and reduced toxin production (Summarized in Table 1).The technique takes advantage of the heritable and stable RNAi phenotypes in plants.

表1。通过RNAi设计的新植物特性的例子。

Trait 靶基因 宿主 应用
Enhanced
营养成分
Lyc Tomato 增加番茄红素(类胡萝卜素抗氧化剂)浓度
DET1 Tomato 类黄酮和B-胡萝卜素含量较高
SBEII 小麦,红薯,玉米 提高直链淀粉水平以控制血糖和促进消化健康
FAD2 油菜,Peanut,Cotton 油酸含量增加
SAD1 Cotton 硬脂酸含量增加
ZLKR/SDH 玉米 Lysine-fortified maize
Reduced alkaloid production CAMXMT1 咖啡 无咖啡因咖啡
慢性阻塞性肺病 Opium poppy Production of non-narcotic alkaloid,而不是吗啡
CYP82E4 Tobacco 降低治愈叶片中致癌物去甲尼古丁的含量
重金属
accumulation
丙烯醛 拟南芥 Arsenic hyperaccumulation for phytoremediation
还原多酚
production
S-卡地宁合酶基因 Cotton 棉籽中棉酚含量较低,为了安全消费
Ethylene
sensitivity
LeETR4 Tomato 早熟番茄
ACC氧化酶基因 Tomato 由于成熟缓慢,保质期更长。
减少
过敏原性
Arah2 花生 Allergen-free peanuts
Lolp1,Lolp2 黑麦草 Hypo-allergenic ryegrass
Reduced production of lachrymatory factor synthase lachrymatory factor synthase gene 洋葱 “无泪”洋葱

RNAi的前景

With RNAi,利用一个经过彻底设计的单一转化结构,可以将多个基因作为沉默的靶基因。此外,RNAi还可以提供广谱的抗高变异性病原体的能力,类病毒9.最近的研究暗示了RNAi相关过程在植物逆境适应中的可能作用。

尽管在过去几年里RNAi领域取得了很大进展,RNAi在作物改良方面的全部潜力仍有待实现。RNAi通路的复杂性,the molecular machineries,and how it relates to plant development are still to be elucidated.

References

  1. Tang G.等。(2007)代谢组学3:357-369
  2. 诺维娜and Sharp P.A.(2004)自然430:161-164
  3. 那不勒斯湾等。(1990)植物细胞4:279-289
  4. 火A等。(1998)自然391:806-811
  5. 弗里茨J.S.等。(2006)科学STKE 339:27
  6. Gupta P.K.(2006) Current Science 91:1443-1446
  7. Mansoor S.等。(2006) Trends in Plant Science 11:559-565
  8. Qu J.等。(2007) Journal of Virology 81: 6690-6699
  9. 黄庚等。(2006) PNAS 103: 14302-1406
  10. 毛依。(2007) Science 25: 1307-1313
  11. Baum J.A.(2007) Nature Biotechnology 25:1322-1326
  12. Nawaz-Ul-Rehman M.S.等。(2007)分子生物技术36:159-165
  13. Sandhu A.S.等。(2007) PNAS 104: 1766-1770
  14. Matthew L(2004)比较基因组学和功能基因组学5:240-244
  15. 佐藤(2005)植物生物技术22:431-442
  16. Burch-Smith T.M.等。(2004) The Plant Journal 39:734-746
  17. 苦萨巴等。(2003) Plant Cell 15: 1455-1467

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