分离叶测定,以简化马铃薯中基因表达研究,在咀嚼昆虫曼杜卡性塔感染
Summary
该方法通过将曼杜卡性幼虫应用于马铃薯分离叶,创造出天然的草食性植物组织受损。植物组织被测定为六个转录因子同源的表达参与早期反应昆虫食草动物。
Abstract
昆虫食草动物基因表达研究的多营养性需要大量的生物复制,因此需要更简单、更简化的食草动物方案。咀嚼昆虫的扰动通常在整个植物系统中进行研究。虽然这整个有机体策略很受欢迎,但如果类似的观察结果可以在单个分离的叶子中复制,则没有必要。假设信号转导所需的基本元素存在于叶本身中。在信号转导的早期事件的情况下,细胞只需要从扰动接收信号,并将该信号传输到被测定为基因表达的相邻细胞。
建议的方法只是改变了分遣队的时间。在整个植物实验中,幼虫被限制在一片叶子中,最终从植物分离并进行基因表达的测定。如果切除的顺序颠倒,从整个植物研究的最后一个,到分离研究的第一个,喂养实验被简化。
索兰图管龙和葡萄球,Kennebec通过节点转移在简单的组织培养基中传播,并转移到土壤中,如果需要,进一步生长。叶子从母植物被切除,并转移到培养皿,在那里喂养测定进行与M.sexta的幼虫阶段。对受损的叶组织进行测定,以表达信号转导中相对早期的事件。基因表达分析确定了侵扰性特异性Cys2-His2(C2H2)转录因子,确认在早期反应研究中使用分离叶的成功。该方法比整个植物的侵扰更容易执行,并且使用更少的空间。
Introduction
Herbivory 启动一系列分子事件,在此期间,植物可以识别攻击并对其生存作出适当反应。植物从咀嚼昆虫中接收两个基本线索;一个来自对组织的物理损害,另一个来自昆虫特有的物质。损伤相关的分子模式 (DAMPs) 被释放,以响应由幼虫口腔部分造成的损伤,并触发一个明确的伤口反应,导致激素茉莉花酸的增加和防御基因1的转录。其中最著名的DamPs是系统蛋白,一种多肽,由较大的亲系统蛋白的裂解形成后,叶受伤2,3。茉莉花酸伤口反应进一步由草食相关分子模式(HAMPs)调节,可以从毛毛虫唾液、肠道内容物(胃肠)和粪便(frass)4中得出。昆虫使用这些物质来增强或逃避防御反应5。转录因子然后通过调节下游防御基因6,7,8在防御反应中传递来自激素信号的信息。
实验室设置中使用的一些植物-昆虫相互作用研究是模拟类型的,目的是接近昆虫的自然喂养方法。模拟食草动物通常是通过各种工具对植物组织造成人工损伤来实现的,这些工具模拟昆虫嘴部的特定机制,足以引起DAMPs的释放并触发防御基因的产生。其他昆虫特异性成分,如口服分泌物或回流剂,经常被添加来复制从HAMPs9,10,11的贡献。创建特定尺寸和类型的伤口以及应用精确数量的 HAMPs 是这些类型的研究的一个优势,并可提供更可重复的结果。自然草本动物研究,其中对植物组织的损害是通过应用田间获得或实验室饲养的昆虫来完成的,往往更具挑战性,因为伤口大小和HAMP量受昆虫行为控制,并增加了变化数据。自然与模拟方法及其优缺点在文献第12、13、14中得到了很好的争论。
为了研究早期信号事件,如转录因子,必须在相对短的时间内食用一定比例的叶子,因此幼虫必须立即开始咀嚼并保持食用,直到叶子被冷冻进行分析。M. sexta是多索拉动物植物的贪婪的喂食器,在其许多幼虫阶段,使它理想的在相对短的时间内给予最大的伤害15。这在研究早期信号事件时很方便,因为昆虫接触叶面16、17后,植物反应几乎立即发生。常用的夹笼围堵方法证明笨拙,因为多个笼子需要在整个实验过程中不断调整,以便去除或添加幼虫。叶子也必须足够大和足够强大,以支持多个昆虫同时觅食。这些类型的马铃薯植物需要大量的空间来观察喂养。幼虫经常搬迁到叶子表面的底面,这也使得喂食观察相当困难。使用整个工厂进行这些实验显然很麻烦。
目前的研究使用分离的叶子隔离在培养皿,而不是整个植物,以简化和简化整个植物方法,研究草本植物。本研究对该方案的应用仅限于观察M.性幼虫在草食性损伤后马铃薯叶早期诱发的一组C2H2转录因子。
Protocol
注: 以下协议是为一个人设置、进行观察和收集样本而设计的。可以组合相同设置的多个运行,以增加生物复制。实验的任何额外重复都应在一天的同一时间进行,以消除对基因表达的可能日影响。该协议旨在为 5 个单独的收获时间点创建 3 个”受感染”的叶子。每个时间点的匹配控制叶总共创建 30 个样本。实验可以进行各种叶片大小和幼虫阶段,但建议叶的大小,幼虫阶段和侵扰时间在整个过程中保持?…
Representative Results
叶消耗定义协议的成功。健康、准确分级的幼虫应在放置在叶面后立即开始喂食,并且在整个侵扰时间继续以相当一致的方式进食。在视频1中,顶部的幼虫在放置后立即开始咀嚼,并在喂食时保持一致的速率。如果对感染后的早期基因表达事件进行分瓶,这一点尤其重要。底部的幼虫不消耗任何叶物质,是一次不成功的侵扰。 <p class="jove_content" fo:keep-together.within-…
Discussion
使用现有的整个植物食草方法对于实现这一特定研究的目标(即筛选一组候选基因以对感染的反应)是不必要的。分离叶细化的明显好处是缩短了进行草本测定所需的时间。整个植物与夹笼的笨拙性质被消除,并更快地进行检测,因为植物只有2周,可以用来收获叶子。在喂食过程中,它也需要更小的占地面积和更少的生长室空间;当这些资源有限时,两者都很重要。
当对防御基因表达?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者要感谢鲍勃·法拉尔和亚历克西斯·帕克提供了在这项研究中使用的昆虫,以及他们在幼虫分期方面的专长。此外,感谢迈克尔·布莱克本和赛卡特·戈什对手稿的批判性评论。
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Materials
| agar substitute | PhytoTechnology Laboratories | G3251 | product is Gelzan |
| containment vessel (6,12 or 24 well dish) | Fisher Scientific | 08-772-49, 08-772-50, 08-72-51 | many other companies sell these products |
| manduca eggs | Carolina Biological Supply Company | 143880 | 30-50 eggs |
| manduca eggs | Great Lakes Hornworm | NA | 50, 100, 250 or 500 eggs |
| manduca larvae | Carolina Biological Supply Company | call for specific larval instar requests | any instar |
| manduca larvae | Great Lakes Hornworm | call for specific larval instar requests | any instar |
| microcentrifuge tubes, 1.7 ml | Thomas Scientific | 1158R22 | these have been tested in liquid N2 and will not explode |
| Murashige & Skoog (MS) Basal Medium w/Vitamins | PhytoTechnology Laboratories | M519 | used to make propagation medium |
| nutrient agar mix | PhytoTechnology Laboratories | M5825 | product is Murashige & Skoog Basal Medium with vitamins, sucrose, and Gelzan |
| paper filter discs | Fisher Scientific | 09-805A | Whatman circles-purchase to fit in petri dish |
| petri dish, 60X15 mm or 100X15 mm | Fisher Scientific | FB0875713A or FB0875712 | purchase size appropriate for leaf size |
| potato tubers | any | B size (not organic) | suggest Maine Farmer’s Exchange |
| pots, 10" | Griffin Greenhouse Supplies, Inc. | 41PT1000CN2 | |
| preservative/biocide | Plant Cell Technology | NA | product is PPM (Plant Preservative Mixture) |
| seed potatoes for explant source | any | B size (not organic) | suggest Maine Farmer’s Exchange |
| slow release fertilizer (14-14-14 ) | any | NA | Osmocote is a popular brand name |
| soft touch forceps | BioQuip | 4750 | |
| soil mix | Griffin Greenhouse Supplies, Inc. | 65-51121 | product is Sunshine LC1 mix |
| sterile culture vessel | PhytoTechnology Laboratories | C2100 | Magenta-type vessel, PTL-100 |
| sterile culture vessel | Fisher Scientific | ICN2672206 | product is MP Biomedicals Plantcon |
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Citar este artigo
Novak, N. G., Perez, F. G., Jones, R. W., Lawrence, S. D. Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta. J. Vis. Exp. (147), e59153, doi:10.3791/59153 (2019).