CN112626071B - 一种RNA抑菌剂miRNA482a及作物病菌抑制剂 - Google Patents
一种RNA抑菌剂miRNA482a及作物病菌抑制剂 Download PDFInfo
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Abstract
本发明提供一种RNA抑菌剂miRNA482a，其具有SEQ ID NO.1所示的RNA序列。本发明还提供一种作物病菌抑制剂，所述抑制剂中至少包含miRNA482a。具体的，所述抑制剂可以为包含所述miRNA482a的喷雾剂，通过喷雾来实现对灰霉病的有效预防和治疗。本发明施加miRNA482a的溶液能显著抑制灰霉菌孢子的萌发以及灰霉菌对植物的感染毒力，可用于蔬菜作物的灰霉病的防治。
Description
技术领域
本发明涉及作物灰霉病菌的保护和治疗，具体涉及RNA抑菌剂miRNA482a及一种作物病菌抑制剂。
背景技术
灰霉病是由灰葡萄孢菌(Botrytis cinerea)引起的植物性真菌病害,可侵染番茄、草莓、辣椒、葡萄、菜豆、韭菜等200多种水果和蔬菜，寄主范围极其广泛。灰霉病主要是通过气流传播，并且传播速度很快，使得该病在番茄的设施农业栽培过程中的防治非常困难。随着设施栽培农业的种植面积不断扩大，塑料大棚、温室、小拱棚等保护设施栽培番茄的灰霉病也随之普遍发生，一般年份减产达到20％～30％，严重时达到50％以上。因此，番茄灰霉病的危害日趋严重，已成为保护地番茄产业发展的主要限制因素。目前生产上对于灰霉病的防治仍然以甲酰胺、嘧霉胺、乙霉威、多菌灵、腐霉利和甲基硫菌灵等化学农药为主，农民为了追求短期防效，过量重复用药，造成了灰霉菌抗药性增加，蔬菜和水果中农药残留量增大，对人类健康产生巨大危害。因此迫切的需要一些可持续且有效，环境友好的新型药物来防治灰霉病。
MiRNA是一类不编码蛋白质的小分子RNA，其广泛存在于动植物的各类细胞中。但是动物miRNA和植物miRNA之间却存在诸多差异，所以人们普遍认为植物中的miRNA很难在动物体内出现，乃至发挥功能。但是，最近的大量研究表明miRNA可以在相互作用的动物和植物、植物和微生物以及动物和微生物之间相互传播，并且可在对方中诱导基因沉默，这种机制被称为“跨物种RNAi”。
在植物与病原菌的相互作用过程中，植物和病原菌通过这种“跨物种RNAi”来杀死病原菌或调解植物宿主的免疫力。2013年，Weiberg等人证明了灰霉菌驱动的miRNA可以转移到拟南芥中，并且可以通过沉默宿主植物的Ago1基因来提高拟南芥对灰霉菌的易感性。Wang等人也发现，灰霉菌可以通过诱导拟南芥中的沉默机制将其miRNA转移到植物中并抑制靶基因的表达，从而增加植物对病原菌的敏感性。从感染了大丽花病的植物组织中回收的黄萎病菌菌丝中已发现大量棉花miRNA，并且一些棉花起源的miRNA可以靶向与之相互作用的真菌毒力基因。蔡等成功地将拟南芥中的miRNA通过细胞外囊泡转移至真菌病原体内，然后在病原体内沉默了相应的真菌靶基因。侯等人还发现拟南芥可以通过细胞外小泡将miRNA传递到疫霉菌中，并使疫霉菌中的靶基因沉默。以上研究表明miRNA的跨界干扰使miRNA在植物与病原菌之间相互传递，并通过RNAi沉默目标宿主中的靶基因。植物通过将miRNA传递到相互作用的微生物中来抑制其毒力的最新发现为植物病害的控制创造了新思路。
将miRNA通过化学方法直接合成，将其称之为miRNA药物。通过以病原体基因为靶标的双链(ds)RNA的转基因表达，诱导宿主基因沉默(HIGS)有可能成为一种重要的疾病控制方法。将dmiRNA或smiRNA直接应用到宿主植物上会导致目标微生物/害虫基因沉默(称为喷雾诱导基因沉默，SIGS)，这种方法对病原菌具有有效的控制能力。有趣的是，局部喷洒的RNA也能抑制远端非喷雾叶片的病原菌毒力，表明这些RNA能够在植物体内全身扩散。纳米粒子技术的最新进展改善了SIGS在植物保护中的潜在应用。小RNA抑制的论证，增加了新一代生物杀虫剂使用的可能性，这种杀菌剂可能可以很有效地同时控制多种疾病。
尽管RNAi的成本效率仍有待研究，但现有研究已经表明，RNAi作为新时代的杀菌剂具有巨大的影响力，比目前基于化学方法的杀菌剂更具有可持续性。而且RNA是我们摄取的几乎所有食物中都存在的生物分子，RNA的局部应用不会在田间或环境中留下有毒残留物，也不会引起作物基因的任何潜在改变。由于这些原因，我们期望新的比化学杀菌剂和转基因生物更便捷、更环保的RNA“杀菌剂”。
发明内容
本发明的一个目的在于提供一种RNA抑菌剂miRNA482a，其具有SEQ ID NO.1所示的RNA序列(UUUCCAAUUCCACCCAUUCCUA)。
本发明的另一个目的在于提供一种作物病菌抑制剂，所述抑制剂中至少包含miRNA482a。
本发明所述抑制剂可抑制的病菌至少包括灰霉菌，及其同属病菌。
在某些实施例中，所述抑制剂为包含所述miRNA482a的喷雾剂，通过喷雾来实现对灰霉病的有效预防和治疗。
本发明的有益效果主要体现在：
1、施加miRNA482a的溶液能显著抑制灰霉菌孢子的萌发以及灰霉菌对植物的感染毒力，可用于蔬菜作物的灰霉病的防治。
2、miRNA482a为植物体内产生，安全可靠。
3、基于SIGS技术防治作物病害的另一个优势在于RNA作为抑菌因子，具有环境友好、专一性强、抑菌效果良好等特性，是未来农药发展的主要方向之一。
附图说明
图1为miRNA482a对灰霉病菌孢子萌发的抑制效果。
图3miRNA482a对灰霉病菌菌丝侵染叶片的抑制效果(A)及病斑直径(B)。
图2miRNA482a对灰霉病菌孢子侵染叶片的抑制效果(A)及病斑直径(B)。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
一、实验准备
(1)miRNA482a药物的合成
在吉玛基因公司(中国上海)合成了miRNA482a药物。每管miRNA482a药物1OD，按照说明书要求加入无RNA酶的ddH2O，miRNA482a药物的最终浓度为10μM。
(2)番茄灰霉菌孢子的培养
选取完整的无伤口的番茄，在超净工作台中用75％的无水乙醇反复擦洗5遍。如果番茄根部有蒂，最好把蒂去除。去除时注意不要造成伤口，伤口会使灰霉菌在培养的过程中容易污染。用灭过菌的手术刀在番茄上切出几个伤口，然后将灰霉菌块覆盖在番茄表面的伤口上。灰霉菌块是培养在固体PDA培养基上。将番茄放在架子上，然后置于盆中。放置前要将架子和盆用75％的无水乙醇多喷洒几遍，保证消毒彻底，可以减少灰霉菌培养过程中的污染。然后把灰霉菌放在22℃的潮湿的培养箱中进行培养。培养时一定要注意灰霉菌的保湿。2周后，番茄表面会长满灰霉菌菌丝及孢子，用作实验。采取灰霉菌孢子时，用刷子轻刷番茄的表面，溶于ddH2O。然后通过玻璃棉过滤得到灰霉菌的孢子。将过滤出来的灰霉菌孢子在无菌蒸馏水中洗涤两次。用细胞计数板进行计数，并调节至5×106分生孢子/mL的浓度。用于实验中的生物测定。
二、miRNA482a对灰霉菌孢子萌发的影响
用Bilir等人描述的方法来进行分生孢子的萌发实验。简单来说，就是将玻璃纸剪成大小为1.0cm X 1.0cm大小，然后在高压灭菌锅下，115℃，灭菌15min，备用。灭菌时，将剪好的玻璃纸放入无菌水中在放进灭菌锅。然后在超净工作台中，将玻璃纸置于无添加抗生素的1/2MS培养基上。然后用移液枪在玻璃纸上滴加5μL的分生孢子的悬浮液，同时加入5μL的miRNA药物(终浓度为：10uM)；
对照组1以等量的NC RNA代替miRNA482a药物，NC RNA是一条长度为21nt的RNA，但它不能靶向番茄和灰霉病菌的任何基因，也就是在番茄和灰霉病菌的RNA上没有结合位点。
对照组2：以等量的miR1001代替miRNA482a药物，miR1001是一条已报道对灰霉病菌生长及植物感染有抑制作用的番茄miRNA。
将培养基放置于24℃的培养箱中进行培养。12h后在光照显微镜下观察分生孢子的萌发情况。结果如图1所示，miRNA482a处理后绝大部分的灰霉菌孢子没有萌发，而未经miRNA482a药物处理的对照组孢子萌发效率极高，长势良好；经miR1001处理的灰霉菌孢子虽有萌发，但萌发效率低，萌发后的菌丝生长慢。这些结果表明施加miRNA482a对灰霉菌孢子的萌发具有明显的抑制效果。
三、miRNA482a对灰霉病菌孢子感染植物叶片的影响
取上述浓度为5×106个/mL的孢子溶液5μL，加入miRNA482a使终浓度达到10μM，混匀后将10μL的miRNA482a-孢子混合液滴加到离体的烟草叶片上；
对照组1以等量的NC RNA代替miRNA482a药物与孢子混合液后滴加到离体的烟草叶片上。
对照组2：以等量的miR1001代替miRNA482a药物与孢子混合液后滴加到离体的烟草叶片上。
实验设3个重复。处理叶片在24℃光照培养箱中保温保湿培养3天后，对灰霉菌的侵染程度进行观测并拍照记录。并且进行了台盼蓝染色处理。侵染叶片时，为了方便拍照和后期的台盼蓝染色处理，我们会采用离体的番茄叶片及烟草叶片。培养时，要注意离体叶片的保湿。我们将离体叶片的根部用湿棉花包裹起来，培养期间小心地往棉花上喷无菌水。喷水时注意不要喷到植物叶片上，防止对实验结果造成影响。结果如图2所示，未经miRNA482a处理，灰霉菌孢子在叶片上的病斑明显大于经miR1001处理的病斑,而miR001处理的病斑也明显大于miRNA482a处理的病斑。经测量病斑直径，NC RNA处理的病斑平均直径约为17mm左右；miR001处理的病斑平均直径约为5mm，而经miRNA482a处理的病斑平均直径非常小，小于2mm左右(图2)。这些结果表明，相比NC RNA和miR1001，施加miRNA482a对灰霉病菌孢子侵染植物叶片的毒力抑制更好。
四、miRNA482a对灰霉菌菌丝侵染番茄叶片的抑制效果
从PDA固体培养基上刮取约10mg灰霉菌菌丝，将其放置于植物的表面，然后往灰霉菌菌丝上添加5μL浓度为10μM的miRNA482a药物，使药物完全覆盖灰霉菌的菌丝。
对照组1中以等量的NC RNA代替miRNA482a药物。
对照组2：以等量的miR1001代替miRNA482a药物。
将上述处理好的样品放在24℃光照培养箱中保温保湿培养3天后，观察叶片菌斑大小。结果如图3所示，经miRNA482a处理后，灰霉病菌孢子在叶片上的病斑明显小于经NCRNA处理的对照1和经miR1001处理的对照2上的病斑。测量病斑直径，对照1组叶片的病斑平均直径约为15mm；对照2的平均直径约为6mm；而经miRNA482a处理的病斑直径小于3mm(图3)，也证实施加siR2对灰霉病菌菌丝的侵染毒力抑制效果好于NC RNA和miR1001。
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
序列表
<110> 浙江理工大学
<120> 一种RNA抑菌剂miRNA482a及作物病菌抑制剂
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213> 未知(Unknown)
<400> 1
uuuccaauuc cacccauucc ua 22
Claims (1)
1. 一种RNA抑菌剂miRNA482a在防治灰霉菌的应用，所述RNA抑菌剂miRNA482a的序列如SEQ ID NO.1所示。
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