CN105779502A - 提高咀嚼式口器昆虫rna干扰效率的方法 - Google Patents
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Abstract
本发明提供一种提高咀嚼式口器昆虫RNA干扰效率的方法，包括：1)用无RNA酶的水将靶基因特异性siRNA配成一定浓度的溶液；2)将人工饲料加工成3～5mm厚、直径1～2cm的圆片，置于24孔板中；3)取1)的溶液加在2)的圆片上，使溶液被饲料完全吸收；4)将初孵的咀嚼式口器昆虫幼虫，放入3)的有饲料圆片的24孔板中，饲喂一段时间后，检测昆虫中靶基因的表达量。本发明通过对饲喂昆虫siRNA的方法进行改进，建立一种提高咀嚼式口器昆虫RNA干扰效率的方法。该方法高效、简单，可操作性强，节约了siRNA的用量，干扰效果明显，为研究咀嚼式口器昆虫基因的功能提供技术支持。
Description
技术领域
本发明涉及生物技术领域，具体地说，涉及一种提高咀嚼式口器昆虫RNA干扰效率的方法。
背景技术
小分子量RNA(smallRNA)是一类长度约为20-30个核苷酸(nt)的非编码RNA分子。自从1995年首次在线虫中发现以来，由于它具有特殊的生物学功能作用，因此越来越受到有关科学工作者的关注。根据合成途径可将小分子量RNA分成小分子量干扰RNA(smallinterferingRNA，siRNA)和微小RNA(microRNA，miRNA)两种类型。当一些小分子量的内源性或外源性双链RNA(double-strandedRNA,dsRNA)分子导入寄主细胞之后，便会诱发与之同源的内源mRNA发生特异性的降解作用，从而高效且特异地阻断了体内同源基因的表达活性，导致靶基因的表达沉默，产生相应的功能表型缺失的现象，即出现RNAi(RNA干扰，RNAinterference)。这是生物界普遍存在的一种在转录后调节基因表达的有效方式。
将siRNA导入细胞或动物体内，使特定基因表达沉默，又被称为基因敲除。目前，已经在线虫、果蝇、小鼠、大鼠等动物体内实现了多种基因的表达抑制，显示了RNAi在功能基因组学和基因治疗等领域的广阔应用前景。目前对昆虫进行RNAi的操作技术已经比较成熟，主要注射、饲喂、浸泡和转基因植物组织培养等方法。较为常用的将siRNA导入昆虫体内的方法主要是注射法和饲喂法，注射法要求有特殊的设备，而且由于注射本身的机械损伤会引起昆虫较高的死亡率；饲喂法较为简便，但存在作用较慢、效率较低的特点。因此，提高饲喂法的干扰效率对于进一步研究相关昆虫基因功能有重要意义。
发明内容
本发明的目的是提供一种提高咀嚼式口器昆虫RNA干扰效率的方法。
为了实现本发明目的，本发明的一种提高咀嚼式口器昆虫RNA干扰效率的方法，包括以下步骤：
1)用无RNA酶的水将靶基因特异性siRNA配成一定浓度的溶液；
2)将人工饲料加工成3～5mm厚、直径1～2cm的圆片，置于24孔板中；
3)取1)的溶液加在2)的圆片上，使溶液被饲料完全吸收；
4)将初孵的咀嚼式口器昆虫幼虫，放入3)的有饲料圆片的24孔板中，饲喂一段时间后，检测昆虫中靶基因的表达量。
前述的方法，步骤1)所述溶剂包括但不限于水。
前述的方法，步骤1)用无RNA酶的水将靶基因特异性siRNA配成浓度0.015nmol/L的溶液。
前述的方法，步骤2)人工饲料经干燥后，加工成3～5mm厚、直径1.5cm的圆片，置于24孔板中。例如，若饲料太湿，可以稍微风干30min,以便于吸收siRNA。
前述的方法，步骤3)具体为：取1)的溶液60-80μl(优选75μl)加在2)的圆片上，使溶液被饲料完全吸收。
前述的方法，步骤4)将初孵的咀嚼式口器昆虫幼虫，放入3)的有饲料圆片的24孔板中，饲喂2～4天(优选2天)后取样，提取总RNA，反转录成cDNA，用荧光定量PCR检测昆虫中靶基因的表达量。
前述的方法，所述靶基因包括但不限于氨肽酶N(APN)基因，其特异性siRNA序列为：5'-GCAACUACUCCAUCGCCAUTT-3'。
前述的方法，所述昆虫包括但不限于棉铃虫。
本发明通过对饲喂昆虫siRNA的方法进行改进，建立一种提高咀嚼式口器昆虫RNA干扰效率的方法。该方法高效、简单，可操作性强，节约了siRNA的用量，干扰效果明显，为研究咀嚼式口器昆虫基因的功能提供技术支持。
附图说明
图1为本发明实施例1中利用荧光定量PCR检测RNA干扰沉默棉铃虫APN基因的表达的结果；其中，CK代表空白对照，GFPsi代表GFP基因siRNA，APNsi代表APN基因siRNA，APNsi-C代表APN基因siRNA的传统饲料混合法对照。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(SambrookJ&RussellDW,MolecularCloning:aLaboratoryManual,2001)，或按照制造厂商说明书建议的条件。
实施例1RNA干扰对棉铃虫APN基因的影响
实验对象：初孵的棉铃虫幼虫，72头，分3次重复。
用无RNA酶的水分别将APN(氨肽酶N)基因siRNA(5'-GCAACUACUCCAUCGCCAUTT-3')和对照绿色荧光蛋白GFP基因siRNA(5'-GCGUUGGGAAGUCAAGUUUTT-3')。
用无RNA酶的水将上述siRNA配成浓度0.015nmol/L的溶液，用无RNA酶的水作为对照；将人工饲料切成约3～5mm的薄片，用直径为1.5cm的打孔器在饲料上打孔，打出的饲料圆片放在24孔板中(若饲料太湿，可以稍微风干30min,以便于吸收siRNA)；取75μlsiRNA溶液加在饲料圆片上，使其被完全吸收；将初孵的棉铃虫幼虫，放入有饲料圆片的24孔板中，饲喂2天后取样，此时棉铃虫已将饲料取食完。提取棉铃虫总RNA，反转录成cDNA，用荧光定量PCR检测基因表达量的差异。同时以传统的饲料混合法为对照进行处理。传统的饲料混合法是称取一定量的棉铃虫饲料，加入适量的siRNA，混合均匀后使用。这种方法siRNA用量大，而且加入siRNA不易完全混匀，因此无法控制被棉铃虫取食掉的饲料量及siRNA量。
以GAPDH和actin为双内参基因，引物和探针设计序列见表1；qPCR反应体系及反应条件如下：
表1引物和探针序列(5′-3′)
| APN-F | TCGACAGCTGGGTCCAGAAC |
| APN-R | GATGACACCTGTGTTGTTGTTACG |
| APN-Probe | CTGGATCTCCCGTCATCAACGTTGC |
| GAPDH-F | CATTGAAGGTCTGATGACCACTGT |
| GAPDH-R | CAGAGGGTCCATCCACTGTCTT |
| GAPDH-Probe | CACGCCACCATTGCCACCCA |
| β-actin-F | GGCCCCGTCCACAATGA |
| β-actin-R | CCGATCCATACGGAGTACTTCCT |
| β-actin-Probe | ATCAAGATCATCGCGCCCCCAGA |
qPCR反应体系：
qPCR反应程序：95℃预变性60s；95℃5s，60℃15s，40个循环。
根据荧光定量PCR结果(图1)，与饲喂水的对照相比，饲喂棉铃虫APN基因的siRNA的基因表达量显著降低了63.9％，而饲喂GFPsiRNA的基因表达量与对照相比没有显著差异。与传统的混合饲料进行RNAi的方法相比，本方法3次生物学重复试验稳定，误差小，且干扰效率明显提高。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (9)
1.提高咀嚼式口器昆虫RNA干扰效率的方法，其特征在于，包括以下步骤：
1)用无RNA酶的水将靶基因特异性siRNA配成一定浓度的溶液；
2)将人工饲料加工成3～5mm厚、直径1～2cm的圆片，置于24孔板中；
3)取1)的溶液加在2)的圆片上，使溶液被饲料完全吸收；
4)将初孵的咀嚼式口器昆虫幼虫，放入3)的有饲料圆片的24孔板中，饲喂一段时间后，检测昆虫中靶基因的表达量。
2.根据权利要求1所述的方法，其特征在于，步骤1)所述溶剂包括水。
3.根据权利要求1或2所述的方法，其特征在于，步骤1)用无RNA酶的水将靶基因特异性siRNA配成浓度0.015nmol/L的溶液。
4.根据权利要求1-3任一项所述的方法，其特征在于，步骤2)人工饲料经干燥后，加工成3～5mm厚、直径1.5cm的圆片，置于24孔板中。
5.根据权利要求1-4任一项所述的方法，其特征在于，步骤3)具体为：取1)的溶液60-80μl加在2)的圆片上，使溶液被饲料完全吸收。
6.根据权利要求1-5任一项所述的方法，其特征在于，步骤4)将初孵的咀嚼式口器昆虫幼虫，放入3)的有饲料圆片的24孔板中，饲喂2～4天后取样，提取总RNA，反转录成cDNA，用荧光定量PCR检测昆虫中靶基因的表达量。
7.根据权利要求1-6任一项所述的方法，其特征在于，所述靶基因包括氨肽酶N基因，其特异性siRNA序列为：5'-GCAACUACUCCAUCGCCAUTT-3'。
8.根据权利要求1-7任一项所述的方法，其特征在于，步骤3)具体为：取1)的溶液75μl加在2)的圆片上，使溶液被饲料完全吸收，步骤4)将初孵的咀嚼式口器昆虫幼虫，放入3)的有饲料圆片的24孔板中，饲喂2天后，检测昆虫中靶基因的表达量。
9.根据权利要求1-8任一项所述的方法，其特征在于，所述昆虫包括棉铃虫。
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| CN106222286A (zh) * | 2016-08-12 | 2016-12-14 | 湖北大学 | 一种研究棉铃虫幼虫rna干扰效率的方法 |
| CN109077028A (zh) * | 2018-08-28 | 2018-12-25 | 中国农业科学院蔬菜花卉研究所 | 对西花蓟马进行rna干扰的方法及装置 |
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| CN101343637A (zh) * | 2007-07-10 | 2009-01-14 | 中山大学 | 饲喂dsRNA抑制昆虫基因表达的方法 |
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| CN101343637A (zh) * | 2007-07-10 | 2009-01-14 | 中山大学 | 饲喂dsRNA抑制昆虫基因表达的方法 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222286A (zh) * | 2016-08-12 | 2016-12-14 | 湖北大学 | 一种研究棉铃虫幼虫rna干扰效率的方法 |
| CN106222286B (zh) * | 2016-08-12 | 2019-06-18 | 湖北大学 | 一种研究棉铃虫幼虫rna干扰效率的方法 |
| CN109077028A (zh) * | 2018-08-28 | 2018-12-25 | 中国农业科学院蔬菜花卉研究所 | 对西花蓟马进行rna干扰的方法及装置 |
| CN109077028B (zh) * | 2018-08-28 | 2023-07-18 | 中国农业科学院蔬菜花卉研究所 | 对西花蓟马进行rna干扰的方法及装置 |
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