CN116732041B - 一种可用于rna干扰防治桃蚜的致死基因、rna干扰序列及其制备方法 - Google Patents
一种可用于rna干扰防治桃蚜的致死基因、rna干扰序列及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种可用于RNA干扰防治桃蚜的致死基因,属于植物病虫防治领域,所述致死基因包括Tret1,所述Tret1的cDNA的核苷酸序列如SEQ ID NO.1所示。可基于该致死基因Tret1设计RNA干扰序列,高效诱发同源mRNA特异性降解,沉默Tret1基因在桃蚜体内的表达,最终导致桃蚜繁殖力降低及桃蚜死亡,为利用RNA干扰技术控制害虫提供了新途径。本发明还提供一种用于防治桃蚜的RNA干扰序列及其制备方法。
Description
技术领域
本发明属于植物病虫防治领域,特别涉及一种可用于RNA干扰防治桃蚜的致死基因、RNA干扰序列及其制备方法。
背景技术
桃蚜Myzus persicae属半翅目(Hemiptera)蚜科(Aphididae),在世界各地广泛分布,是十字花科蔬菜、烟草、辣椒、马铃薯、茄子等数百种作物的主要害虫之一,还可传播多种病毒病,给农业生产造成很大损失。目前桃蚜及其传播的病毒病主要依靠大面积使用农药来防控,给农业生产带来了严重的安全性问题。因此,生产上迫切需要一种安全绿色的防控新技术或策略,能够弥补化学防治弊端。
RNA干扰(RNA interference,RNAi)是指由内源或外源的双链RNA引发的mRNA高效特异性降解,导致特异性阻碍靶标基因表达的现象。根据RNA干扰原理,筛选桃蚜的致死基因,通过制备RNA干扰制剂,能够安全高效地对桃蚜起到防治作用,是当前发展趋势下解决化学防治问题的良好途径,被誉为新一代害虫防治新技术。该技术是针对桃蚜的特异性基因设计,不会改变桃蚜的基因组,仅对桃蚜具有杀灭效果,具有特异性强、安全性高、致死率高、环境友好等优点,可实现桃蚜的绿色精准防控,能够有效减少化学农药对生态环境的破坏。
发明内容
为了解决农药防控桃蚜带来的安全性问题,本发明提供了一种可用于RNA干扰防治桃蚜的致死基因,可基于该致死基因Tret1设计RNA干扰序列,高效诱发同源mRNA特异性降解,沉默Tret1基因在桃蚜体内的表达,最终导致桃蚜繁殖力降低及桃蚜死亡,为利用RNA干扰技术控制害虫提供了新途径。
本发明还提供了一种用于防治桃蚜的RNA干扰序列及其制备方法。
本发明通过以下技术方案实现:
本发明提供一种可用于RNA干扰防治桃蚜的致死基因,所述致死基因包括Tret1,所述Tret1的cDNA的核苷酸序列如SEQ ID NO.1所示。
基于同一发明构思,本发明还提供一种用于防治桃蚜的RNA干扰序列,所述RNA干扰序列为根据致死基因Tret1合成的dsRNA,所述RNA干扰序列的核苷酸序列如SEQ ID NO.2所示。
基于同一发明构思,本发明还提供一种用于防治桃蚜的RNA干扰序列的制备方法,包括:
克隆桃蚜Tret1基因的全长cDNA;
在Tret1基因的全长cDNA序列中选取RNAi的靶标区域,设计所述RNAi的靶标区域的PCR扩增引物Tret1-F/Tret1-R;
提取桃蚜总RNA,反转录合成cDNA,用所述Tret1-F/Tret1-R对所述RNAi的靶标区域进行PCR扩增,获得目的基因片段,将所述目的基因片段克隆到质粒载体并转化至感受态细胞中,提取质粒;
在所述Tret1-F/Tret1-R的5’端加上T7 promoter序列,获得引物dsTret1-F/dsTret1-R,以提取的质粒为模板进PCR扩增,获得带T7序列的目的基因,后利用MEGAscriptT7Transcription Kit合成双链RNA,得到用于防治桃蚜的RNA干扰序列。
进一步的,所述克隆桃蚜Tret1基因的全长cDNA,具体包括:
提取桃蚜总RNA,合成cDNA;
基于桃蚜转录组数据库获得的Tret1基因序列设计Tret1基因上下游引物Tret1-F1/Tret1-R1或Tret1-F2/Tret1-R2,以cDNA为模板进行PCR扩增,纯化产物获得桃蚜Tret1基因的全长cDNA;
其中,所述Tret1-F1的核苷酸序列如SEQ ID NO.3所示,所述Tret1-R1的核苷酸序列如SEQ ID NO.4所示,所述Tret1-F2的核苷酸序列如SEQ ID NO.5所示,所述Tret1-R2的核苷酸序列如SEQ ID NO.6所示。
进一步的,所述以cDNA为模板进行PCR扩增,纯化产物获得桃蚜Tret1基因的全长cDNA,具体包括:
以cDNA为模板进行PCR扩增,PCR反应体系为:Premix Taq酶12.5μL、cDNA模板1μL,正反向引物各1μL、ddH2O 9.5μL,反应程序为:95℃30s;94℃30s,55℃30s,72℃1min,35个循环,72℃10min;
将PCR产物进行琼脂糖凝胶电泳分离,纯化回收目的片段,获得桃蚜Tret1基因的全长cDNA。
进一步的,所述Tret1-F的核苷酸序列如SEQ ID NO.7所示,所述Tret1-R的核苷酸序列如SEQ ID NO.8所示。
进一步的,所述dsTret1-F的核苷酸序列如SEQ ID NO.9所示,所述dsTret1-R的核苷酸序列如SEQ ID NO.10所示。
基于同一发明构思,本发明还提供一种用于防治桃蚜的RNA干扰试剂,包括dsRNA与纳米载体,所述dsRNA为上述用于防治桃蚜的RNA干扰序列,所述dsRNA与所述纳米载体的质量比为1:1。
基于同一发明构思,本发明还提供一种可用于RNA干扰防治桃蚜的致死基因在桃蚜防治中的用途。
基于同一发明构思,本发明还提供一种用于防治桃蚜的RNA干扰序列在桃蚜防治中的用途。
本发明实施例中的一个或多个技术方案,至少具有如下技术效果或优点:
1.本发明一种可用于RNA干扰防治桃蚜的致死基因,所述致死基因为桃蚜Tret1基因,该基因编码海藻糖转运蛋白,可基于该致死基因Tret1设计RNA干扰序列,诱发同源mRNA特异性降解,沉默Tret1基因在桃蚜体内的表达,影响桃蚜体内海藻糖的跨细胞膜转运,从而阻断了有机体能量代所需的源料物质,最终导致桃蚜繁殖力降低及桃蚜死亡,为利用RNA干扰技术控制害虫提供了新途径。
2.本发明一种用于防治桃蚜的RNA干扰序列,该RNA干扰序列为根据致死基因Tret1合成的dsRNA,能够高效诱发同源mRNA特异性降解,沉默Tret1基因在桃蚜体内的表达,最终导致桃蚜繁殖力降低及桃蚜死亡,具有特异性强、安全性高、致死率高、环境友好等优点,有效减少化学农药对生态环境的破坏。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作一简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图。
图1为本发明克隆获得的桃蚜Tret1基因全长cDNA序列及推导的氨基酸序列。
图2为本发明纳米载体介导不同浓度dsRNA对桃蚜Tret1基因的沉默效果。
图3为本发明RNA干扰对桃蚜的致死表型图。图4为本发明RNA干扰对桃蚜存活率的影响结果。
图5为本发明RNA干扰对桃蚜繁殖力的影响结果。
具体实施方式
下文将结合具体实施方式和实施例,具体阐述本发明,本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解,这些具体实施方式和实施例是用于说明本发明,而非限制本发明。
在整个说明书中,除非另有特别说明,本文使用的术语应理解为如本领域中通常所使用的含义。因此,除非另有定义,本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾,本说明书优先。
除非另有特别说明,本发明中用到的各种原材料、试剂、仪器和设备等,均可通过市场购买得到或者可通过现有方法制备得到。
本发明整体思路如下:
为了解决农药防控桃蚜带来的安全性问题,本发明一方面提供一种通过RNA干扰可使桃蚜致死的基因Tret1,Tret1基因编码海藻糖转运蛋白(trehalose transporter 1)。海藻糖(Trehalose)被称为“昆虫的血糖”,是昆虫生长发育过程所需能量代谢的源料物质,在昆虫的生长发育方面具有重要作用。然而,海藻糖不能像水或者脂肪一样直接穿过细胞膜,而需要在特殊的物质——糖转运蛋白(sugar transporter,ST)的帮助下才能顺利地进出细胞,并发挥具体的功能。糖转运蛋白属于MFS(major facilitator superfamily)超家族,是动物中最丰富的小分子转运体,海藻糖转运蛋白是其中的一种。在昆虫中,海藻糖转运蛋白1(trehalose transporter1,Tret1)可响应细胞内和细胞外梯度转运海藻糖。Tret1基因表达受阻后会影响昆虫体内海藻糖的跨细胞膜转运,从而阻断了有机体能量代所需的源料物质,并影响昆虫正常的生长发育。
另一方面提供了桃蚜RNA干扰序列及其制备方法,基于致死特异基因Tret1设计dsRNA,该dsRNA可高效诱发同源mRNA特异性降解,沉默Tret1基因在桃蚜体内的表达,最终导致桃蚜繁殖力降低及桃蚜死亡,该发明为利用RNA干扰技术控制害虫提供了新途径。
下面将结合实施例及实验数据对本申请一种可用于RNA干扰防治桃蚜的致死基因、RNA干扰序列及其制备方法进行详细说明。
实施例1
一种用于防治桃蚜的RNA干扰序列的制备方法,包括以下步骤:
(1)克隆桃蚜Tret1基因的全长cDNA
采用Super总RNA提取试剂盒(Shanghai Promega公司)法提取桃蚜总RNA,合成cDNA。基于桃蚜转录组数据库获得的Tret1基因序列设计引物,Tret1基因上下游引物Tret1-F1/Tret1-R1,或者Tret1-F2/Tret1-R2,以cDNA为模板,进行PCR扩增,PCR反应体系为:Premix Taq酶12.5μL、cDNA模板1μL,正反向引物各1μL、ddH2O 9.5μL;反应程序为:95℃30s;94℃30s,55℃30s,72℃1min,35个循环,72℃10min。
将经PCR扩增获得的PCR产物进行琼脂糖凝胶电泳分离,纯化回收目的片段,即桃蚜Tret1基因的全长cDNA。将回收目的片段克隆至pMDTM 19-T vector后转化至感受态细胞DH5α。通过菌落PCR进行验证后进行测序。
Tret1基因上下游引物序列如下:
Tret1-F1:GCCGTTGTCCTGCAAAATGT
Tret1-R1:ACAGAATTGCGGTGACTCGT
Tret1-F2:CGTCAGCCTCATGTTCCAGT
Tret1-R2:ACAGAATTGCGGTGACTCGT。
(2)在Tret1基因的全长cDNA序列中选取RNAi的靶标区域,并利用SnapDragon-dsRNA Design在线工具设计设计所述RNAi的靶标区域的PCR扩增引物Tret1-F/Tret1-R,采用绿色荧光蛋白(GFP)基因作为对照,同样设计靶标区域的PCR扩增引物。
各个靶标区域的PCR扩增引物序列如下:
Tret1-F:GCCGTTCCTGTTTTACGTGT
Tret1-R:GATGACCTGAACCCGTCAGT
GFP-F:GCCAACACTTGTCACTACTT
GFP-R:GGAGTATTTTGTTGATAATGGTCTG。
(3)提取桃蚜总RNA,反转录合成cDNA,然后用靶标区域扩增引物对cDNA中含有Tret1基因进行PCR扩增,PCR反应体系为:CloneAmp HiFi PCR Premix酶12.5μL、cDNA模板1μL,正反向引物各1μL、ddH2O 9.5μL;反应程序为:98℃10s,55℃30s,72℃30s,35个循环;纯化产物获得目的基因。将Tret1基因片段克隆到pMDTM 19-T vector(Takara)中,并转化到感受态细胞DH5α中。提取质粒并通过测序验证。
(4)对步骤(2)中的靶标区域特异性引物5’-端加上T7 promoter序列:
dsTret1-F:TAATACGACTCACTATAGGGGCCGTTCCTGTTTTACGTGT
dsTret1-R:TAATACGACTCACTATAGGGGATGACCTGAACCCGTCAGT
dsGFP-F:TAATACGACTCACTATAGGGGCCAACACTTGTCACTACTT
dsGFP-R:TAATACGACTCACTATAGGGGGAGTATTTTGTTGATAATGGTCTG。
(5)以步骤(3)中提取的质粒为模板进PCR扩增,PCR反应体系与步骤(2)相同,反应程序为:98℃10s,65℃30s,72℃30s,35个循环;纯化产物获得带T7序列目的基因,然后利用MEGAscript T7 Transcription Kit合成双链RNA。合成的dsRNA序列如下:
Tret1干扰靶序列:
GCCGTTCCTGTTTTACGTGTTCTTCGCAATGTTCGCCGTGCTGCCGTTACCGTGGAGCGCGTGCGGCGAGATGTTCCCGATGGCTGTAAAGGGCACCATGAACGGCGTCATGTACTCGTGCGGTTACGAGCTCATGTTCGCCGCCATCAAGGTGTACCCGATGCTGGTGGACACGTTCGGCATCCGTGTCGTGTGGACAGCGTCAGCGTGCACGTGCTTGATCACTTCCCTGTTCGGCGCGTTCGTCCTGCCCGAGACCACTGGCAAGACGTTAAATGAGATAACTGACGGGTTCAGGTC
GFP干扰靶序列:
GCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTCTCAAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTACAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAAATGGAATACAACTATAACTCACATAATGTATACATCATGGGAGACAAACCAAAGAATGGCATCAAAGTTAACTTCAAAATTAGACACAACATTAAAGATGGAAGCGTTCAATTAGCAGACCATTATCAACAAAATACTCC
实施例2
桃蚜致死基因Tret1的RNA干扰效率及应用检测
1.RNA干扰试验步骤如下:
1)dsRNA与纳米载体(nanocarrier)以质量比1:1混合,使dsRNA的最终浓度为40、160和500ng/μL三个水平,加入0.5%的表面活性剂(detergent),形成dsRNA/nanocarrier/detergent复合物,所述的纳米载体为一种星形阳离子聚合物(Star Polycation)。
2)将0.3μL dsRNA/nanocarrier/detergent和dsGFP/nanocarrier/detergent(对照)点滴至桃蚜3龄若虫腹背面,干扰处理24h后,随机选取10头活虫,用于检测RNA干扰对Tret1基因沉默的效果。
3)使用NCBI Primer-BLAST设计Tret1基因qRT-PCR引物,以cDNA为模板,进行qRT-PCR扩增检测基因表达水平,PCR反应体系为:TB Premix Ex Taq II 10μL、cDNA模板1μL、正反向引物各1μL、ddH2O 7μL。选用桃蚜β-actin和18s作为内参基因。采用2-ΔΔCT法测定相对转录水平,选取两个内控基因的几何平均值进行归一化处理。
各个PCR扩增引物序列如下:
qTret1-F:CATCGGTTCGCTGGTGTTTG
qTret1-R:TGTACACGTAGATCACGCCG
β-actin-F:AGTGCGACGTTGACATCAGA
β-actin-R:GCTTGGAGCTAAGGCAGTGA
18s-F:TCAACACGGGAAACCTCACCA
18s-R:CACCACCCACCGAATCAAGAA
4)采用2)中RNA干扰方法,选择桃蚜3龄若虫进行RNAi处理,每天统计其蜕皮量及存活量,连续统计6d,以GFP处理作为对照,每个处理30头桃蚜,重复3次。
5)采用IBM SPSS Statistics for Windows,version 19.0通过单因素进行方差分析,采用Tukey法进行多重比较,p<0.05被认为具有统计学意义。
2.RNA干扰试验结果与分析
2.1桃蚜Tret1基因的克隆
克隆测序得到1567bp序列,经NCBI的Blast工具分析发现其核苷酸序列与其他同源昆虫的Tret1序列具有很高的相似性,并确定该序列为蚜虫Tret1基因。Tret1基因的开放阅读框(ORF)为1389bp,编码462个氨基酸,5'非编码区为18bp(5'-UTR),3'非编码区(3'-UTR)为160bp。编码蛋白相对分子量为50.57kD,等电点(pI)为8.29,分子式为C2333H3598N562O623S34;N-端氨基酸为蛋氨酸(M,Met),推测的蛋白质半衰期为30h;含有带负电荷的氨基酸残基(Asp+Glu)31个,带正电荷的氨基酸残基(Arg+Lys)35个;稳定性系数为35.13,即该蛋白质性质稳定;总平均疏水指数(GRAVY)为0.572,表明其为疏水性蛋白质;脂肪族氨基酸指数为97.53,利用ScanProsite工具进行的氨基酸分析得到个主要促进者超家族(MFS)概要,即(第1-434位残基)(图1)
2.2 RNA干扰对桃蚜Tret1基因的沉默效果
测定了纳米载体介导不同浓度dsRNA(40、160和500ng/μL)沉默桃蚜Tret1基因的效果,结果表明,dsTret1(即RNA干扰序列)可有效降低Tret1基因的表达量,干扰48h后沉默效果分别达到42.57%、70.88%和68.69%,以160ng/μL dsRNA沉默效果最佳(如图2)。
2.3 RNA干扰对桃蚜存活率的影响
RNA干扰桃蚜Tret1基因后发现桃蚜3龄若虫的发育蜕皮困难,导致蜕皮畸形而死亡(图3)。进一步测定了RNA干扰桃蚜Tret1基因后连续6天内桃蚜的存活率,结果表明,沉默Tret1基因可有效降低桃蚜的存活率,沉默Tret1基因后第3天桃蚜存活率迅速降低,到第6天时存活率为32.22%,比对照GFP下降了50.36%(如图4)。
2.4 RNA干扰对桃蚜繁殖力的影响
测定了RNA干扰桃蚜Tret1基因后桃蚜的繁殖力,结果表明,沉默Tret1基因可有效降低桃蚜的繁殖能力,RNA干扰桃蚜3龄若虫6天内单产若虫总量(2.02头)与对照(7.33头)相比显著降低(如图5)。
综上可知,本发明通过RNA干扰桃蚜Tret1基因的表达,能够显著降低Tret1基因的表达量,有效降低桃蚜的存活率,并降低桃蚜的繁殖力,为利用RNA干扰技术控制害虫提供了新途径。
最后,还需要说明的是,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (2)
1.一种可用于RNA干扰防治桃蚜的致死基因在桃蚜(Myzus persicae)防治中的用途,其特征在于,所述致死基因包括Tret1,所述Tret1的cDNA的核苷酸序列如SEQ ID NO.1所示。
2.一种用于防治桃蚜的RNA干扰分子在桃蚜防治中的用途,其特征在于,所述RNA干扰分子为根据致死基因Tret1合成的dsRNA,所述Tret1的cDNA的核苷酸序列如SEQ ID NO.1所示,所述RNA干扰分子的核苷酸序列如SEQ ID NO.2所示。
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