CN115927301B - RNAi-based spodoptera frugiperda control method - Google Patents
RNAi-based spodoptera frugiperda control method Download PDFInfo
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- CN115927301B CN115927301B CN202210793393.0A CN202210793393A CN115927301B CN 115927301 B CN115927301 B CN 115927301B CN 202210793393 A CN202210793393 A CN 202210793393A CN 115927301 B CN115927301 B CN 115927301B
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Abstract
Description
技术领域Technical field
本发明涉及基因技术技术领域,具体而言,涉及一种基于RNAi的草地贪夜蛾防治方法。The present invention relates to the technical field of gene technology, and specifically to an RNAi-based control method for Spodoptera frugiperda.
背景技术Background technique
草地贪夜蛾(Spodoptera frugiperda)是一种杂食性农业害虫,取食350多种植物,分布范围广,适应能力极强,对农业生产造成了严重危害。化学农药在防治农业有害生物的过程中发挥着重要的作用,但因杀虫剂的盲用、滥用等不合理操作,使有害生物产生耐药性的同时,也对粮食安全、生态环境及生物多样性等产生了负面影响。目前,为了应对上述问题,除了大力推广有害生物综合防治(IPM)的相关措施之外,主要依赖于不断研发安全高效且具有新型作用机制的绿色农药。随着对RNAi现象及机制的深入研究,RNAi技术已渐渐走入农业害虫防治以及新型农药开发等领域,相较于传统化学农药,RNAi具有更明确的作用靶标和作用机制,对哺乳动物等非靶标生物安全,且不存在残留问题,符合当今社会对于农产品质量安全和生态安全的要求,是极具潜力的新型绿色害虫防治策略。核酸农药借助碱基序列的互补配对原则实现与靶标的相互作用,最终从mRNA水平阻断靶标蛋白产生的源头,进而表现出与小分子抑制剂类似的调节效果,成为一类新型的转录后基因沉默调控工具。同时,因大部分害虫与植物具有完善的RNAi系统,RNAi在这些生物群体中介导的基因沉默信号会发生级联放大,最终导致该靶标基因的不表达从而影响生物生长发育。核酸农药是以昆虫生长发育必需基因为靶标,通过沉默关键基因以实现致死级的杀虫效果,具有剂量低、高效和可持续的特点。近年来,RNAi目前在实验室条件下已经成功应用于半翅目(Hemiptera)、鳞翅目(Lepidoptera)、鞘翅目(Coleoptera)、双翅目(Diptera)以及直翅目(Orthoptera)等种类的害虫中。U6启动子是一类能够介导siRNA表达的启动子,由RNA聚合酶III来进行识别和转录。该类型的启动子在合适的条件下可以驱动shRNA在表达载体内合成大量的siRNA,进而沉默目的基因。昆虫杆状病毒具有专一性,仅感染节肢动物。可侵入昆虫体内,并且在昆虫的细胞核内进行复制和转录,由于其基因可以与外源基因相连接并且能很好的融合,使其成为理想的载体。将U6启动子加shRNA的siRNA表达体系构建至昆虫杆状病毒基因组中在病毒增殖过程中可以持续产生siRNA,从而实现对目的基因的持续性干扰。将杆状病毒与RNAi相结合的方式研发的新型生物农药,即规避了传统核酸农药存在的成本高、易降解、不持续等弊端问题,也能够加快病毒侵染寄主后死亡的速度,且由于杆状病毒能够可在害虫种群之间进行传染,并能够遗传给下一代幼虫,使其达到一次施用长久有效的目的,符合当今社会对于农产品质量安全和生态安全的要求,因此是潜在的新型绿色植保产品。Spodoptera frugiperda is an omnivorous agricultural pest that feeds on more than 350 species of plants. It has a wide distribution range and strong adaptability, causing serious harm to agricultural production. Chemical pesticides play an important role in the prevention and control of agricultural pests. However, due to the blind use and abuse of pesticides and other irrational practices, harmful organisms develop drug resistance, which also affects food security, the ecological environment and biological processes. Diversity, etc. have had a negative impact. At present, in order to deal with the above problems, in addition to vigorously promoting the relevant measures of integrated pest management (IPM), it mainly relies on the continuous development of safe, efficient and green pesticides with new mechanisms of action. With in-depth research on the phenomenon and mechanism of RNAi, RNAi technology has gradually entered the fields of agricultural pest control and new pesticide development. Compared with traditional chemical pesticides, RNAi has clearer targets and mechanisms of action. The target biomass is safe and there are no residue problems. It meets the requirements of today's society for the quality and safety of agricultural products and ecological safety. It is a new green pest control strategy with great potential. Nucleic acid pesticides rely on the principle of complementary pairing of base sequences to interact with targets, ultimately blocking the source of target protein production at the mRNA level, and then exhibiting regulatory effects similar to small molecule inhibitors, becoming a new type of post-transcriptional gene Silence control tool. At the same time, because most pests and plants have complete RNAi systems, the gene silencing signals mediated by RNAi in these biological groups will cascade amplify, eventually leading to the non-expression of the target gene and affecting the growth and development of organisms. Nucleic acid pesticides target genes essential for insect growth and development and achieve lethal insecticidal effects by silencing key genes. They are characterized by low dosage, high efficiency and sustainability. In recent years, RNAi has been successfully used in Hemiptera, Lepidoptera, Coleoptera, Diptera, Orthoptera and other species under laboratory conditions. among pests. The U6 promoter is a type of promoter that can mediate siRNA expression and is recognized and transcribed by RNA polymerase III. This type of promoter can drive shRNA to synthesize a large amount of siRNA in the expression vector under appropriate conditions, thereby silencing the target gene. Insect baculoviruses are specific and only infect arthropods. It can invade the body of insects and be replicated and transcribed in the nucleus of the insect. Because its genes can be connected to foreign genes and can be well fused, it becomes an ideal vector. The siRNA expression system of U6 promoter plus shRNA is constructed into the genome of insect baculovirus, which can continuously produce siRNA during the virus proliferation process, thereby achieving continuous interference with the target gene. The new biopesticide developed by combining baculovirus and RNAi not only avoids the disadvantages of traditional nucleic acid pesticides such as high cost, easy degradation, and non-sustainability, but also can speed up the death of the virus after infecting the host, and because Baculovirus can be transmitted between pest populations and can be passed on to the next generation of larvae, making it effective for a long time after one application. It meets the requirements of today's society for the quality and safety of agricultural products and ecological safety, so it is a potential new green Plant protection products.
现有的RNAi作用方式主要通过注射、饲喂、喷洒以及浸泡等体外施用核酸分子的方式进行害虫防治。但常规dsRNA存在易降解、成本高、不持续等问题,不适合大面积推广应用。杆状病毒存在如杀虫速度慢,不能及时得到明显的效果等不足,这些不足导致多数杆状病毒杀虫剂难以大面积推广。The existing modes of action of RNAi mainly control pests through the in vitro application of nucleic acid molecules such as injection, feeding, spraying and soaking. However, conventional dsRNA has problems such as easy degradation, high cost, and non-sustainability, and is not suitable for large-scale promotion and application. Baculovirus has shortcomings such as slow insecticide speed and inability to obtain obvious effects in time. These shortcomings make it difficult for most baculovirus insecticides to be promoted on a large scale.
发明内容Contents of the invention
本发明涉及一种siRNA,其包含第一链和第二链,所述第一链和第二链互补共同形成RNA二聚体,并且所述第一链的序列为SEQ ID NO:1或2所示。The present invention relates to a siRNA, which includes a first strand and a second strand, the first strand and the second strand are complementary and jointly form an RNA dimer, and the sequence of the first strand is SEQ ID NO: 1 or 2 shown.
本发明还涉及一种shRNA,其包含正义链片段和反义链片段,以及连接所述正义链片段和反义链片段的茎环结构,所述正义链片段和反义链片段的序列互补,并且所述正义链的序列为SEQ ID NO:1或2所示。The present invention also relates to an shRNA, which includes a sense strand fragment and an antisense strand fragment, and a stem-loop structure connecting the sense strand fragment and the antisense strand fragment, and the sequences of the sense strand fragment and the antisense strand fragment are complementary, And the sequence of the sense strand is shown in SEQ ID NO: 1 or 2.
本发明还提供了上述shRNA的DNA片段、基因表达盒、载体以及宿主细胞。The invention also provides DNA fragments, gene expression cassettes, vectors and host cells of the above-mentioned shRNA.
本发明还提供了昆虫病毒的制备方法。The invention also provides a method for preparing insect viruses.
本发明的再一方面还提供了宿主细胞,其含有如上所述的DNA片段,或被如上所述的载体所转化。Yet another aspect of the invention also provides a host cell containing the DNA fragment as described above, or transformed by the vector as described above.
本发明还提供了包含如上所述的siRNA、如上所述的shRNA或如上所述的载体的用于防治草地贪夜蛾的组合物,以及应用该组合物防治草地贪夜蛾的方法。The present invention also provides a composition for controlling Spodoptera frugiperda, which contains the above-mentioned siRNA, the above-mentioned shRNA, or the above-mentioned vector, and a method for using the composition to control Spodoptera frugiperda.
本发明所提供的siRNA和shRNA,其靶点是草地贪夜蛾生长发育过程中发挥重要功能的V-ATPase,V-ATPase主要生理功能是以ATP为能量介导H+跨膜转运,维持细胞内外正常的pH梯度,形成跨膜电势差,进一步介导其他物质的主动运输,进而参与生物体其他重要的生理活动,其功能受到抑制会导致草地贪夜蛾生长发育受到抑制进而死亡。The target of the siRNA and shRNA provided by the present invention is V-ATPase, which plays an important role in the growth and development of Spodoptera frugiperda. The main physiological function of V-ATPase is to use ATP as energy to mediate H + transmembrane transport and maintain cells. The normal pH gradient inside and outside forms a transmembrane potential difference, which further mediates the active transport of other substances, and then participates in other important physiological activities of the organism. Inhibition of its function will cause the growth and development of Spodoptera Frugiperda to be inhibited and then die.
附图说明Description of drawings
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,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他的附图。In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings that need to be used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description The drawings illustrate some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting any creative effort.
图1为候选启动子的转录活性比较结果;图为转染后72h sf9细胞荧光。Figure 1 shows the comparison results of transcription activities of candidate promoters; the picture shows the fluorescence of sf9 cells 72h after transfection.
图2为U61-shA/B基因的合成电泳图,泳道M为2K Plus DNA Marker;泳道1为U61-shA,泳道2为U61-shB。Figure 2 shows the synthetic electropherogram of the U61-shA/B gene. Lane M is 2K Plus DNA Marker; lane 1 is U61-shA, and lane 2 is U61-shB.
图3为3龄草地贪夜蛾取食重组昆虫杆状病毒核酸农药后的死亡率比较,小写字母表示处理与对照有显著差异(p<0.05)。Figure 3 shows the comparison of mortality rates of 3-instar Spodoptera Frugiperda after feeding on recombinant insect baculovirus nucleic acid pesticides. Lowercase letters indicate significant differences between the treatment and the control (p<0.05).
图4为取食重组昆虫杆状病毒核酸农药后的死亡率以及成虫羽化率比较,小写字母表示处理与对照有显著差异(p<0.05)。Figure 4 shows the comparison of mortality and adult emergence rate after feeding on recombinant insect baculovirus nucleic acid pesticides. Lowercase letters indicate significant differences between the treatment and the control (p<0.05).
图5为注射重组昆虫杆状病毒核酸农药后的目的基因表达量情况,小写字母表示处理与对照有显著差异(p<0.05)。Figure 5 shows the expression level of the target gene after injection of recombinant insect baculovirus nucleic acid pesticide. Lowercase letters indicate significant differences between the treatment and the control (p<0.05).
图6为取食重组昆虫杆状病毒核酸农药后的中毒症状图。Figure 6 is a diagram of poisoning symptoms after ingesting recombinant insect baculovirus nucleic acid pesticides.
图7为注射重组昆虫杆状病毒对草地贪夜蛾幼虫生长发育抑制情况图。Figure 7 shows the inhibition of growth and development of Spodoptera frugiperda larvae by injection of recombinant insect baculovirus.
具体实施方式Detailed ways
现将详细地提供本发明实施方式的参考,其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上,对本领域技术人员而言,显而易见的是,可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如,作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中,来产生更进一步的实施方式。Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment, to yield still further embodiments.
除非另有说明,用于披露本发明的所有术语(包括技术和科学术语)的意义与本发明所属领域普通技术人员所通常理解的相同。通过进一步的指导,随后的定义用于更好地理解本发明的教导。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。Unless otherwise defined, all terms (including technical and scientific terms) used to disclose the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing specific embodiments only and is not intended to limit the invention.
本文所使用的术语“和/或”、“或/和”、“及/或”的选择范围包括两个或两个以上相关所列项目中任一个项目,也包括相关所列项目的任意的和所有的组合,所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。需要说明的是,当用至少两个选自“和/或”、“或/和”、“及/或”的连词组合连接至少三个项目时,应当理解,在本申请中,该技术方案毫无疑问地包括均用“逻辑与”连接的技术方案,还毫无疑问地包括均用“逻辑或”连接的技术方案。比如,“A及/或B”包括A、B和A+B三种并列方案。又比如,“A,及/或,B,及/或,C,及/或,D”的技术方案,包括A、B、C、D中任一项(也即均用“逻辑或”连接的技术方案),也包括A、B、C、D的任意的和所有的组合,也即包括A、B、C、D中任两项或任三项的组合,还包括A、B、C、D的四项组合(也即均用“逻辑与”连接的技术方案)。The terms "and/or", "or/and" and "and/or" used in this article include any one of two or more related listed items, and also include any of the related listed items. and all combinations, including any two of the related listed items, any more of the related listed items, or a combination of all of the related listed items. It should be noted that when at least three items are connected in combination with at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that in this application, the technical solution It undoubtedly includes technical solutions that are all connected by "logical AND", and it also undoubtedly includes technical solutions that are all connected by "logical OR". For example, "A and/or B" includes three parallel solutions: A, B and A+B. For another example, the technical solution of "A, and/or, B, and/or, C, and/or, D" includes any one of A, B, C, and D (that is, they are all connected with "logical OR" technical solution), also includes any and all combinations of A, B, C, and D, that is, including combinations of any two or any three of A, B, C, and D, and also includes A, B, C , four combinations of D (that is, technical solutions that are all connected by "logical AND").
本发明中所使用的术语“含有”、“包含”和“包括”是同义词,其是包容性或开放式的,不排除额外的、未被引述的成员、元素或方法步骤。The terms "comprising", "comprising" and "including" as used in this invention are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.
本发明中用端点表示的数值范围包括该范围内所包含的所有数值及分数,以及所引述的端点。Numerical ranges expressed by endpoints herein include all numbers and fractions included within the range, as well as the recited endpoints.
本发明中涉及浓度数值,其含义包括在一定范围内的波动。比如,可以在相应的精度范围内波动。比如2%,可以允许±0.1%范围内波动。对于数值较大或无需过于精细控制的数值,还允许其含义包括更大波动。比如100mM,可以允许±1%、±2%、±5%等范围内的波动。The present invention refers to concentration values, and their meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuation within the range of ±0.1%. For values that are large or do not require too fine control, the meaning is also allowed to include larger fluctuations. For example, 100mM can allow fluctuations within the range of ±1%, ±2%, ±5%, etc.
本发明中,涉及“多个”、“多种”等描述,如无特别限定,指在数量上指大于等于2。In the present invention, descriptions such as "plurality" and "multiple" refer to a quantity greater than or equal to 2 unless otherwise specified.
本发明中,以开放式描述的技术特征中,包括所列举特征组成的封闭式技术方案,也包括包含所列举特征的开放式技术方案。In the present invention, the technical features described in open terms include closed technical solutions composed of the listed features, and also include open technical solutions including the listed features.
本发明中,“优选”、“更好”、“更佳”、“为宜”仅为描述效果更好的实施方式或实施例,应当理解,并不构成对本发明保护范围的限制。本发明中,“可选地”、“可选的”、“可选”,指可有可无,也即指选自“有”或“无”两种并列方案中的任一种。如果一个技术方案中出现多处“可选”,如无特别说明,且无矛盾之处或相互制约关系,则每项“可选”各自独立。In the present invention, "preferable", "better", "better" and "suitable" are only used to describe implementations or examples with better effects. It should be understood that they do not limit the scope of protection of the present invention. In the present invention, "optionally", "optional" and "optional" mean that it is optional, that is, it refers to any one selected from the two parallel solutions of "with" or "without". If there are multiple "optionals" in a technical solution, each "optional" will be independent unless otherwise specified and there is no contradiction or mutual restriction.
本发明涉及一种siRNA,其包含第一链和第二链,所述第一链和第二链互补共同形成RNA二聚体,并且所述第一链的序列为SEQ ID NO:1或2所示。The present invention relates to a siRNA, which includes a first strand and a second strand, the first strand and the second strand are complementary and jointly form an RNA dimer, and the sequence of the first strand is SEQ ID NO: 1 or 2 shown.
本发明还涉及一种shRNA,其包含正义链片段和反义链片段,以及连接所述正义链片段和反义链片段的茎环结构,所述正义链片段和反义链片段的序列互补,并且所述正义链的序列为SEQ ID NO:1或2所示。The present invention also relates to an shRNA, which includes a sense strand fragment and an antisense strand fragment, and a stem-loop structure connecting the sense strand fragment and the antisense strand fragment, and the sequences of the sense strand fragment and the antisense strand fragment are complementary, And the sequence of the sense strand is shown in SEQ ID NO: 1 or 2.
在本发明中,所述siRNA,即小干扰RNA(small interfering RNA)是一种双链小RNA分子,其由完全互补的第一链和第二链组成,由Dicer(RNAaseⅢ家族中对双链RNA具有特异性的酶)加工而成。所述第一链和所述第二链互补共同形成RNA二聚体,并且所述第一链的序列与草地贪夜蛾V-ATPaseA/B基因中的靶序列相同,或者为与该靶序列在高等严紧条件下杂交的序列。siRNA是siRISC的主要成员,激发与之互补的目标mRNA的沉默。In the present invention, the siRNA, that is, small interfering RNA, is a double-stranded small RNA molecule, which is composed of a completely complementary first strand and a second strand. RNA is processed by specific enzymes). The first strand and the second strand are complementary and jointly form an RNA dimer, and the sequence of the first strand is the same as the target sequence in the Spodoptera frugiperda V-ATPaseA/B gene, or is identical to the target sequence. Sequences that hybridize under high stringency conditions. siRNA is the main component of siRISC and triggers the silencing of its complementary target mRNA.
在本发明中,所述shRNA,即小发卡或短发卡RNA(small hairpin RNA orshorthairpin RNA,shRNA)是一段具有紧密发卡环(tight hairpin turn)的RNA序列,其包含正义链片段、反义链片段以及连接正义链片段和反义链片段的茎环结构,常被用于RNA干扰沉默靶基因的表达。其中正义链和反义链的序列互补,并且所述正义链片段的序列与草地贪夜蛾V-ATPaseA/B基因中的靶序列相同。shRNA的发卡结构可被细胞机制切割成siRNA,然后siRNA结合到RNA诱导沉默复合物上(RNA-inducedsilencing complex,RISC),该复合物能够结合到目的mRNAs并将其降解。In the present invention, the shRNA, that is, small hairpin RNA or short hairpin RNA (shRNA), is an RNA sequence with a tight hairpin turn, which includes a sense strand fragment and an antisense strand fragment. As well as the stem-loop structure connecting the sense strand fragment and the antisense strand fragment, it is often used for RNA interference to silence the expression of target genes. The sequences of the sense strand and the antisense strand are complementary, and the sequence of the sense strand fragment is the same as the target sequence in the Spodoptera frugiperda V-ATPaseA/B gene. The hairpin structure of shRNA can be cleaved into siRNA by cellular machinery, and then the siRNA binds to the RNA-induced silencing complex (RISC), which can bind to target mRNAs and degrade them.
RNA干扰(RNAinterference,RNAi)是指内源性或外源性双链RNA(dsRNA)介导的细胞内mRNA发生特异性降解,从而导致靶基因的表达沉默,产生相应的功能表型缺失的现象。RNA interference (RNAi) refers to the specific degradation of intracellular mRNA mediated by endogenous or exogenous double-stranded RNA (dsRNA), resulting in the expression silencing of target genes and the corresponding loss of functional phenotypes. .
在本发明中,“互补”是指在严格条件下能够进行杂交。“杂交条件”根据测量杂交时所用条件的“严紧性”程度来分类。严紧性程度可以以例如核酸结合复合物或探针的解链温度(Tm)为依据。例如,“最大严紧性”典型地发生在约Tm-5℃(低于探针Tm 5℃);“高等严紧性”发生在Tm以下约5℃~10℃;“中等严紧性”发生在探针Tm以下约10℃~20℃;“低严紧性”发生在Tm以下约20℃~5℃。作为替代,或者进一步地,杂交条件可以以杂交的盐或离子强度条件和/或一或多次的严紧性洗涤为依据。例如,6×SSC=极低严紧性;3×SSC=低至中等严紧性;1×SSC=中等严紧性;0.5×SSC=高等严紧性。从功能上说,可以采用最大严紧性条件确定与杂交探针严紧同一或近严紧同一的核酸序列;而采用高等严紧性条件确定与该探针有约80%或更多序列同一性的核酸序列。In the present invention, "complementary" means capable of hybridizing under stringent conditions. "Hybridization conditions" are classified according to the degree of "stringency" of the conditions used in measuring hybridization. The degree of stringency may be based, for example, on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, “maximum stringency” typically occurs at about Tm-5°C (5°C below the probe Tm); “high stringency” occurs at about 5°C to 10°C below the Tm; and “medium stringency” occurs at about 5°C to 10°C below the probe Tm. The needle Tm is about 10°C to 20°C; "low stringency" occurs at about 20°C to 5°C below the Tm. Alternatively, or in addition, hybridization conditions may be based on salt or ionic strength conditions of hybridization and/or one or more stringency washes. For example, 6×SSC = very low stringency; 3×SSC = low to medium stringency; 1×SSC = medium stringency; 0.5×SSC = high stringency. Functionally speaking, maximum stringency conditions can be used to determine nucleic acid sequences that are strictly identical or nearly identical to the hybridization probe; high stringency conditions can be used to determine nucleic acid sequences that have about 80% or more sequence identity with the probe. .
对于要求高选择性的应用,典型地期望采用相对严紧的条件来形成杂交体,例如,选择相对低的盐和/或高温度条件。Sambrook等(Sambrook,J.等(1989)分子克隆,实验室手册,Cold Spring Harbor Press,Plainview,N.Y.)提供了包括中等严紧性和高等严紧性在内的杂交条件。For applications requiring high selectivity, it is typically desirable to employ relatively stringent conditions for hybrid formation, for example, selecting relatively low salt and/or high temperature conditions. Hybridization conditions including medium stringency and high stringency are provided by Sambrook et al. (Sambrook, J. et al. (1989) Molecular Cloning, Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).
为便于说明,用于检测本发明的多核苷酸与其它多核苷酸杂交的合适的中度严紧条件包括:用5×SSC、0.5%SDS、1.0mM EDTA(pH8.0)溶液预洗;在50-65℃下在5×SSC中杂交过夜;随后用含0.1%SDS的2×、0.5×和0.2×SSC在65℃下各洗涤两次20分钟。本领域技术人员应当理解,能容易地操作杂交严紧性,如改变杂交溶液的含盐量和/或杂交温度。例如,在另一个实施方案中,合适的高度严紧杂交条件包括上述条件,不同之处在于杂交温度升高到例如60℃~65℃或65℃~70℃。For convenience of explanation, suitable moderately stringent conditions for detecting the hybridization of the polynucleotide of the present invention with other polynucleotides include: pre-washing with 5×SSC, 0.5% SDS, 1.0mM EDTA (pH8.0) solution; Hybridize overnight at 50-65°C in 5x SSC; then wash twice with 2x, 0.5x and 0.2x SSC containing 0.1% SDS for 20 min each at 65°C. Those skilled in the art will appreciate that hybridization stringency can be readily manipulated, such as by changing the salt content of the hybridization solution and/or the hybridization temperature. For example, in another embodiment, suitable highly stringent hybridization conditions include the conditions described above, except that the hybridization temperature is increased to, for example, 60°C to 65°C or 65°C to 70°C.
进一步地,所述shRNA的茎环结构的序列可为本领域的常规选择,例如选自以下任一种序列:UUCAAGAGA、AUG、CCC、UUCG、CCACC、CUCGAG、AAGCUU和CCACACC。Furthermore, the sequence of the stem-loop structure of the shRNA can be conventionally selected in the art, for example, selected from any one of the following sequences: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CUCGAG, AAGCUU and CCACACC.
本发明还涉及编码如上所述shRNA的DNA片段。The invention also relates to DNA fragments encoding shRNA as described above.
本发明还包含如上所述DNA片段的基因表达盒。The present invention also includes gene expression cassettes of DNA fragments as described above.
基因表达盒中可包含基因工程中常用的调控元件,例如增强子、启动子、内部核糖体进入位点(IRES)和其他表达控制元件(例如转录终止信号,或者多腺苷酸化信号和多聚U序列等)。The gene expression cassette may contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (such as transcription termination signals, or polyadenylation signals and polymerization signals). U sequence, etc.).
在一些实施方式中,基因表达盒中用于驱动所述DNA片段表达的启动子的核苷酸序列如SEQ ID NO:5所示。In some embodiments, the nucleotide sequence of the promoter used to drive expression of the DNA fragment in the gene expression cassette is shown in SEQ ID NO: 5.
该启动子为发明人筛选得到的效果优异的草地贪夜蛾的U6启动子,其能有效提高目的基因的表达效率。This promoter is the excellent U6 promoter of Spodoptera frugiperda screened by the inventor, which can effectively improve the expression efficiency of the target gene.
另外,需要说明的是,在一个方面,本发明中所涉及的有用的序列包括与SEQ IDNO:1~5所示的核酸片段具有大于80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%同一性的核苷酸序列。术语“%同一性”在两个或更多个核苷酸序列或氨基酸序列的上下文中,指的是相同或具有特定百分比的相同氨基酸残基或核苷酸的两个或多个序列或子序列,当比较和比对以用于最大对应时,如使用以下序列比较算法之一或通过目视检查来测量的。例如,%同一性是相对于要比较的序列的编码区域的整个长度。In addition, it should be noted that, in one aspect, useful sequences involved in the present invention include nucleic acid fragments shown in SEQ ID NO: 1 to 5 that have greater than 80%, 85%, 90%, 91%, 92%, Nucleotide sequences that are 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. The term "% identity", in the context of two or more nucleotide sequences or amino acid sequences, refers to two or more sequences or sub-sequences that are identical or have a specific percentage of identical amino acid residues or nucleotides. Sequences, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example, % identity is relative to the entire length of the coding region of the sequences being compared.
对于序列比较,通常一个序列用作参考序列,测试序列与该序列进行比较。当使用序列比较算法时,测试序列和参考序列被输入到计算机中,如果需要,指定子序列坐标,并且指定序列算法程序参数。然后,序列比较算法根据指定的程序参数计算测试序列相对于参考序列的百分比序列同一性。可使用搜索算法例如BLAST和PSI-BLAST(Altschul etal.,1990、JMol Biol 215:3,403-410;Altschul et al.,1997,Nucleic Acids Res25:17,3389-402)确定百分比同一性。For sequence comparisons, typically one sequence is used as a reference sequence and test sequences are compared to this sequence. When using a sequence comparison algorithm, test and reference sequences are input into the computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. Percent identity can be determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25:17, 3389-402).
有用的序列还包括经过修饰的核酸,常见修饰包括4-乙酰胞苷、5-(羧羟甲基)尿苷、二氢尿苷、2'-O-甲基假尿苷、β,D-半乳糖Q核苷、2'-O-甲基鸟苷、肌苷、N6-异戊烯基腺苷、1-甲基腺苷、1-甲基假尿苷、1-甲基肌苷、2'2-二甲基腺苷、2-甲基腺苷、2-甲基鸟苷、5-甲基尿苷、3-甲基胞苷、5-甲基胞苷、N6-甲基腺苷、7-甲基鸟苷、5-甲基氨基甲基尿苷、5-羧甲基氨甲基尿苷、5-羧甲基氨甲基-2-硫代尿苷、β,D-甘露糖Q核苷、5-甲氧基羰基甲基-2-硫代尿苷、5-甲氧基羰基甲基尿苷、5-甲氧基尿苷、2-硫代甲基-N6-异戊烯基腺苷、N-((9-β-D-呋喃核糖基-2-硫代甲基嘌呤-6-Yl)氨基甲酰)苏氨酸、N-((9-β-D-呋喃核糖嘌呤-6-yl)N-甲基氨基甲酰)苏氨酸、尿苷-5-氧化乙酸-甲基酯、尿苷-5-氧化乙酸、wybutoxosine、假尿苷、Q核苷、2-硫代胞苷、5-甲基-2硫代尿苷、2-硫代尿苷、4-硫代尿苷、5-硫代尿苷、N-((9-β-D-呋喃核糖基-6-基)-氨基甲酰)苏氨酸、2'-O-甲基腺苷-5甲基尿苷、2'-O-甲基腺苷、2'-O-甲基胞苷、wybutosine、3-(3-氨基-3-羧基-丙基)尿苷、N6-乙酰基腺苷以及2-甲硫基-N6-甲基腺苷中的一种、两种或更多种。Useful sequences also include modified nucleic acids. Common modifications include 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, 2'-O-methylpseudouridine, β,D- Galactose Q nucleoside, 2'-O-methylguanosine, inosine, N 6 -prenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylinosine , 2'2-dimethyladenosine, 2-methyladenosine, 2-methylguanosine, 5-methyluridine, 3-methylcytidine, 5-methylcytidine, N 6 -methyl Gadenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2-thiouridine, β, D-mannose Q nucleoside, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-thiomethyl- N 6 -Prenyladenosine, N-((9-β-D-ribofuranosyl-2-thiomethylpurine-6-Yl)carbamoyl)threonine, N-((9- β-D-ribofuranosyl purine-6-yl)N-methylcarbamoyl)threonine, uridine-5-oxyacetate-methyl ester, wybutoxosine, pseudouridine, Q nucleoside, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-thiouridine, N-((9-β -D-ribofuranosyl-6-yl)-carbamoyl)threonine, 2'-O-methyladenosine-5-methyluridine, 2'-O-methyladenosine, 2'-O - One of methylcytidine, wybutosine, 3-(3-amino-3-carboxy-propyl)uridine, N 6 -acetyl adenosine and 2-methylthio-N 6 -methyladenosine , two or more kinds.
本发明还涉及含有如上所述基因表达盒的载体。The present invention also relates to vectors containing gene expression cassettes as described above.
当基因表达盒(或称核酸构建体)能使插入的多核苷酸编码的转录或翻译,或单纯的用于保存,或增加其感染宿主细胞的能力时,其也被称为载体。载体可以通过转化,转导或者转染导入宿主细胞,使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的,包括但不限于:质粒;噬菌粒;柯斯质粒;人工染色体,例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC);噬菌体如λ噬菌体或M13噬菌体及动物病毒等。在本发明中,优选的载体是病毒,更优选为昆虫病毒,特别是各种能感染草地贪夜蛾的病毒,优选为昆虫杆状病毒。When a gene expression cassette (or nucleic acid construct) can enable the transcription or translation of the inserted polynucleotide encoding, or is simply used for preservation, or to increase its ability to infect host cells, it is also called a vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc. In the present invention, the preferred vector is a virus, more preferably an insect virus, especially various viruses that can infect Spodoptera frugiperda, preferably an insect baculovirus.
本发明还涉及宿主细胞,其含有如上所述的DNA片段,或被如上所述的载体所转化。The invention also relates to host cells containing a DNA fragment as described above, or transformed with a vector as described above.
宿主细胞优选为昆虫细胞系,例如sf9、Mimic sf9、sf21、HighFive等;在一些具体的实施方式中,所述宿主细胞为sf9细胞。The host cell is preferably an insect cell line, such as sf9, Mimic sf9, sf21, HighFive, etc.; in some specific embodiments, the host cell is an sf9 cell.
本发明还涉及如上所述昆虫病毒的制备方法,包括在合适的条件下培养如上所述的宿主细胞(特别是昆虫宿主细胞),并从培养液上清或细胞裂解液中收集如此得到的昆虫病毒。The present invention also relates to a method for preparing insect viruses as described above, which includes culturing the host cells (especially insect host cells) as described above under appropriate conditions, and collecting the insects thus obtained from the culture supernatant or cell lysate. Virus.
本发明还涉及一种用于防治草地贪夜蛾的组合物,其活性成分包含如上所述的siRNA、如上所述的shRNA或如上所述的载体。The present invention also relates to a composition for controlling Spodoptera Frugiperda, the active ingredient of which includes the above-mentioned siRNA, the above-mentioned shRNA or the above-mentioned vector.
组合物中的核酸组分(如siRNA、shRNA和质粒等)可结合(缀合)到合成载体上,以增加其生物稳定性和/或生物利用度,从而引起RNA干扰。合成载体可以是具有天然或改造亲和力的惰性化合物,在某些方面,合成载体包含惰性化学物质或纳米颗粒的组合,其在结合时和/或单独具有净正电荷或一般亲和力以结合带负电荷的dsRNA。代表性实例包括壳聚糖、脂质体、碳量子点、植物(例如椰糠或谷物粉等)或土壤(例如钙化粘土)来源的可生物降解颗粒等。优选的,其活性成分为昆虫病毒。The nucleic acid components (such as siRNA, shRNA, plasmids, etc.) in the composition can be combined (conjugated) to a synthetic carrier to increase its biological stability and/or bioavailability, thereby causing RNA interference. Synthetic carriers can be inert compounds with natural or engineered affinity. In certain aspects, synthetic carriers comprise a combination of inert chemicals or nanoparticles that when bound and/or alone have a net positive charge or general affinity to bind negatively charged dsRNA. Representative examples include chitosan, liposomes, carbon quantum dots, biodegradable particles derived from plants (such as coconut bran or cereal flour, etc.) or soil (such as calcified clay), and the like. Preferably, the active ingredient is insect virus.
本发明还涉及一种防治草地贪夜蛾的方法,包括:The invention also relates to a method for preventing and controlling Spodoptera Frugiperda, which includes:
将如上所述的组合物施用以使得其被草地贪夜蛾取食;Applying a composition as described above such that it is eaten by Spodoptera frugiperda;
所述施用的方法包括:The methods of administration include:
对植物局部或整株用药,或涂覆在植物种子上,或通过肥料传播,或通过灌溉传播,或上述施用方法的组合。Apply the medicine locally or to the whole plant, or coat the plant seeds, or spread it through fertilizer, or spread it through irrigation, or a combination of the above application methods.
草地贪夜蛾属于杂食性害虫,其可危害多种植物,因而容易理解,所述植物包括草地贪夜蛾的所有宿主,优选为经济作物,例如花生、甜菜、大豆、木瓜、草莓、地瓜、苋菜等超过80多种植物。在一些实施方式中,所述植物选自禾本科植物,更优选水稻、甘蔗、小麦、大麦、高粱、黑麦草、苏丹草和玉米。Spodoptera frugiperda is an omnivorous pest that can harm a variety of plants, so it is easy to understand. The plants include all hosts of Spodoptera frugiperda, preferably cash crops, such as peanuts, sugar beets, soybeans, papayas, strawberries, sweet potatoes, Amaranth and more than 80 kinds of plants. In some embodiments, the plant is selected from the group consisting of grasses, more preferably rice, sugarcane, wheat, barley, sorghum, ryegrass, sudangrass and corn.
下面将结合实施例对本发明的实施方案进行详细描述。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,优先参考本发明中给出的指引,还可以按照本领域的实验手册或常规条件,还可以参考本领域已知的其它实验方法,或者按照制造厂商所建议的条件。The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. For experimental methods that do not indicate specific conditions in the following examples, priority is given to the guidelines given in the present invention. You can also follow the experimental manuals or conventional conditions in the field. You can also refer to other experimental methods known in the field, or according to the manufacturing methods. Conditions recommended by the manufacturer.
下述的具体实施例中,涉及原料组分的量度参数,如无特别说明,可能存在称量精度范围内的细微偏差。涉及温度和时间参数,允许仪器测试精度或操作精度导致的可接受的偏差。In the following specific examples, the measurement parameters of raw material components are involved. Unless otherwise specified, there may be slight deviations within the range of weighing accuracy. Temperature and time parameters are involved, allowing for acceptable deviations due to instrument testing accuracy or operating accuracy.
实施例1Example 1
本实施例利用V-ATPase基因片段的siRNA与草地贪夜蛾的U6启动子连接持续产生可干扰V-ATPase的核酸农药,可极大地抑制草地贪夜蛾幼虫的生长发育。In this example, siRNA of the V-ATPase gene fragment is connected to the U6 promoter of Spodoptera frugiperda to continuously produce nucleic acid pesticides that can interfere with V-ATPase, which can greatly inhibit the growth and development of Spodoptera frugiperda larvae.
其具体的构建方法如下:Its specific construction method is as follows:
U6启动子基因的克隆:Cloning of U6 promoter gene:
基因组DNA的提取:利用Omega Bio-Tek公司的E.Z.N.A.Insect DNA Kit提取草地贪夜蛾基因组DNA,DNA稀释10倍后作为目标基因扩增的模板。Extraction of genomic DNA: Use Omega Bio-Tek's E.Z.N.A.Insect DNA Kit to extract Spodoptera frugiperda genomic DNA. The DNA was diluted 10 times and used as a template for target gene amplification.
设计引物:将家蚕U6 snRNA基因全序列(AY6493 81.1)与果蝇的U6snRNA基因全序列(AH004871.1)经在线软件ClustalOmega(http://www.ebi.ac.uk/Tools/msa/clustalo/)比对,分析它们的同源区域,并在草地贪夜蛾基因组数据库中检索草地贪夜蛾候选的U6启动子,选择分别取5'端27bp U6snRNA及其转录起始位点上游500-2000bp作为草地贪夜蛾全长U6启动子候选序列,并在设计引物时将EFFP基因的shRNA序列添加至U6启动子下游分别命名为u61-shEGFP、u62-shEGFP、u63-shEGFP和u64-shEGFP。根据NCBI primerblast设计引物如表1所示:Design primers: The complete sequence of U6 snRNA gene of Bombyx mori (AY6493 81.1) and the complete sequence of U6 snRNA gene of Drosophila melanogaster (AH004871.1) were used through the online software ClustalOmega (http://www.ebi.ac.uk/Tools/msa/clustalo/ ), analyze their homologous regions, and search Spodoptera frugiperda candidate U6 promoters in the Spodoptera frugiperda genome database, and select the 5'-end 27bp U6 snRNA and the 500-2000bp upstream of the transcription start site. As a candidate sequence for the full-length U6 promoter of Spodoptera frugiperda, when designing primers, the shRNA sequence of the EFFP gene was added to the downstream of the U6 promoter and named u61-shEGFP, u62-shEGFP, u63-shEGFP and u64-shEGFP respectively. The primers designed according to NCBI primerblast are shown in Table 1:
表1Table 1
PCR扩增:PCR amplification:
PCR扩增体系如表2所示:The PCR amplification system is shown in Table 2:
表2Table 2
PCR扩增程序为:The PCR amplification procedure is:
①94℃ 3min①94℃ 3min
②94℃ 30s②94℃ 30s
③55℃ 30s 35cycles③55℃ 30s 35cycles
步骤②和③重复35个循环Repeat steps ② and ③ for 35 cycles
④72℃ 2min④72℃ 2min
⑤72℃ 10min⑤72℃ 10min
扩增产物经2.0%琼脂糖电泳,利用Omega Bio-Tek公司的DNA纯化胶回收试剂盒(Gel Extraction Kit)回收PCR产物,具体为:The amplified product was subjected to 2.0% agarose electrophoresis, and the PCR product was recovered using the DNA purification gel extraction kit (Gel Extraction Kit) of Omega Bio-Tek Company, specifically:
1.将含有DNA条带的胶条切下放入离心管中,称重。(需先称空离心管的重量,在放入切下的凝胶后再次称量,两次重量相减,即得到凝胶的重量)。1. Cut the strip containing the DNA band and put it into a centrifuge tube and weigh it. (You need to weigh the empty centrifuge tube first, weigh it again after placing the cut gel, and subtract the two weights to get the weight of the gel).
2.加入与胶等体积的Binding buffer(凝胶重为0.1g,则其体积视为100μL),55-65℃水浴融化7min,期间2-3min混匀一次。2. Add the same volume of Binding buffer as the gel (the gel weighs 0.1g, then its volume is regarded as 100 μL), melt in a water bath at 55-65°C for 7 minutes, and mix once every 2-3 minutes.
3.吸取700μL步骤2中得到的混合液,加入HiBindTM DNA柱中(吸附柱放入2mL收集管中),室温下12 000rpm离心1min,倒掉收集管中的废液,不断重复直至加入全部2中样品。3. Take 700 μL of the mixed solution obtained in step 2, add it to the HiBind TM DNA column (put the adsorption column into a 2 mL collection tube), centrifuge at 12,000 rpm for 1 min at room temperature, pour out the waste liquid in the collection tube, and repeat until all is added. 2 samples.
4.加入300μL Binding buffer于吸附柱,室温下12 000rpm离心1min,倒掉收集管中的废液。4. Add 300 μL Binding buffer to the adsorption column, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the waste liquid in the collection tube.
5.加入700μL spw wash buffer于吸附柱中,室温下12 000rpm离心1min,弃滤液。5. Add 700 μL spw wash buffer to the adsorption column, centrifuge at 12 000 rpm for 1 min at room temperature, and discard the filtrate.
6.重复步骤5。6. Repeat step 5.
7.将吸附柱重新放回收集管中,12 000rpm空转离心1min。7. Put the adsorption column back into the collection tube, and centrifuge idling at 12,000 rpm for 1 minute.
8.将吸附柱转移进一个干净且灭过菌的离心管中,在吸附膜的中间部位加入15-30μL预热的ddH2O,12 000rpm离心1min。8. Transfer the adsorption column into a clean and sterilized centrifuge tube, add 15-30 μL of preheated ddH 2 O to the middle of the adsorption membrane, and centrifuge at 12,000 rpm for 1 min.
9.将上一步离心到1.5mL离心管内的溶液全部吸取后加入到吸附柱中,离心1min。即得到纯化的DNA产物。测浓度后在-20℃保存。9. Absorb all the solution centrifuged in the previous step into a 1.5 mL centrifuge tube, add it to the adsorption column, and centrifuge for 1 minute. That is, the purified DNA product is obtained. After measuring the concentration, store at -20°C.
连接载体:Connection carrier:
取PCR产物连接到pFastBAC1载体(购自淼灵质粒平台)中。The PCR product was ligated into the pFastBAC1 vector (purchased from Miaoling Plasmid Platform).
连接体系如下所示:The connection system is as follows:
将连接体系在16℃连接12h得到连接产物。The connection system was connected at 16°C for 12 hours to obtain the connection product.
转化:Conversion:
1.冰上解冻DH5α感受态细胞。1. Thaw DH5α competent cells on ice.
2.将10μL重组产物轻轻混匀加至解冻后的100μL感受态中,在冰上孵育30min,42℃热激60s后,立即置于冰上冷却2-3min。2. Gently mix 10 μL of recombinant product and add to 100 μL of thawed competent cells, incubate on ice for 30 minutes, heat shock at 42°C for 60 seconds, and immediately cool on ice for 2-3 minutes.
3.加入1mL不含抗生素的LB液体培养基,37℃摇菌1h。3. Add 1 mL of LB liquid culture medium without antibiotics and shake the culture medium at 37°C for 1 hour.
4.5000rpm离心5min,弃800μL上清培养基后轻轻摇匀,涂平板(平板需提前加终浓度为100μg/mL氨苄青霉素钠(Amp),37℃倒置培养12h。4. Centrifuge at 5000 rpm for 5 minutes, discard 800 μL of supernatant culture medium, shake gently, and spread on a plate (the plate needs to be added with a final concentration of 100 μg/mL ampicillin sodium (Amp) in advance, and incubate upside down at 37°C for 12 hours.
提取质粒:随机挑取LB固体培养基上5个白色阳性克隆至3.0mL含50mg/mL卡那霉素的LB液体培养基中,200rpm,37℃摇菌10h,将阳性克隆进行扩增验证,将测序结果正确的菌液通过(OMEGA)Plasmid mini Kit提取质粒。Extract plasmids: Randomly pick 5 white positive clones on LB solid medium into 3.0 mL of LB liquid medium containing 50 mg/mL kanamycin, shake at 200 rpm and 37°C for 10 hours, and amplify the positive clones for verification. Use the (OMEGA) Plasmid mini Kit to extract the plasmid from the bacterial solution with correct sequencing results.
具体步骤为:The specific steps are:
1.取1.5mL离心管加入1 400μL菌液,室温下12 000rpm离心2min。弃上清,不断重复直至样品完全。1. Take a 1.5mL centrifuge tube, add 1 400 μL of bacterial solution, and centrifuge at 12 000 rpm for 2 minutes at room temperature. Discard the supernatant and repeat until the sample is complete.
2.加入250μL SolutionⅠ混合液,涡旋至无白色菌体沉淀。2. Add 250 μL of SolutionⅠ mixture and vortex until no white bacteria precipitate.
3.向步骤2中得到的悬浮的混合液体中加入250μL SolutionⅡ,轻柔的颠倒将混合液混匀5次(此时瓶口有粘丝)。此操作要轻柔,避免剧烈震荡且裂解时间不能超过5min。3. Add 250 μL Solution II to the suspended mixed liquid obtained in step 2, and mix the mixed liquid by gently inverting it 5 times (there is sticky thread on the bottle mouth at this time). This operation should be gentle, avoid violent shaking, and the lysis time should not exceed 5 minutes.
4.加入350μL SolutionⅢ,后立即进行10-20次的旋转颠倒直至有白色的絮状物沉淀形成。此过程要避免剧烈震荡。4. Add 350 μL Solution III, and immediately rotate and invert 10-20 times until white floc precipitates. Avoid violent vibrations during this process.
5.室温下,12 000rpm离心12min。5. Centrifuge at 12 000 rpm for 12 minutes at room temperature.
6.转移上清至滤柱中(套上2mL收集管),室温12 000rpm离心15min,弃滤液。6. Transfer the supernatant to the filter column (put on a 2mL collection tube), centrifuge at room temperature 12,000rpm for 15min, and discard the filtrate.
7.加入500μL的HB Buffer到过滤柱中,12 000rpm离心1.5min,弃滤液。7. Add 500 μL of HB Buffer to the filter column, centrifuge at 12,000 rpm for 1.5 min, and discard the filtrate.
8.加入700μL DNA Wash Buffer到过滤柱中,转速设置为12 000rpm,进行离心1min,弃滤液。8. Add 700 μL DNA Wash Buffer to the filter column, set the rotation speed to 12 000 rpm, centrifuge for 1 min, and discard the filtrate.
9.重复步骤8。9. Repeat step 8.
10.空转离心管,12 000rpm离心2.5min。10. Idle the centrifuge tube and centrifuge at 12,000 rpm for 2.5 minutes.
11.在新离心管上放置上空转后的过滤柱,加入65℃预热的无菌水,静置1min后12000rpm离心1.5min。提取质粒得到的产物通过1%的琼脂糖凝胶电泳检测后,验证质粒条带大小,于-20℃保存。11. Place the idling filter column on a new centrifuge tube, add sterile water preheated at 65°C, let stand for 1 minute, and then centrifuge at 12,000 rpm for 1.5 minutes. The product obtained by extracting the plasmid was detected by 1% agarose gel electrophoresis to verify the size of the plasmid band and stored at -20°C.
测序:质粒pFastBAC1-U6N-shEGFP进行测序(由北京擎科信业生物技术有限公司完成),含U6N-shEGFP基因的pFastBAC1-U6N-shEGFP质粒模板序列如下所示:Sequencing: Plasmid pFastBAC1-U6N-shEGFP was sequenced (completed by Beijing Qingke Xinye Biotechnology Co., Ltd.). The pFastBAC1-U6N-shEGFP plasmid template sequence containing the U6N-shEGFP gene is as follows:
草地贪夜蛾U61启动子序列:SEQ ID NO:5所示;Spodoptera frugiperda U61 promoter sequence: SEQ ID NO: 5;
草地贪夜蛾U62启动子序列:SEQ ID NO:6所示;Spodoptera frugiperda U62 promoter sequence: SEQ ID NO: 6;
草地贪夜蛾U63启动子序列:SEQ ID NO:7所示;Spodoptera frugiperda U63 promoter sequence: SEQ ID NO: 7;
草地贪夜蛾U64启动子序列:SEQ ID NO:8所示;Spodoptera frugiperda U64 promoter sequence: SEQ ID NO: 8;
shEGFP序列:SEQ ID NO:9所示。shEGFP sequence: SEQ ID NO:9.
转化DH10Bac感受态细胞:Transform DH10Bac competent cells:
将得到的产物pFastBAC1-U6N-shEGFP转入DH10Bac感受态细胞,具体步骤如下:Transfer the obtained product pFastBAC1-U6N-shEGFP into DH10Bac competent cells. The specific steps are as follows:
1.在冰上解冻整管DH10Bac感受态细胞。1. Thaw the entire tube of DH10Bac competent cells on ice.
2.在感受态细胞中加入5μL(1ng/μL)的pFastBAC1-U6N-shEGFP质粒DNA,轻轻混匀。2. Add 5 μL (1ng/μL) of pFastBAC1-U6N-shEGFP plasmid DNA to the competent cells and mix gently.
3.冰孵细胞30min,42℃热激细胞45s,切勿摇晃,立即将试管转移至冰上,冷浴2min。3. Incubate the cells on ice for 30 minutes, and heat-shock the cells at 42°C for 45 seconds. Do not shake. Immediately transfer the test tube to ice and keep in a cold bath for 2 minutes.
4.加入900μL的不含抗生素的液体LB培养基。4. Add 900 μL of liquid LB medium without antibiotics.
5.37℃摇床225rpm培养4h。5. Incubate for 4 hours on a shaker at 37°C and 225 rpm.
6.使用LB液体培养基(不含抗生素)制备,将100μL的细胞接种在含有卡那霉素(50μg/mL)、庆大霉素(7μg/mL),四环素(10μg/mL),BLuo-gal(100μg/mL)和IPTG(40μg/mL)的LB琼脂平板上。6. Use LB liquid medium (without antibiotics) to prepare, inoculate 100 μL of cells in a medium containing kanamycin (50 μg/mL), gentamicin (7 μg/mL), tetracycline (10 μg/mL), BLuo- gal (100 μg/mL) and IPTG (40 μg/mL) on LB agar plates.
7.将平板置于37℃下孵育48h,冰箱1h呈色。7. Incubate the plate at 37°C for 48 hours and in the refrigerator for 1 hour to develop color.
8.摇菌:平板37℃下培养48h挑选纯正白斑单菌落,加入含有50μg/mL卡那霉素,70μg/mL庆大霉素,10μg/mL四环素的LB液体培养基中,37℃,200rpm摇床培养过夜。8. Shake the bacteria: Cultivate the plate at 37°C for 48 hours to select pure single colonies of white spots, add LB liquid culture medium containing 50 μg/mL kanamycin, 70 μg/mL gentamicin, and 10 μg/mL tetracycline, 37°C, 200 rpm Incubate overnight on a shaker.
9.利用通用引物M13F和M13R进行PCR鉴定,鉴定阳性Bacmid DNA。9. Use universal primers M13F and M13R for PCR identification to identify positive Bacmid DNA.
利用PCR验证重组杆粒中是否含有目的基因(使用通用引物M13核测),检测成功后转染昆虫sf9细胞。Use PCR to verify whether the recombinant bacmid contains the target gene (use universal primer M13 for nuclear detection), and then transfect insect sf9 cells after successful detection.
细胞转染验证候选U6转录活性:Cell transfection to verify candidate U6 transcriptional activity:
为比较U61/U62/U63/U64候选启动子的转录活性,将载体Bacmid-EGFP-U61-shEGFP、Bacmid-EGFP-U62-shEGFP、Bacmid-EGFP-U63-shEGFP和Bacmid-EGFP-U64-shEGFP分别与Bacmid-EGFP共转染sf9细胞,72h后将各孔细胞置于倒置荧光显微镜下,观察同一视野下细胞生长情况及各组荧光强度(图1)。具体操作如下:To compare the transcriptional activities of U61/U62/U63/U64 candidate promoters, the vectors Bacmid-EGFP-U61-shEGFP, Bacmid-EGFP-U62-shEGFP, Bacmid-EGFP-U63-shEGFP and Bacmid-EGFP-U64-shEGFP were used, respectively. sf9 cells were co-transfected with Bacmid-EGFP. After 72 hours, the cells in each well were placed under an inverted fluorescence microscope to observe the cell growth and fluorescence intensity of each group in the same field of view (Figure 1). The specific operations are as follows:
在6孔板进行sf9细胞的转染,将构建的Bacmid-U6N-shEGFP及Bacmid-EGFP重组载体共转染sf9细胞(具体方法见表3)。操作如下:Transfect sf9 cells in a 6-well plate, and co-transfect sf9 cells with the constructed Bacmid-U6N-shEGFP and Bacmid-EGFP recombinant vectors (see Table 3 for specific methods). Here's how to do it:
1.培养悬浮细胞:将处于对数生长期的细胞培养瓶中原来的培养基和悬浮细胞倒掉,加入12mL新鲜Sf-900TMⅢSFM培养基。用弯头玻璃吸管轻轻将贴壁细胞吹下。1. Cultivate suspended cells: Pour out the original culture medium and suspended cells in the cell culture flask in the logarithmic growth phase, and add 12 mL of fresh Sf-900 TM III SFM culture medium. Use an elbow glass pipette to gently blow off the adherent cells.
2.细胞贴壁:在6孔板中加入2mL细胞悬液,置于27℃恒温箱中,细胞贴壁培养至少1h,使细胞数量达到2×106细胞/孔。2. Cell adhesion: Add 2 mL of cell suspension to a 6-well plate, place it in a 27°C incubator, and culture the cells for at least 1 hour until the number of cells reaches 2 × 10 6 cells/well.
3.准备转染混合液3. Prepare transfection mixture
①吸取1.5μg的Bacmid质粒加入150μL的Sf-900TMⅢSFM培养基中,轻柔的吹打湿匀。① Add 1.5 μg of Bacmid plasmid to 150 μL of Sf-900 TM III SFM medium, and gently pipet to wet it evenly.
②吸取10μL的轻染试剂cellfection12加入到150μL的Sf-900TMⅢSFM培养基中,轻柔的吹打混匀。② Take 10 μL of light staining reagent cellfection 12 and add it to 150 μL of Sf-900 TM III SFM medium, and mix gently by pipetting.
③将稀释好的①和②轻轻混匀,室温静置30min。③Mix diluted ① and ② gently and let stand at room temperature for 30 minutes.
④轻染:30min后将轻染混合液加入6孔板中,每孔100μL,十字交叉混匀,27℃培养。④ Light staining: After 30 minutes, add the light staining mixture into the 6-well plate, 100 μL per well, mix crosswise, and incubate at 27°C.
表3转染组分及用量Table 3 Transfection components and dosage
评判标准:U6启动子启动效率越高,shEGFP对荧光蛋白EGFP的干扰效率就越高,EGFP的表达量就越低,在显微镜下荧光就越弱;反之,U6启动子启动效率越差,shEGFP对荧光蛋白EGFP的干扰效率就越低,EGFP的表达量就越高,在显微镜下荧光就越强。Evaluation criteria: The higher the U6 promoter startup efficiency, the higher the interference efficiency of shEGFP on the fluorescent protein EGFP, the lower the expression of EGFP, and the weaker the fluorescence under the microscope; conversely, the worse the U6 promoter startup efficiency, shEGFP The lower the interference efficiency with the fluorescent protein EGFP, the higher the expression level of EGFP, and the stronger the fluorescence under the microscope.
结果发现Bacmid-EGFP-U61-shEGFP+Bacmid-EGFP的荧光信号强度显著降低并在四个候选启动子中最低,说明EGFP基因的表达量有所下降,进一步表明U6候选启动子能够驱动shEGFP在sf9细胞中成功表达,即证明草地贪夜蛾U61候选启动子对于发夹结构RNA具有一定的转录活性。因此,选取草地贪夜蛾6号染色体上的U61候选启动子用于后续重组载体的构建试验。The results showed that the fluorescence signal intensity of Bacmid-EGFP-U61-shEGFP+Bacmid-EGFP was significantly reduced and was the lowest among the four candidate promoters, indicating that the expression of the EGFP gene had decreased, further indicating that the U6 candidate promoter can drive shEGFP in sf9 Successful expression in cells proves that the Spodoptera frugiperda U61 candidate promoter has certain transcriptional activity for hairpin structured RNA. Therefore, the U61 candidate promoter on chromosome 6 of Spodoptera frugiperda was selected for subsequent construction experiments of recombinant vectors.
pFastBac1-EGFP转移载体的酶切:用XhoI和HindIII对质粒进行双酶切,酶切后的载体命名为pFastBac1-EGFP-,对酶切后的质粒进行切胶回收,回收产物置于-20℃备用。Enzyme digestion of the pFastBac1-EGFP transfer vector: Double-digest the plasmid with XhoI and HindIII. The digested vector is named pFastBac1-EGFP-. The digested plasmid is gel-cut and recovered, and the recovered product is placed at -20°C. spare.
V-ATPase A/B亚基发夹结构的设计:用在线软件siDirect(http://design.RNA.jp/)对草地贪夜蛾V-ATP A/B亚基的最佳干扰最佳片段ATPaseA-2和ATPaseB-2进行预测分析,获得潜在的siRNA并根据预测出的siRNA设计发夹结构。Design of hairpin structure of V-ATPase A/B subunit: optimal interference fragment of Spodoptera frugiperda V-ATP A/B subunit using online software siDirect (http://design.RNA.jp/) ATPaseA-2 and ATPaseB-2 perform predictive analysis to obtain potential siRNA and design a hairpin structure based on the predicted siRNA.
草地贪夜蛾基因V-ATPaseA/B的最佳干扰片段The best interference fragment of Spodoptera frugiperda gene V-ATPaseA/B
dsV-ATPaseA-2 378bpdsV-ATPaseA-2 378bp
AAGACTGTCGTCTCACAGGCTCTGTCCAAGTACTCCAACTCTGACGTCATCATCTACGTCGGATGCGGTGAACGTGGTAACGAGATGTCTGAGGTACTGCGTGACTTCCCCGAGCTGACGGTGGAGATCGAGGGCATGACCGAGTCCATCATGAAGCGTACCGCGCTCGTCGCCAACACCTCCAACATGCCTGTAGCCGCCCGAGAGGCTTCCATCTACACCGGTATCACCCTCTCCGAGTACTTCCGTGACATGGGTTACAACGTGTCCATGATGGCTGACTCCACCTCTCGTTGGGCCGAGGCTCTTCGTGAGATCTCAGGTCGTCTGGCTGAGATGCCTGCCGACTCCGGTTACCCCGCCTACCTGGGAGCCCGTAAGACTGTCGTCTCACAGGCTCTGTCCAAGTACTCCAACTCTGACGTCATCATCTACGTCGGATGCGGTGAACGTGGTAACGAGATGTCTGAGGTACTGCGTGACTTCCCCGAGCTGACGGTGGAGATCGAGGGCATGACCGAGTCCATCATGAAGCGTTACCGCGCTCGTCGCCAACACCTCCAACATGCCTGTAGCCGCCCGAGAGGCTTCCATCTACACCGGTATCACCCTCTCCGAGTACTTCCGTGACATGGGTTA CAACGTGTCCATGATGGCTGACTCCACCTCTCGTTGGGCCGAGGCTCTTCGTGAGATCTCAGGTCGTCTGGCTGAGATGCCTGCCGACTCCGGTTACCCCGCCTACCTGGGAGCCCGT
dsV-ATPaseB-2 408bpdsV-ATPaseB-2 408bp
AACTCCATCGCTCGTGGTCAGAAGATCCCCATCTTCTCCGCTGCTGGTCTGCCCCACAACGAAATTGCCGCCCAGATCTGTAGACAGGCCGGTCTTGTCAAGATCCCCGGCAAATCAGTGTTGGATGACCACGAGGACAACTTCGCCATCGTGTTCGCCGCTATGGGTGTGAACATGGAAACCGCCCGGTTCTTCAAACAGGACTTCGAAGAGAACGGTTCCATGGAGAACGTGTGCCTGTTCTTGAACTTGGCCAACGACCCTACCATTGAGAGAATTATCACACCCCGTCTGGCTCTTACTGCCGCCGAGTTCTTGGCCTACCAGTGCGAGAAACACGTGTTGGTCATCTTGACTGACATGTCCTCATACGCCGAGGCTCTGCGTGAGGTATCCGCCGCCCGTGAGAACTCCATCGCTCGTGGTCAGAAGATCCCCATCTTCTCCGCTGCTGGTCTGCCCCACAACGAAATTGCCGCCCAGATCTGTAGACAGGCCGGTCTTGTCAAGATCCCCGGCAAATCAGTGTTGGATGACCACGAGGACAACTTCGCCATCGTGTTCGCCGCTATGGGTGTGAACATGGAAACCGCCCGGTTCTTCAAACAGGACTTCGAAGAGAACGGTTCCATGGAGAACGTGTGCCTGTTCTTGAACTTGGCCAACGACCCTACCATT GAGAGAATTATCACACCCCGTCTGGCTCTTACTGCCGCCGAGTTCTTGGCCTACCAGTGCGAGAAACACGTGTTGGTCATCTTGACTGACATGTCCTCATACGCCGAGGCTCTGCGTGAGGTATCCGCCGCCCGTGAG
shRNA序列如下:The shRNA sequence is as follows:
U6-shA/B的克隆。Cloning of U6-shA/B.
根据草地贪夜蛾U61启动子序列及V-ATPase A/B亚基的发夹结构序列设计包含质粒同源臂的特异性引物克隆U6-shA/B下表所示,引物由北京擎科生物技术有限公司合成。Based on the Spodoptera frugiperda U61 promoter sequence and the hairpin structure sequence of V-ATPase A/B subunits, specific primers containing plasmid homology arms were designed to clone U6-shA/B as shown in the table below. The primers were provided by Beijing Qingke Biotechnology Technology Co., Ltd. Synthetic.
表4 U6-shA/B引物系列Table 4 U6-shA/B primer series
注:正义链同源臂为CGAGCTGTACAAGTTCTAGCTCGAG,反义链同源臂为CTAGTACTTCTCGACAAGCTT。Note: The homology arm of the sense strand is CGAGCTGTACAAGTTCTAGCTCGAG, and the homology arm of the antisense strand is CTAGTACTTCTCGACAAGCTT.
重组杆状病毒转移载体Bacmid-U61-EGFP-shA/B的构建:Construction of recombinant baculovirus transfer vector Bacmid-U61-EGFP-shA/B:
将XhoI和HindIII酶切后的pFastBac1-EGFP-与U61-shA/B连接,得到pFastBac1-EGFP-U61-shA/B重组载体,转化后进行菌液PCR验证及测序。将测序正确的阳性质粒转入DH10Bac感受态细胞中,提取含目的基因的重组质粒Bacmid-U61-shA/B并用M13通用引物再次验证目的基因。The pFastBac1-EGFP- digested by XhoI and HindIII was connected to U61-shA/B to obtain the pFastBac1-EGFP-U61-shA/B recombinant vector. After transformation, bacterial liquid PCR verification and sequencing were performed. Transfer the correctly sequenced positive plasmid into DH10Bac competent cells, extract the recombinant plasmid Bacmid-U61-shA/B containing the target gene, and use the M13 universal primer to verify the target gene again.
重组昆虫杆状病毒的获得:利用Bac To Bac System进行重组昆虫杆状病毒转染试验,在细胞培养瓶中进行Bacmid-U61-shA/B重组质粒向sf9细胞的转染,转染后2-3d待细胞开始破裂时,收集培养液至10mL离心管中,22℃,500g离心,5min,在5mL离心管中装入移取的上清液,置4℃避光保存,即为p1代。在6孔板中准备贴壁细胞,分别取P1代病毒300μL逐滴加入,置于27℃恒温箱中,2-3d待细胞开始破裂时,收集p2代病毒(方法同p1代)。在含有贴壁细胞6孔板中逐滴加入Pn代病毒,培养2-3d,收集Pn+1代病毒。Obtaining the recombinant insect baculovirus: Use the Bac To Bac System to conduct the transfection test of the recombinant insect baculovirus, and transfect the Bacmid-U61-shA/B recombinant plasmid into sf9 cells in a cell culture flask. 2- When the cells begin to rupture on 3 days, collect the culture medium into a 10 mL centrifuge tube, centrifuge at 22°C at 500g for 5 min, put the transferred supernatant into a 5 mL centrifuge tube, and store it at 4°C in the dark, which is the p1 generation. Prepare adherent cells in a 6-well plate, add 300 μL of P1 generation virus drop by drop, and place it in a 27°C incubator. When the cells begin to rupture for 2-3 days, collect the p2 generation virus (the method is the same as that of p1 generation). Add Pn-generation viruses dropwise to a 6-well plate containing adherent cells, culture for 2-3 days, and collect Pn+1-generation viruses.
实施例2核酸杀虫剂在杀灭草地贪夜蛾的应用Example 2 Application of nucleic acid pesticides in killing Spodoptera frugiperda
其应用方法为:Its application method is:
(1)分别将重组昆虫杆状病毒以及空载体杆状病毒,按每3g烘干饲料1mL杆状病毒的量饲喂三龄草地贪夜蛾幼虫。(1) Feed the recombinant insect baculovirus and the empty vector baculovirus to third-instar Spodoptera frugiperda larvae at a dosage of 1 mL of baculovirus per 3 g of dry feed.
(2)每天记录各处理组幼虫死亡率,直到幼虫化蛹,还记录了幼虫化蛹后记录不同处理组下的化蛹率和成虫羽化率。(2) The mortality rate of larvae in each treatment group was recorded every day until the larvae pupated. After the larvae pupated, the pupation rate and adult emergence rate under different treatment groups were also recorded.
(3)向3龄试虫注射100nL重组杆状病毒shA/shB、空载体杆状病毒以及清水,每天观察幼虫形态变化和存活情况,并定期取出幼虫检测基因表达情况,并做好记录。(3) Inject 100 nL of recombinant baculovirus shA/shB, empty vector baculovirus and clean water into the third-instar test worms. Observe the morphological changes and survival of the larvae every day, take out the larvae regularly to detect gene expression, and keep records.
图3为草地贪夜蛾取食含有病毒核酸农药后的死亡率比较,小写字母表示处理与对照有显著差异(p<0.05)。从图中可知看出:重组杆状病毒对草地贪夜蛾具有生长发育抑制作用,能够显著降低幼虫存活率,饲喂空载体杆状病毒的存活率为43.75%,而饲喂重组杆状病毒shA的幼虫存活率为18.77%。饲喂重组杆状病毒shB的幼虫存活率为41.05%,空白处理组的存活率为72.2%。Figure 3 shows the comparison of mortality rates of Spodoptera Frugiperda after feeding on pesticides containing viral nucleic acids. Lowercase letters indicate significant differences between treatments and controls (p<0.05). It can be seen from the figure that the recombinant baculovirus has an inhibitory effect on the growth and development of Spodoptera frugiperda and can significantly reduce the survival rate of larvae. The survival rate of feeding empty vector baculovirus is 43.75%, while the survival rate of feeding recombinant baculovirus The larval survival rate of shA was 18.77%. The survival rate of larvae fed recombinant baculovirus shB was 41.05%, and the survival rate of blank treatment group was 72.2%.
图4为草地贪夜蛾取食含有病毒核酸农药后的化蛹率和成虫羽化率比较,重组杆状病毒shA及shB处理组分别有18.75%和35.40%的幼虫能够化蛹,而在空载体杆状病毒处理中有43.75%的幼虫化蛹,清水处理组幼虫的化蛹率为72.20%;与对照组72.20%的成虫羽化率相比,杆状病毒处理组的成虫羽化率均有显著下降。且在空载体杆状病毒处理中观察到35.40%的羽化率,而重组杆状病毒shA及shB处理中分别观察到最低的成虫羽化率为18.75%和22.95%,显著低于空载体杆状病毒。Figure 4 shows the comparison of the pupation rate and adult emergence rate of Spodoptera frugiperda after feeding on pesticides containing viral nucleic acids. 18.75% and 35.40% of the larvae in the recombinant baculovirus shA and shB treatment groups were able to pupate respectively, while in the empty vector In the baculovirus treatment, 43.75% of the larvae pupated, and the pupation rate of the larvae in the clean water treatment group was 72.20%; compared with the 72.20% adult emergence rate in the control group, the adult emergence rate in the baculovirus treatment group was significantly reduced. . And in the empty vector baculovirus treatment, an emergence rate of 35.40% was observed, while the lowest adult emergence rates of 18.75% and 22.95% were observed in the recombinant baculovirus shA and shB treatments, respectively, which were significantly lower than the empty vector baculovirus. .
图5为注射杆状病毒后目的基因表达量情况。Figure 5 shows the expression level of the target gene after injection of baculovirus.
图6为取食重组昆虫杆状病毒核酸农药后的中毒症状图。Figure 6 is a diagram of poisoning symptoms after ingesting recombinant insect baculovirus nucleic acid pesticides.
表5为注射重组昆虫杆状病毒后草地贪夜蛾体重体长变化比较,小写字母表示处理与对照有显著差异(p<0.05)。Table 5 shows the comparison of changes in body weight and length of Spodoptera frugiperda after injection of recombinant insect baculovirus. Lowercase letters indicate significant differences between the treatment and the control (p<0.05).
表5table 5
图7为注射重组昆虫杆状病毒对草地贪夜蛾幼虫生长发育抑制情况图。Figure 7 shows the inhibition of growth and development of Spodoptera frugiperda larvae by injection of recombinant insect baculovirus.
注射昆虫杆状病毒能够引起幼虫大量死亡,注射重组昆虫杆状病毒shA和shB处理组幼虫均在第3d开始出现大量死亡,4d全部死亡;空载体组在第4d没有幼虫死亡,第5d部分幼虫出现死亡现象,直至第6d全部死亡;空白对照组在观察期间则没有出现死亡现象。Injection of insect baculovirus can cause massive death of larvae. In the treatment groups injected with recombinant insect baculovirus shA and shB, large numbers of larvae began to die on the 3rd day, and all larvae died on the 4th day; in the empty vector group, no larvae died on the 4th day, and some larvae died on the 5th day. Death occurred until the 6th day. There was no death in the blank control group during the observation period.
注射杆状病毒可以显著降低幼虫的体重和体长增长。在注射第一天各处理组与空白对照组的体重、体长没有显著差异;第三天出现显著差异(P<0.05),其中空白对照组体重平均增长0.1396g、体长平均增长0.817cm;空载体组体重平均增长0.01465g、体长平均增长0.305cm;重组载体shA体重平均下降0.0063g、体长平均下降0.305cm;重组载体shB体重平均下降0.00631g、体长平均下降0.243cm。Injection of baculovirus significantly reduced larval body weight and body length growth. On the first day of injection, there was no significant difference in body weight and body length between each treatment group and the blank control group; significant differences appeared on the third day (P<0.05), in which the average weight and body length increase of the blank control group was 0.1396g and 0.817cm; The weight of the empty vector group increased on average by 0.01465g, and the body length increased by 0.305cm on average; the weight of the recombinant vector shA decreased on average by 0.0063g, and the body length decreased by 0.305cm on average; the weight of the recombinant vector shB decreased on average by 0.00631g, and the body length decreased by 0.243cm on average.
本实施例的方法并不仅限于以V-ATPase A/B亚基为靶标的核酸农药,其他昆虫的致死基因也,可采用本实施例的方法,并能达到同样的技术效果。The method of this embodiment is not limited to nucleic acid pesticides targeting V-ATPase A/B subunits. The method of this embodiment can also be used for lethal genes of other insects, and the same technical effect can be achieved.
根据本实施例,通过饲喂和注射草地贪夜蛾重组昆虫杆状病毒,病毒进入草地贪夜蛾体内后导致靶标基因的沉默,影响植物害虫的功能,最终导致其死亡,从而达到防治植物害虫的目的。本发明的方法为绿色环保的核酸农药,相比化学药剂更加环保安全,靶标性也更强,另一方面本方法较喷施dsRNA更有利于防治草地贪夜蛾。According to this embodiment, by feeding and injecting the recombinant insect baculovirus of Spodoptera frugiperda, the virus enters the body of Spodoptera frugiperda and causes the silencing of the target gene, affecting the function of plant pests and eventually leading to their death, thereby achieving the prevention and control of plant pests. the goal of. The method of the present invention is a green and environmentally friendly nucleic acid pesticide, which is more environmentally friendly and safer than chemical agents, and has stronger targeting properties. On the other hand, the method is more conducive to controlling Spodoptera Frugiperda than spraying dsRNA.
本实施例的方法并不仅限于昆虫杆状病毒表达载体,可侵染其他有害生物的表达载体,也可采用本实施例的方法,并能达到同样的技术效果。The method of this embodiment is not limited to insect baculovirus expression vectors. Expression vectors that can infect other harmful organisms can also use the method of this embodiment, and can achieve the same technical effect.
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准,说明书及附图可以用于解释权利要求的内容。The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be determined by the appended claims, and the description and drawings can be used to interpret the content of the claims.
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