技术领域Technical Field
本发明涉及基因工程以及害虫防控技术领域，具体涉及一种对番茄红螨高效致死的靶标基因及其应用。The invention relates to the technical field of genetic engineering and pest control, and in particular to a target gene highly effective in killing tomato red mites and an application thereof.
背景技术Background technique
番茄红螨，又名伊氏叶螨Tetranychusevansi，隶属节肢动物门Arthropoda、螯肢动物亚门Chelicerata、蛛形纲Arachnida、蜱螨亚纲Acari、真螨总目Acariformes、前气门亚目Prostigmata、叶螨总科Tetranychidae、叶螨属Tetranychus。番茄红螨发育共历经卵、幼螨、前若螨、后若螨、成螨5个阶段，每个发育阶段均会经历一次蜕皮，其生活史为不完全变态发育。番茄红螨体型较小，其成螨仅有零点几毫米。幼螨有三对足，若螨和成螨则有四对足。雌成螨的外形呈椭圆形，体色多数呈红黑色，雄成螨的体型较细长，腹部呈锥状，体型较雌成螨略小，体色仍呈红黑色。Tomato red mite, also known as Tetranychusevansi, belongs to Arthropoda, Chelicerata, Arachnida, Acari, Acariformes, Prostigmata, Tetranychidae, Tetranychus. The development of tomato red mite goes through five stages: egg, larva, pronymph, postnymph, and adult. Each development stage will experience a molt, and its life history is incomplete metamorphosis. Tomato red mite is small in size, and its adult mite is only a few tenths of a millimeter. The larvae have three pairs of legs, while the nymphs and adults have four pairs of legs. The female adult mite is oval in shape, and the body color is mostly red and black. The male adult mite is slender, with a cone-shaped abdomen, slightly smaller than the female adult mite, and the body color is still red and black.
番茄红螨是典型的入侵害螨，也是我国重要的检疫对象。番茄红螨起源于南美洲，是取食茄科作物的专食性害虫。目前，已在我国多地发现，由于番茄红螨适应能力强，缺少天敌，正在逐渐取代当地的土著叶螨种群，逐步成为当地寄主植物上的主要害螨种群，并给当地的农业生产造成了巨大的损失。在新的气候变化及单一作物规模化种植的影响下，番茄红螨有了更多的适生地，可能遭到入侵的区域也相应的扩大。尤其我国东南部的气候条件非常适合番茄红螨的生存，在其进入当地后极易爆发成灾。The tomato red mite is a typical invasive mite and an important quarantine target in my country. Originated from South America, the tomato red mite is a specialized pest that feeds on Solanaceae crops. At present, it has been found in many places in my country. Due to its strong adaptability and lack of natural enemies, the tomato red mite is gradually replacing the local indigenous spider mite population and gradually becoming the main pest mite population on local host plants, causing huge losses to local agricultural production. Under the influence of new climate change and large-scale planting of single crops, the tomato red mite has more suitable habitats, and the areas that may be invaded have also expanded accordingly. In particular, the climatic conditions in southeastern my country are very suitable for the survival of the tomato red mite, and it is very easy to cause a disaster after entering the local area.
番茄红螨是r—对策害虫，具有体型小、发育历期短、繁殖能力强、世代交替严重、爬行迅速的特点，并在环境不适时会结网随风传播，规避自然灾害的能力较强。番茄红螨通过口针刺入植物叶肉细胞，摄取细胞内容物，造成取食部位塌陷及出现白色危害斑点。番茄红螨除了具有较强的解毒代谢能力，还会分泌唾液蛋白抑制番茄等寄主植物的防御，并增加同一生态位其他有害生物的适应性。当番茄红螨大爆发时，会大量聚集在植物叶片、枝干、果实等进行危害，严重时可导致植物叶片、茎秆萎蔫失绿、焦枯甚至死亡，严重制约了我国的农业发展。Tomato red mites are r-strategy pests with the characteristics of small size, short development period, strong reproductive ability, severe generation alternation, rapid crawling, and will weave webs to spread with the wind when the environment is unsuitable. They have a strong ability to avoid natural disasters. Tomato red mites penetrate plant mesophyll cells through their mouth needles and ingest cell contents, causing the feeding site to collapse and white harmful spots to appear. In addition to having strong detoxification and metabolic capabilities, tomato red mites also secrete salivary proteins to inhibit the defense of host plants such as tomatoes, and increase the adaptability of other pests in the same ecological niche. When tomato red mites break out, they will gather in large numbers on plant leaves, branches, fruits, etc. to cause damage. In severe cases, they can cause plant leaves and stems to wilt, lose green, scorch, or even die, which seriously restricts the development of agriculture in my country.
为减弱害螨在农业生产上造成的严重损失，一系列防治方法应运而生。虽然近年来以利用捕食螨为主的生物防治取得了显著成效，但化学防治仍占据主要地位。由于长时间、大规模、单一等不科学地使用化学农药，加之害螨典型的r-对策生活史，致使农药“3R”问题非常严重。同时，由于叶螨繁殖力强、世代历期短，加剧了害螨抗药性的积累，循环往复致使其出现了极高的抗药性水平。目前，在二斑叶螨中已发现抗药性记录417例，位列节肢动物抗药性记录第一，且叶螨已对多种杀螨剂产生了交互抗性。因此寻找新型防控靶标、研制环保有效的新型叶螨防控技术是害螨持续控制的必由之路。In order to reduce the serious losses caused by pest mites in agricultural production, a series of prevention and control methods have emerged. Although biological control based on predatory mites has achieved remarkable results in recent years, chemical control still occupies a major position. Due to the long-term, large-scale, and single unscientific use of chemical pesticides, coupled with the typical r-strategy life history of pest mites, the "3R" problem of pesticides is very serious. At the same time, due to the strong reproductive capacity and short generation period of spider mites, the accumulation of pest mites' resistance has been aggravated, and the cycle has led to an extremely high level of resistance. At present, 417 cases of resistance have been found in two-spotted spider mites, ranking first in the record of arthropod resistance, and spider mites have developed cross-resistance to a variety of acaricides. Therefore, finding new control targets and developing new environmentally friendly and effective spider mite control technologies are the only way to continuously control pest mites.
RNA干扰(RNAinterference)是一种保守的转录后序列特异性基因沉默机制。RNA干扰主要是通过短发夹RNA或双链小RNA对靶标mRNA的特异性降解或调控其表达，使得相应靶基因沉默、生物体正常功能被扰乱。由于其极具高效性、特异性和便捷性，RNAi技术已广泛应用于基因功能研究、高通量目的基因筛选、基因治疗、药物靶标预测、新药开发和农业病虫害防治等领域。RNA interference is a conservative post-transcriptional sequence-specific gene silencing mechanism. RNA interference mainly uses short hairpin RNA or double-stranded small RNA to specifically degrade or regulate the expression of target mRNA, resulting in the silencing of the corresponding target gene and the disruption of the normal function of the organism. Due to its high efficiency, specificity and convenience, RNAi technology has been widely used in gene function research, high-throughput target gene screening, gene therapy, drug target prediction, new drug development and agricultural pest control.
RNAi作为绿色环保的控虫技术已被广泛认可，且未来在叶螨的控制方面具有重要的开发应用潜力。但高效的RNAi靶标的缺少是限制其应用的关键因素之一。近年来的入侵叶螨—番茄红螨在我国呈扩散趋势，但关于该螨的RNAi研究十分匮乏。因此，筛选高效的番茄红螨RNAi防控靶标，对番茄红螨绿色防控技术的开发具有重要意义。RNAi has been widely recognized as a green and environmentally friendly insect control technology, and has important development and application potential in the control of spider mites in the future. However, the lack of efficient RNAi targets is one of the key factors limiting its application. In recent years, the invasive spider mite, the tomato red mite, has been spreading in my country, but RNAi research on this mite is very scarce. Therefore, screening efficient RNAi control targets for tomato red mites is of great significance for the development of green control technology for tomato red mites.
发明内容Summary of the invention
本发明首先通过鉴定番茄红螨9个基因，作为RNAi靶基因，发现番茄红螨RNAi响应显著，基于人工饲料法介导的dsRNA递送可在基因表达水平有效沉默番茄红螨TeCOPB2、TeVATPase、TeSlp三个基因。同时，干扰9个基因后番茄红螨的存活率均出现不同程度的显著下降，其中TeRpt3致死效果最好，10d的总死亡率达到了66.10％，具有很好的作为RNAi靶标田间防控的潜力。基于此，本发明提供了如下技术方案：The present invention first identified 9 genes of tomato red mite as RNAi target genes, and found that the RNAi response of tomato red mite was significant. The dsRNA delivery mediated by artificial diet method can effectively silence the three genes of tomato red mite TeCOPB2, TeVATPase, and TeSlp at the gene expression level. At the same time, after interfering with the 9 genes, the survival rate of tomato red mite showed a significant decrease to varying degrees, among which TeRpt3 had the best lethal effect, and the total mortality rate of 10 days reached 66.10%, which has a good potential as an RNAi target for field control. Based on this, the present invention provides the following technical solutions:
番茄红螨TeRpt3基因，其核苷酸序列如SEQ ID NO.27所示，以该基因为RNAi靶基因，能对番茄红螨高效致死。The nucleotide sequence of the tomato red mite TeRpt3 gene is shown in SEQ ID NO.27. Using this gene as an RNAi target gene can effectively kill the tomato red mite.
上述的TeRpt3基因或TeRpt3基因编码的蛋白质作为RNAi靶标在防控番茄红螨中的应用或者在制备防控番茄红螨的产品中的应用。The above-mentioned TeRpt3 gene or the protein encoded by the TeRpt3 gene is used as an RNAi target in the prevention and control of tomato red mites or in the preparation of a product for the prevention and control of tomato red mites.
一种用于扩增上述的番茄红螨TeRpt3基因的引物组，所述引物组的上、下游引物的核苷酸序列如下：A primer set for amplifying the above-mentioned tomato red mite TeRpt3 gene, the nucleotide sequences of the upstream and downstream primers of the primer set are as follows:
上游引物(SEQ ID NO.36)：5’-CAGCTAGTGTTGCCTTGCAT-3’；Upstream primer (SEQ ID NO. 36): 5′-CAGCTAGTGTTGCCTTGCAT-3′;
下游引物(SEQ ID NO.37)：5’-CGACGATCTGGTAACGGGAAT-3’。Downstream primer (SEQ ID NO.37): 5’-CGACGATCTGGTAACGGGAAT-3’.
一种上述的番茄红螨TeRpt3基因的扩增方法，包括如下步骤：以番茄红螨总RNA反转录得到的cDNA为模板，用上述的上、下游引物进行PCR扩增。A method for amplifying the TeRpt3 gene of tomato red mite mentioned above comprises the following steps: using the cDNA obtained by reverse transcription of the total RNA of tomato red mite as a template, and performing PCR amplification using the above-mentioned upstream and downstream primers.
上述的番茄红螨TeRpt3基因的扩增方法中，PCR反应体系为：浓度为10μM的上、下游引物各1.0μL、cDNA模板2.0μL、ddH2O 8.5μL、2×taq Mixture 12.5μL；In the above-mentioned method for amplifying the TeRpt3 gene of tomato red mite, the PCR reaction system is: 1.0 μL of each of the upstream and downstream primers with a concentration of 10 μM, 2.0 μL of cDNA template, 8.5 μL of ddH2 O, and 12.5 μL of 2×taq Mixture;
PCR反应程序为：95℃预变性3min；95℃变性30s，引物Tm值退火30s，72℃延伸1min/1000bp，35个循环；72℃再延伸10min。The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, primer Tm value annealing for 30 s, extension at 72°C for 1 min/1000 bp, 35 cycles; and extension at 72°C for another 10 min.
一种番茄红螨TeRpt3基因的dsRNA，以如SEQ ID NO.27所示的番茄红螨TeRpt3基因片段为模板转录获得。A dsRNA of the tomato red mite TeRpt3 gene is obtained by transcription using the tomato red mite TeRpt3 gene fragment shown in SEQ ID NO.27 as a template.
优选地，所述dsRNA的核苷酸序列如SEQ ID NO.26所示。Preferably, the nucleotide sequence of the dsRNA is as shown in SEQ ID NO.26.
本发明还提供了上述的番茄红螨TeRpt3基因的dsRNA在防控番茄红螨中的应用或者在制备防控番茄红螨的产品中的应用。The present invention also provides the use of the dsRNA of the tomato red mite TeRpt3 gene in preventing and controlling tomato red mites or in preparing products for preventing and controlling tomato red mites.
本发明还提供了一种番茄红螨TeRpt3基因的dsRNA的合成方法，包括如下步骤：以番茄红螨总RNA反转录得到的cDNA为模板，以序列为SEQ ID NO.9的上游引物和以序列为SEQ ID NO.10的下游引物进行PCR扩增，PCR扩增产物进行电泳后回收产物，以胶回收得到的DNA产物为模板合成得到番茄红螨TeRpt3基因的dsRNA。The present invention also provides a method for synthesizing dsRNA of the tomato red mite TeRpt3 gene, comprising the following steps: using cDNA obtained by reverse transcription of tomato red mite total RNA as a template, performing PCR amplification with an upstream primer with a sequence of SEQ ID NO.9 and a downstream primer with a sequence of SEQ ID NO.10, recovering the PCR amplification product after electrophoresis, and using the DNA product recovered by gel as a template to synthesize the dsRNA of the tomato red mite TeRpt3 gene.
优选地，dsRNA合成反应体系为：5×Transcript Aid Reaction Buffer 4.0μL、ATP/CTP/GTP/UTP分别加入2.0μL、TranscriptAid Enzyme Mix 2.0μL、DNA模板1.0μg、ddH2O补充至20.0μL；将混合液置于37℃下反应6h或者过夜。Preferably, the dsRNA synthesis reaction system is: 5×Transcript Aid Reaction Buffer 4.0 μL, ATP/CTP/GTP/UTP 2.0 μL, TranscriptAid Enzyme Mix 2.0 μL, DNA template 1.0 μg, ddH2 O to 20.0 μL; the mixture is reacted at 37° C. for 6 hours or overnight.
本发明的有益效果是：首次鉴定到番茄红螨TeRpt3基因，根据番茄红螨TeRpt3基因序列设计得到其dsRNA，经实验证实，该dsRNA对番茄红螨的致死效果优异，番茄红螨TeRpt3基因具有很好的作为RNAi靶标田间防控的潜力，应用前景好，而本发明设计筛选到的TeRpt3基因的dsRNA则具有作为新型杀虫剂的良好应用前景，有望应用于番茄红螨防控，而不对环境和人产生毒害作用。The present invention has the following beneficial effects: the TeRpt3 gene of tomato red mite is identified for the first time, and its dsRNA is designed according to the sequence of the TeRpt3 gene of tomato red mite. Experiments have confirmed that the dsRNA has an excellent lethal effect on tomato red mite. The TeRpt3 gene of tomato red mite has a good potential as an RNAi target for field control and has a good application prospect. The dsRNA of the TeRpt3 gene designed and screened by the present invention has a good application prospect as a new type of insecticide and is expected to be used in the control of tomato red mite without causing toxic effects on the environment and humans.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是9个番茄红螨RNAi致死靶标基因的系统发育树分析。FIG1 is a phylogenetic tree analysis of nine tomato red mite RNAi lethal target genes.
图2是dsTeCOPB2、dsTeVATPase、dsTeSlp处理番茄红螨后相应基因的沉默效率结果。Figure 2 shows the silencing efficiency results of the corresponding genes after tomato red mites were treated with dsTeCOPB2, dsTeVATPase, and dsTeSlp.
图3是分别沉默9个番茄红螨RNAi候选靶标基因后试螨的生存曲线。FIG. 3 is a survival curve of the test mites after silencing 9 candidate target genes of tomato red mite RNAi respectively.
具体实施方式Detailed ways
下面结合实施例对本发明作进一步说明，但并不因此而限制本发明。The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.
下述实施例中的实验方法，如无特别说明，均为常规方法；所用化学、生物试剂，如无特殊说明，均为本领域常规试剂。The experimental methods in the following examples are all conventional methods unless otherwise specified; the chemical and biological reagents used are all conventional reagents in the art unless otherwise specified.
实施例1Example 1
1实验材料1 Experimental Materials
1.1供试螨源1.1 Test mite source
实验所使用的番茄红螨种群由西南大学昆虫学及害虫控制工程重庆市重点实验室提供，其饲养条件如下：温度为27±1℃、光周期为14L：10D、相对湿度为60±10％，主要利用千禧番茄进行饲养。定期给番茄植株浇水以防止植物脱水死亡。The tomato red mite population used in the experiment was provided by the Chongqing Key Laboratory of Entomology and Pest Control Engineering of Southwest University. The breeding conditions were as follows: temperature 27±1℃, photoperiod 14L:10D, relative humidity 60±10%, mainly using Millennium tomatoes for breeding. Tomato plants were watered regularly to prevent dehydration and death of the plants.
统一番茄红螨的龄期：挑取雌成螨至新的番茄植株上产卵，产卵1日后将雌成螨挑走，待卵发育到成螨后即可用于后续试验。Standardize the age of tomato red mites: pick female adult mites to lay eggs on new tomato plants, pick them away one day after laying eggs, and wait for the eggs to develop into adult mites before using them for subsequent tests.
1.2实验试剂1.2 Experimental Reagents
2实验方法2 Experimental methods
2.1基因鉴定2.1 Gene identification
基于课题组测序获得的番茄红螨转录组数据，通过软件tbtools鉴定靶标番茄红螨基因，分别对番茄红螨和二斑叶螨中的靶标基因进行保守结构域鉴定，最后通过NCBI查找相应靶标基因的同源基因序列，得到多个物种的同源基因序列。利用Mega 7.0软件构建系统发育树，所用建树方法为最大似然法，最终通过二斑叶螨中靶标基因鉴定对应的番茄红螨中的基因。Based on the transcriptome data of tomato red mite obtained by sequencing, the target tomato red mite gene was identified by the software tbtools, and the conserved domains of the target genes in tomato red mite and two-spotted spider mite were identified respectively. Finally, the homologous gene sequences of the corresponding target genes were searched through NCBI, and the homologous gene sequences of multiple species were obtained. The phylogenetic tree was constructed using Mega 7.0 software, and the tree construction method used was the maximum likelihood method. Finally, the corresponding genes in tomato red mite were identified through the target genes in two-spotted spider mite.
2.2靶标序列克隆2.2 Target sequence cloning
通过生物信息学分析鉴定番茄红螨潜力靶标基因，利用Primer-Blast在线设计引物，包括9条扩增片段为600bp的dsRNA引物以及3条基因扩增长度为200bp左右的qPC R引物，在dsRNA正反引物前加上T7启动子序列(SEQ ID NO.25:TAATACGACTCA CTATAGGG)，用于番茄红螨中的9条潜力靶基因序列扩增，引物序列如表1所示，其中qTeSlp、qTeCOPB2、qTeVATPase为荧光定量引物序列，其余为dsRNA引物序列。The potential target genes of tomato red mite were identified by bioinformatics analysis, and primers were designed online using Primer-Blast, including 9 dsRNA primers with an amplification fragment of 600 bp and 3 qPCR primers with a gene amplification length of about 200 bp. The T7 promoter sequence (SEQ ID NO.25:TAATACGACTCA CTATAGGG) was added before the dsRNA forward and reverse primers to amplify the 9 potential target gene sequences in tomato red mite. The primer sequences are shown in Table 1, among which qTeSlp, qTeCOPB2, and qTeVATPase are fluorescence quantitative primer sequences, and the rest are dsRNA primer sequences.
表1番茄红螨RNAi靶基因引物序列表Table 1 Tomato red mite RNAi target gene primer sequence list
先对所设计的qPCR引物进行评估，利用无核酶水作为阴性对照，将cDNA模板依次稀释五个梯度，各梯度间稀释倍数为5倍，将扩增效率为90％-110％的qPCR引物保存备用，以进行后续定量分析实验。First, the designed qPCR primers were evaluated. Nuclease-free water was used as a negative control. The cDNA template was diluted into five gradients in sequence. The dilution factor between each gradient was 5 times. The qPCR primers with an amplification efficiency of 90%-110% were saved for use in subsequent quantitative analysis experiments.
PCR反应加样体系为：上游引物(10μM/L)1.0μL、下游引物(10μM/L)1.0μL、cDNA模板2.0μL、ddH2O 8.5μL、2×taq Mixture 12.5μL。The sample loading system for PCR reaction was: 1.0 μL of upstream primer (10 μM/L), 1.0 μL of downstream primer (10 μM/L), 2.0 μL of cDNA template, 8.5 μL of ddH2 O, and 12.5 μL of 2×taq Mixture.
PCR反应程序为：95℃预变性3min；95℃变性30s，引物Tm值退火30s，72℃延伸1min/1000bp，35个循环；72℃再延伸10min；12℃保存。The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, primer Tm value annealing for 30 s, extension at 72°C for 1 min/1000 bp, 35 cycles; extension at 72°C for another 10 min; and storage at 12°C.
PCR反应结束后，利用琼脂糖凝胶电泳对合成的DNA进行检测，利用2000bp DNAMarker指示所合成的DNA分子量大小。After the PCR reaction was completed, the synthesized DNA was detected by agarose gel electrophoresis, and the molecular weight of the synthesized DNA was indicated by a 2000 bp DNA Marker.
2.3目的片段的回收2.3 Recovery of target fragments
按照Agarose Gel DNAExtraction Mini Kit说明书(Takara)操作，切下胶块上的目的条带，加入溶解液溶解胶块，吸取反应液(至多600μL)至吸附柱中，待其充分吸附DNA产物后，在室温下12,000g离心1min，用洗涤剂洗涤两次，空离一次，待洗涤液风干后，用洗脱液洗脱纯化好的DNA产物，利用浓度仪测定目的片段浓度。According to the instructions of Agarose Gel DNA Extraction Mini Kit (Takara), the target band on the gel block was cut off, and the dissolving solution was added to dissolve the gel block. The reaction solution (up to 600 μL) was aspirated into the adsorption column. After the DNA product was fully adsorbed, it was centrifuged at 12,000 g for 1 min at room temperature. The column was washed twice with detergent and evacuated once. After the washing solution was air-dried, the purified DNA product was eluted with elution solution, and the concentration of the target fragment was determined using a concentration meter.
2.4靶DNA片段与载体的体外链接2.4 In vitro ligation of target DNA fragments and vectors
按照pGEM-T Easy vector克隆试剂盒说明书(Promega)操作，反应体系为：2×ligBuffer5μL、胶回收产物3μL、pGEM-T vector 1μL、T4 DNAligase 1μL。将混合液置于4℃或者16℃过夜。According to the instructions of the pGEM-T Easy vector cloning kit (Promega), the reaction system is: 2×ligBuffer 5μL, gel recovery product 3μL, pGEM-T vector 1μL, T4 DNA ligase 1μL. The mixture was placed at 4℃ or 16℃ overnight.
2.5重组质粒转入大肠杆菌DH5α细胞2.5 Transformation of recombinant plasmid into E. coli DH5α cells
(1)DH5α超级感受态细胞置于-80℃冰箱内，使用时分装为3管。待感受态细胞融化后，吸取链接反应液旋转式缓慢加入感受态细胞中，4℃孵育40min，后将样品置于42℃水浴锅中热激1min，再置于冰上冷激2min。(1) DH5α super competent cells were placed in a -80℃ refrigerator and divided into 3 tubes for use. After the competent cells were thawed, the linkage reaction solution was slowly added to the competent cells by rotation and incubated at 4℃ for 40min. The sample was then placed in a 42℃ water bath for 1min for heat shock and then placed on ice for 2min for cold shock.
(2)在(1)中的样品里加入LB液体培养基(无抗生素)200μL，于37℃摇床中摇晃3-4h。(2) Add 200 μL of LB liquid culture medium (without antibiotics) to the sample in (1) and shake in a shaker at 37°C for 3-4 h.
(3)将涂布器置于酒精灯火焰下消毒备用，将70μL菌液、7μLX-gal和7μLIPTG加入含氨苄抗生素的固体培养基上，待涂布器冷却后将混合液均匀涂布于固体培养基表面，倒置于37℃下培养12h。(3) Place the applicator under the flame of an alcohol lamp for disinfection and set aside. Add 70 μL of bacterial solution, 7 μL of X-gal and 7 μL of IPTG to the solid culture medium containing ampicillin antibiotics. After the applicator cools down, evenly spread the mixture on the surface of the solid culture medium and invert it at 37°C for incubation for 12 h.
2.6蓝白斑筛选及重组质粒检测2.6 Blue-white screening and recombinant plasmid detection
使用移液枪枪头一次性挑取培养基上的白斑菌落至1.5mL离心管，在其中加入500μL含有氨苄抗生素的液体培养基，置于37℃下培养3-5h，利用PCR对培养后的菌液进行检测。Use a pipette tip to pick up the white spot colonies on the culture medium into a 1.5 mL centrifuge tube, add 500 μL of liquid culture medium containing ampicillin antibiotics, and culture at 37°C for 3-5 hours. Use PCR to detect the cultured bacterial liquid.
2.7克隆产物的测序和序列分析2.7 Sequencing and sequence analysis of cloned products
样品经PCR检测成功后，吸取200μL送至擎科生物公司进行序列测定，测序结果与目的序列一致即为克隆成功，将克隆成功的菌液置于4℃下长期保存。After the sample is successfully tested by PCR, 200 μL is taken and sent to Qingke Biotechnology Co., Ltd. for sequence determination. If the sequencing result is consistent with the target sequence, the cloning is successful. The successfully cloned bacterial solution is stored at 4°C for a long time.
2.8 dsRNA的合成与纯化2.8 Synthesis and purification of dsRNA
2.8.1 dsRNA合成2.8.1 dsRNA synthesis
按照TranscriptAidT7 HighYield Transcription Kit(Thermo Scientific)试剂盒的说明书进行，反应体系如下：5×TranscriptAid Reaction Buffer 4.0μL、ATP/CTP/GTP/UTP分别加入2.0μL、TranscriptAid Enzyme Mix 2.0μL、DNA模板1.0μg、RNase-Free水补充至20.0μL。将混合液置于37℃下反应6h或者过夜。According to the instructions of the TranscriptAidT7 HighYield Transcription Kit (Thermo Scientific), the reaction system is as follows: 4.0 μL of 5×TranscriptAid Reaction Buffer, 2.0 μL of ATP/CTP/GTP/UTP, 2.0 μL of TranscriptAid Enzyme Mix, 1.0 μg of DNA template, and RNase-Free water to 20.0 μL. The mixture is placed at 37°C for 6 hours or overnight.
2.8.2 dsRNA纯化2.8.2 dsRNA purification
dsRNA纯化操作均于通风橱中进行，步骤如下：The dsRNA purification operation was performed in a fume hood, and the steps were as follows:
(1)将合成的dsRNA产物取出，加入2μLDNase e I，将样品置于37℃中孵育15min，再加入2μLEDTA，将样品置于65℃中反应10min，将上一步反应液移取至1.5mL离心管中，加入115μL RNAse-Free Water和15μL 3M SodiumAcetate的混合液，需提前混配好相应溶液再加入到反应液中。(1) Take out the synthesized dsRNA product, add 2μL DNase e I, incubate the sample at 37°C for 15 min, then add 2μL ETA, and react the sample at 65°C for 10 min. Transfer the reaction solution from the previous step to a 1.5mL centrifuge tube, and add a mixture of 115μL RNAse-Free Water and 15μL 3M Sodium Acetate. The corresponding solutions must be mixed in advance and then added to the reaction solution.
(2)将75mL苯酚和75mL三氯甲烷混配均匀后加入到(1)中的溶液，再加入300μL氯仿，震荡，将样品置于离心机中离心10min(12,000g，4℃)。(2) Mix 75 mL of phenol and 75 mL of chloroform and add them to the solution in (1). Then add 300 μL of chloroform and shake. Centrifuge the sample for 10 min (12,000 g, 4° C.).
(3)取出离心后的样品，dsRNA处于上清液中，将上清液移入新的1.5ml离心管，再加入350μL无水乙醇，放入-20℃备用。(3) Remove the sample after centrifugation. The dsRNA is in the supernatant. Transfer the supernatant into a new 1.5 ml centrifuge tube, add 350 μL of anhydrous ethanol, and store at -20°C for later use.
(4)-20℃放置2h后，将样品置于离心机中离心10min(12,000g，4℃)，弃去上清液，吸出残余液体。(4) After standing at -20°C for 2 h, the sample was centrifuged for 10 min (12,000 g, 4°C), the supernatant was discarded, and the residual liquid was aspirated.
(5)在(4)中制得的dsRNA沉淀中加入500μL冰冻状态的70％乙醇溶液，置于离心机中离心5min(7,500g，4℃)，弃去上清液，吸出残余液体，风干沉淀20min。(5) Add 500 μL of ice-cold 70% ethanol solution to the dsRNA precipitate prepared in (4), centrifuge for 5 min (7,500 g, 4°C), discard the supernatant, aspirate the residual liquid, and air-dry the precipitate for 20 min.
(6)在(5)中的dsRNA沉淀中加入40μL无核酶水，混匀，吸取1μL以测定制得的dsRNA溶液浓度，吸取1μg上样电泳检测其完整性，后于-80℃条件下保存备用。(6) Add 40 μL of nuclease-free water to the dsRNA precipitate in (5), mix well, aspirate 1 μL to determine the concentration of the prepared dsRNA solution, aspirate 1 μg to load on electrophoresis to detect its integrity, and then store at -80°C for future use.
2.9 dsRNA的递送2.9 dsRNA delivery
(1)在培养皿内部围好湿棉花，用毛笔挑取初羽化的番茄红螨雌成螨至培养皿干燥处，利用保鲜膜将培养皿封闭严实，饥饿番茄红螨12h。(1) Place wet cotton wool around the inside of a culture dish, use a brush to pick up newly emerged female adult tomato red mites and place them in a dry place in the culture dish. Seal the dish tightly with plastic wrap and starve the tomato red mites for 12 hours.
(2)将饥饿处理过的番茄螨转至饲喂装置上(装置制作流程：先将封口膜封在小培养皿上，剪1cm×1cm的纱布，加入40μL1000ng/ul的dsRNA溶液，加入2ul植物源蓝色染料以便确认螨取食dsRNA的情况，后附封口膜于其上。(2) The starved tomato mites were transferred to the feeding device (device production process: first seal the parafilm on the small culture dish, cut 1 cm × 1 cm gauze, add 40 μL 1000 ng/ul dsRNA solution, add 2 ul plant-derived blue dye to confirm that the mites feed on the dsRNA, and then attach the parafilm on it.
(3)待螨取食24h后将番茄红螨转至番茄叶片上观察其表型或检测沉默效率，观察表型时需每隔一天更换一次番茄叶片，每天统计一次死亡情况，连续统计10d。(3) After the mites have been feeding for 24 hours, transfer the tomato red mites to tomato leaves to observe their phenotype or detect the silencing efficiency. When observing the phenotype, the tomato leaves need to be replaced every other day, and the mortality rate should be counted once a day for 10 consecutive days.
2.10 RNA提取及cDNA合成2.10 RNA extraction and cDNA synthesis
2.10.1总RNA提取2.10.1 Total RNA extraction
RNA提取全程在通风橱内进行The entire RNA extraction process was performed in a fume hood.
(1)用毛笔挑取适量番茄红螨至1.5ml离心管中，加入液氮以速冻样品，将样品置于冰上，加入200μLTRIzol研磨1min，再加入200μLTRIzol在1.5ml离心管中以洗脱研磨棒上附着的RNA，震荡5min使叶螨样品裂解，充分释放RNA。(1) Use a brush to pick up an appropriate amount of tomato red mites and put them into a 1.5 ml centrifuge tube. Add liquid nitrogen to freeze the sample quickly. Place the sample on ice and add 200 μL TRIzol to grind for 1 min. Then add another 200 μL TRIzol in a 1.5 ml centrifuge tube to elute the RNA attached to the grinding rod. Shake for 5 min to lyse the spider mite sample and fully release the RNA.
(2)在(1)中的反应液中加入200μL氯仿，震荡30s使其充分混匀，将样品置于室温下孵育3min以萃取RNA，后置于离心机中离心15min(12,000g，4℃)。(2) Add 200 μL of chloroform to the reaction solution in (1), shake for 30 seconds to mix thoroughly, incubate the sample at room temperature for 3 minutes to extract RNA, and then centrifuge for 15 minutes (12,000 g, 4°C).
(3)取出离心后的样品，RNA被萃取至上清液中，将上清液移入新的1.5ml离心管，加入400μL异丙醇以沉淀RNA，颠倒混匀后室温孵育10min，后置于离心机中离心10min(12,000g，4℃)，弃去上清液，用移液枪吸出残余液体。(3) Remove the sample after centrifugation. RNA is extracted into the supernatant. Transfer the supernatant to a new 1.5 ml centrifuge tube. Add 400 μL of isopropanol to precipitate the RNA. Invert to mix and incubate at room temperature for 10 min. Centrifuge for 10 min (12,000 g, 4°C). Discard the supernatant and remove the remaining liquid with a pipette.
(4)在(3)得到的RNA沉淀中加入500μL冰冻状态的75％乙醇溶液，轻微震荡，后将样品置于离心机离心5min(7,500g，4℃)，弃去上清液，吸出残余液体。(4) Add 500 μL of ice-cold 75% ethanol solution to the RNA precipitate obtained in (3), shake gently, and then centrifuge the sample for 5 min (7,500 g, 4°C). Discard the supernatant and aspirate the residual liquid.
(5)风干(4)中得到的RNA样品10min，加入15μL无核酶水使RNA沉淀充分溶解。(5) Air-dry the RNA sample obtained in (4) for 10 min, and add 15 μL of nuclease-free water to fully dissolve the RNA precipitate.
(6)将RNA溶液置于冰上，吸取1μL溶液测定其浓度，吸取1μgRNA通过琼脂糖凝胶电泳确认其完整性。对完整性较好、浓度达标的RNA进行去基因组、反转录，将合成的cDNA放入-20℃备用，将剩余的RNA放入-80℃冰箱长期保存。(6) Place the RNA solution on ice, take 1 μL of the solution to determine its concentration, and take 1 μg of RNA to confirm its integrity by agarose gel electrophoresis. For RNA with good integrity and concentration that meets the standard, perform genome removal and reverse transcription, store the synthesized cDNA at -20°C for standby use, and store the remaining RNA in a -80°C refrigerator for long-term storage.
2.10.2总RNA中gDNA去除2.10.2 Removal of gDNA from total RNA
使用RT reagent Kit(TaKaRa)试剂盒(037A)去除样品中gDNA。初步混合液在37℃下孵育30min，然后加1μL Stop Solution终止反应，再放入PCR仪器中65℃孵育10min使DNase失活，得到的cDNA模板在-20℃保存备用。具体加样体系如下：use RT reagent Kit (TaKaRa) kit (037A) removes gDNA from the sample. The initial mixture was incubated at 37°C for 30 minutes, and then 1 μL Stop Solution was added to terminate the reaction. The mixture was then placed in a PCR instrument and incubated at 65°C for 10 minutes to inactivate DNase. The obtained cDNA template was stored at -20°C for later use. The specific sample addition system is as follows:
2.10.3cDNA合成2.10.3 cDNA synthesis
反转录过程中RNA一直保持在冰盒中，使用方法详见RT reagent Kit(TaKaRa)试剂盒(037A)说明书，反应加样体系如下：During the reverse transcription process, the RNA was kept in an ice box. For details, see RT reagent Kit (TaKaRa) kit (037A) instructions, the reaction sample system is as follows:
2.11实时荧光定量PCR反应2.11 Real-time fluorescence quantitative PCR reaction
使用NovoStartTM试剂盒进行荧光定量PCR反应，操作步骤详见试剂盒说明书，加样体系如下：Use NovoStartTM kit for fluorescence quantitative PCR reaction. The operation steps are detailed in the kit manual. The sample loading system is as follows:
2.12统计分析2.12 Statistical analysis
利用Graphpad软件进行生存曲线绘制，并对各处理下的生存情况进行显著性分析。Graphpad software was used to draw the survival curve and perform significance analysis on the survival status under each treatment.
利用SPSS软件对基因表达量差异情况通过独立t检验分析其显著性。SPSS software was used to analyze the significance of the differences in gene expression levels through independent t-test.
3结果与分析3 Results and analysis
3.1番茄红螨靶标鉴定3.1 Tomato red mite target identification
3.1.1RNAi靶标库的候选基因初筛3.1.1 Preliminary screening of candidate genes in the RNAi target library
选取了9个靶标基因作为番茄红螨RNAi预选靶基因，如表2所示。Nine target genes were selected as tomato red mite RNAi pre-selected target genes, as shown in Table 2.
表2番茄红螨九条候选RNAi靶基因鉴定情况Table 2 Identification of nine candidate RNAi target genes for tomato red mite
3.1.2利用生物信息学鉴定番茄红螨候选RNAi靶基因3.1.2 Identification of candidate RNAi target genes for tomato red mite using bioinformatics
(1)番茄红螨RNAi靶基因的鉴定：将9条二斑叶螨的基因序列与番茄红螨转录组进行序列比对，最终比对到了相应的9条番茄红螨基因序列。如表2所示，二斑叶螨与番茄红螨中的9条基因序列相似度均达到了85％以上，除TuCOPB2外。其余基因序列相似度均在90％以上，其中TuSrp54与Unigene12349_TE最为相似，相似度达到了96.72％。(1) Identification of RNAi target genes of tomato red mite: The gene sequences of 9 two-spotted spider mites were aligned with the transcriptome of tomato red mite, and finally the corresponding 9 tomato red mite gene sequences were aligned. As shown in Table 2, the similarity of the 9 gene sequences of two-spotted spider mites and tomato red mite reached more than 85%, except TuCOPB2. The similarity of the remaining gene sequences was more than 90%, among which TuSrp54 was the most similar to Unigene12349_TE, with a similarity of 96.72%.
(2)保守结构域鉴定：分别鉴定了9个二斑叶螨基因与9个番茄红螨基因的保守结构域，筛选保守结构域相同的基因序列。鉴定结果如表2所示，9个番茄红螨RNAi靶基因与相对应的二斑叶螨基因均有相同的保守结构域，这预示着它们有着相似的基因功能。(2) Identification of conserved domains: The conserved domains of 9 two-spotted spider mite genes and 9 tomato red mite genes were identified, and gene sequences with the same conserved domains were screened. The identification results are shown in Table 2. The 9 tomato red mite RNAi target genes and the corresponding two-spotted spider mite genes all have the same conserved domains, which indicates that they have similar gene functions.
(3)建立系统发育树：基于番茄红螨的9条RNAi靶标基因序列，利用NCBI网站将番茄红螨靶标序列与其他物种基因序列进行比对，筛选出与番茄红螨基因同源性较高的其他物种基因序列，利用这些序列构建系统发育树。如图1所示，系统发育分析显示，二斑叶螨、番茄红螨、柑橘全爪螨和苹果全爪螨聚为一个分支，而其他与番茄红螨基因同源性较高的物种聚类于另一支上，表明四种叶螨相应基因之间的亲缘关系较近。其中，来自两种叶螨相对应的两条基因亲缘关系最近，推测在两种叶螨中发挥着相似的作用。(3) Establishment of phylogenetic tree: Based on the 9 RNAi target gene sequences of tomato red mite, the target sequences of tomato red mite were compared with the gene sequences of other species using the NCBI website, and the gene sequences of other species with high homology to tomato red mite genes were screened out, and the phylogenetic tree was constructed using these sequences. As shown in Figure 1, the phylogenetic analysis showed that two-spotted spider mite, tomato red mite, citrus mites and apple mites were clustered into one branch, while other species with high homology to tomato red mite genes were clustered on another branch, indicating that the corresponding genes of the four spider mites were closely related. Among them, the two genes corresponding to the two spider mites were the closest in relationship, and it was speculated that they played similar roles in the two spider mites.
将鉴定成功的番茄红螨基因参照二斑叶螨中的同源基因名称进行命名，如在二斑叶螨中的基因TuVATPase在番茄红螨中同源基因Unigene714_TE，命名为TeVATPase，类似的，其余基因的名称分别为TeSlp(Unigene8572_TE)、TeSnapα(Unigene5520_TE)、TeCOPB2(Unigene10721_TE)、TeCOPE(CL2276.Contig2_TE)、TeRpt3(Unigene14978_TE)、TeSrp54(Unigene12349_TE)、TeRPS4(CL2444.Contig2_TE)、TeAQ9(CL1002.Contig1_TE)。3.2番茄红螨RNAi靶基因潜力评估The successfully identified tomato red mite genes were named with reference to the names of homologous genes in two-spotted spider mites. For example, the gene TuVATPase in two-spotted spider mites is named TeVATPase, which is the homologous gene Unigene714_TE in tomato red mite. Similarly, the names of the remaining genes are TeSlp (Unigene8572_TE), TeSnapα (Unigene5520_TE), TeCOPB2 (Unigene10721_TE), TeCOPE (CL2276.Contig2_TE), TeRpt3 (Unigene14978_TE), TeSrp54 (Unigene12349_TE), TeRPS4 (CL2444.Contig2_TE), and TeAQ9 (CL1002.Contig1_TE). 3.2 Evaluation of the potential of RNAi target genes for tomato red mite
3.2.1dsRNA处理后靶基因表达量检测3.2.1 Detection of target gene expression after dsRNA treatment
通过人工装置的饲喂法分别饲喂番茄红螨3个靶标基因的dsRNA24 h后，检测基因表达量。结果如图2所示，与饲喂dsGFP对照组相比，番茄红螨取食dsTeCOPB2、dsTeVATPase和dsTeSlp 24h后，3个基因的沉默效率分别为35.6％(P＝0.004)、47.61％(P＝0.0123)、48.07％(P＝0.0026)，表明饲喂法RNAi可有效沉默番茄红螨的靶标基因。After feeding dsRNA of three target genes of tomato red mite by artificial device for 24 hours, the gene expression was detected. The results are shown in Figure 2. Compared with the control group fed with dsGFP, the silencing efficiency of dsTeCOPB2, dsTeVATPase and dsTeSlp for 24 hours after tomato red mite fed with dsTeCOPB2, dsTeVATPase and dsTeSlp was 35.6% (P=0.004), 47.61% (P=0.0123) and 48.07% (P=0.0026), respectively, indicating that RNAi by feeding method can effectively silence the target genes of tomato red mite.
3.2.2 dsRNA处理后番茄红螨死亡率3.2.2 Tomato red mite mortality after dsRNA treatment
番茄红螨的RNAi靶标潜力评估结果如图3与表3所示，相较对照组，不同dsRNA处理组后番茄红螨的死亡率出现不同程度的上升，其中dsTeSlp、dsTeCOPB2、dsTeVATPase、dsTeSnapα、dsTeRPS4、dsTeSrp54、dsTeCOPE以及dsTeAQ9处理番茄红螨后10d的死亡率分别为46.43％(P＝0.0004)、50.85％(P<0.001)、54.29％(P<0.0001)、41.86％(P＝0.0105)、48.94％(P＝0.0008)、40.82％(P＝0.0032)、45.10％(P＝0.0002)、37.78％(P＝0.0093)。而dsTeRpt3对番茄红螨致死效果最为显著，在dsRNA处理第5d时死亡率达到了44.29％，10d的总死亡率达到了66.10％(P<0.001)，说明TeRpt3对番茄红螨的生存具有重要作用，极具RNAi高效防控靶标的开发潜力。The results of RNAi target potential evaluation of tomato red mite are shown in Figure 3 and Table 3. Compared with the control group, the mortality of tomato red mite increased to varying degrees after different dsRNA treatment groups. The mortality rates of tomato red mite 10 days after treatment with dsTeSlp, dsTeCOPB2, dsTeVATPase, dsTeSnapα, dsTeRPS4, dsTeSrp54, dsTeCOPE and dsTeAQ9 were 46.43% (P=0.0004), 50.85% (P<0.001), 54.29% (P<0.0001), 41.86% (P=0.0105), 48.94% (P=0.0008), 40.82% (P=0.0032), 45.10% (P=0.0002) and 37.78% (P=0.0093), respectively. The lethal effect of dsTeRpt3 on tomato red mites was the most significant, with a mortality rate of 44.29% on the 5th day after dsRNA treatment and a total mortality rate of 66.10% on the 10th day (P<0.001), indicating that TeRpt3 plays an important role in the survival of tomato red mites and has great potential for the development of RNAi efficient control targets.
dsTeRpt3的核苷酸序列(SEQ ID NO.26)如下：The nucleotide sequence of dsTeRpt3 (SEQ ID NO.26) is as follows:
CAGCTAGTGTTGCCTTGCATAAGCATAGTAATGCTTTGGTTGATGTTTTACCACCTGAAGCTGACTCTTCTATTGCTATGTTGAGAGCGGATGAAAAGCCTGATGTTAGTTATGCAGATATTGGTGGTCTCGACATTCAAAAGCAAGAAATTCGAGAAGCAGTAGAGCTTCCATTAACTCATTTTGAATTATATAAACAAATTGGTATTGATCCTCCTCGTGGTGTTCTTATGTATGGTCCTCCAGGCTGTGGTAAAACTATGTTAGCCAAAGCAGTTGCTCATCATACCACTGCATCTTTCATTCGCGTTGTTGGATCAGAATTCGTCCAAAAGTATCTTGGTGAAGGTCCTAGGATGGTTCGAGATGTGTTCAGGCTAGCGCGAGAAAATGCCCCAGCTATTATATTTATTGATGAAATCGATGCTATAGCAACCAAGAGATTTGATGCACAAACTGGAGCTGACAGGGAGGTTCAAAGAATCTTGTTAGAATTATTGAATCAAATGGATGGGTTTGATCAAAGTACTAATGTTAAAGTAGTTATGGCCACTAATCGGGCAGATACTTTGGATCCTGCTTTACTCCGTCCAGGACGTTTAGATAGGAAAATTGAATTCCCGTTACCAGATCGTCG。CAGCTAGTGTTGCCTTGCATAAGCATAGTAATGCTTTGGTTGATGTTTTACCACCTGAAGCTGACTCTTCTATTGCTATGTTGAGCGGATGAAAAGCCTGATGTTAGTTATGCAGATATTGGTGGTCTCGACATTCAAAAGCAAGAAATTCGAGAAGCAGTAGAGCTTCCATTAACTCATTTTGAATTATATAAACAAATTGGTATTGATCCTCCTCGTGGTGTTCTTATGTATGGTCCTCCAGGCTGTGGTAAAACTATGTTAGCCAAAGCAGTTGCTCATCATACCACTGCATCTTTCATTCGCGTTGTTGGATC AGAATTCGTCCAAAAGTATCTTGGTGAAGGTCCTAGGATGGTTCGAGATGTGTTCAGGCTAGCGCGAGAAAATGCCCCAGCTATTATATTTATTGATGAAATCGATGCTATAGCAACCAAGAGATTTGATGCACAAACTGGAGCTGACAGGGAGGTTCAAAGAATCTTGTTAGAATTATTGAATCAAATGGATGGGTTTGATCAAAGTACTAATGTTAAAGTAGTTATGGCCACTAATCGGGCAGATACTTTGGATCCTGCTTTACTCCGTCCAGGACGTTTAGATAGGAAAATTGAATTCCCGTTACCAGATCGTCG.
番茄红螨的TeRpt3基因的核苷酸序列如SEQ ID NO.27所示。The nucleotide sequence of the TeRpt3 gene of tomato red mite is shown in SEQ ID NO.27.
dsTeSlp、dsTeCOPB2、dsTeVATPase、dsTeSnapα、dsTeRPS4、dsTeSrp54、dsTeCOPE以及dsTeAQ9的核苷酸序列依次如SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ IDNO.31、SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35所示。The nucleotide sequences of dsTeSlp, dsTeCOPB2, dsTeVATPase, dsTeSnapα, dsTeRPS4, dsTeSrp54, dsTeCOPE and dsTeAQ9 are shown as SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34 and SEQ ID NO.35 respectively.
表3分别沉默9个番茄红螨RNAi候选靶标基因后试螨十天内的死亡率Table 3 Mortality of test mites within ten days after silencing nine candidate target genes of tomato red mite RNAi
3.3结论3.3 Conclusion
通过评价9个RNAi靶基因，发现番茄红螨RNAi响应显著，基于人工饲料法介导的dsRNA递送可在基因表达水平有效沉默番茄红螨TeCOPB2、TeVATPase、TeSlp三个基因。同时，干扰9个基因后番茄红螨的存活率均出现不同程度的显著下降，其中TeRpt3致死效果最好，10d的总死亡率达到了66.10％，具有很好的作为RNAi靶标田间防控的潜力。By evaluating 9 RNAi target genes, it was found that the RNAi response of tomato red mite was significant. The dsRNA delivery mediated by artificial diet method can effectively silence the three genes of tomato red mite TeCOPB2, TeVATPase, and TeSlp at the gene expression level. At the same time, the survival rate of tomato red mite decreased significantly to varying degrees after interfering with 9 genes, among which TeRpt3 had the best lethal effect, with a total mortality rate of 66.10% in 10 days, which has great potential as an RNAi target for field control.
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