WO2021232536A1 - 一种体外大量获得白蜡虫dsRNA的体系构建方法 - Google Patents

一种体外大量获得白蜡虫dsRNA的体系构建方法 Download PDF

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WO2021232536A1
WO2021232536A1 PCT/CN2020/098184 CN2020098184W WO2021232536A1 WO 2021232536 A1 WO2021232536 A1 WO 2021232536A1 CN 2020098184 W CN2020098184 W CN 2020098184W WO 2021232536 A1 WO2021232536 A1 WO 2021232536A1
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dsrna
white wax
constructing
gene
white
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陈航
凌晓霏
陆沁
张金稳
王伟伟
柳鹏飞
汪伟
陈晓鸣
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中国林业科学研究院资源昆虫研究所
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Definitions

  • the invention belongs to the technical field of genetic engineering, and specifically relates to a system construction method for obtaining a large amount of white wax insect dsRNA in vitro.
  • White wax worm is a scale insect with great economic value.
  • the second-instar male larva of white wax worm lives on the branches of host plants and secretes white waxy covering.
  • the wax secreted by this insect becomes white wax (Cerachinensis, also known as white wax). ), that is, the secretion of white wax worm, which is a Chinese specialty.
  • White wax is a natural polymer compound with high melting point and chemical stability. It has the characteristics of moisture-proof, lubrication, and light. It is an important chemical raw material and is widely used in the chemical, machinery, food, pharmaceutical, cosmetic, and agricultural industries.
  • RNA interference refers to the phenomenon that double-stranded RNA (dsRNA) induces homologous mRNA degradation and silences the expression of target genes in eukaryotes.
  • dsRNA transfection mainly includes injection, feeding, soaking, virus infection and gene gun. However, if the research object is too small to move, the infection method of feeding, soaking, and body surface absorption may cause insufficient dsRNA prepared in the kit.
  • the insects in this study are white wax worms.
  • the insects are small in size and have piercing and sucking mouthparts. They grow and reproduce by absorbing the juice of the host S. sylvestris. They cannot move most of the time, and they infect them by absorption by the skin. Due to body surface absorption and large dosage, dsRNA is unstable and easily degraded in the outside world. It is necessary to spray and absorb the surface skin of white wax insects many times.
  • the synthetic dsRNA yield of the kit is low (ul meter), which is not enough for spraying.
  • the method of transfection and the synthesis of a large number of kits make the experiment cost expensive. Therefore, in some RNA interference experiments, how to prepare high-yield dsRNA is a problem that needs to be solved by genetic engineering.
  • the present invention overcomes the shortcomings in the prior art. , To provide a system construction method for obtaining large amounts of white wax insect dsRNA in vitro, which can provide a reference for how to obtain large amounts of dsRNA at low cost.
  • a method for constructing a system for obtaining a large amount of white wax insect dsRNA in vitro including the following steps:
  • Step (1) extract the total RNA of white wax worm and reverse transcribed it into cDNA and then carry out PCR amplification.
  • the amplified product and the L4440 vector plasmid were digested with SacI and SalI respectively, and the product was digested with T4 ligase overnight at 4°C Connect to obtain a recombinant plasmid 302a1-L4440 containing the 302a1 gene fragment;
  • Step (2) Transform the 302a1-L4440 recombinant plasmid obtained in step (1) into the E. coli competent cell HT115 (DE3) strain, and apply a solid medium to screen a single colony to obtain 302a1-L4440-HT115 bacteria;
  • step (4) the dsRNA bacterial solution containing the white waxworm 302a1 gene fragment obtained in step (3) is concentrated, and the bacterial solution is used for the RNA interference experiment.
  • the total RNA of the white wax worm comprises the total RNA of the white wax worm 302a1 gene.
  • the PCR amplification method is used to design primers for the 302a1 gene (primer name is ECEGT01) containing SacI and SalI double restriction sites, wherein
  • ECEGT01-F 5'-atgagctccgctctggtataaattcatttggac-3' (SEQ ID NO.6);
  • ECEGT01-R 5'-atgtcgacgcctgttgaatcattatcactccta-3' (SEQ ID NO.7).
  • the PCR amplification system is:
  • the PCR amplification procedure is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 5 min, and storage at 4°C.
  • step (1) the condition system of restriction enzyme digestion is:
  • the digestion time is 10 minutes and the temperature is 37°C.
  • connection system is:
  • the product after ligation is transformed into competent cells DH5 ⁇ , and then coated and cultured on a SOC solid medium containing 100 ⁇ g/mL ampicillin.
  • the single colony grown is expanded and multiplied and the plasmid is extracted to obtain 302a1-L4440 Recombinant plasmid.
  • the single bacteria grow out later. Use an inoculating loop to pick a single colony into a centrifuge tube containing 100 ⁇ g/mL ampicillin in SOC liquid medium, and culture for 10 hours on a shaker. Extract the plasmid.
  • step (2) transform the 302a1-L4440 recombinant plasmid obtained in step (1) into the competent cell strain HT115 (DE3) of Escherichia coli, and coat it with 100 ⁇ g/mL ampicillin And 50 ⁇ g/mL tetracycline SOC solid medium, culture for 12h in the dark, and pick a single colony to obtain 302a1-L4440-HT115 bacteria;
  • the invention provides a method for constructing a system for obtaining a large amount of dsRNA in a single-cell cloning manner, which mediates the RNAi of the white wax worm to cause the expression of the target gene to be silenced.
  • This method takes the white waxworm RNAi interference as an example.
  • the target gene fragment of white waxworm 302a1 is obtained by PCR using the white waxworm cDNA as a template.
  • the two ends of the gene fragment are modified by restriction enzymes to form sticky ends.
  • Dicer was modified to form a linear L4440 plasmid ligation to form a plasmid 302a1-L4440 recombinant plasmid containing the target gene fragment of the white waxworm.
  • the 302a1-L4440 recombinant plasmid was transformed into the competent strain of Escherichia coli HT115 (DE3) to form the 302a1-L4440-HT1115 strain.
  • the combined strain is resistant to both ampicillin and tetracycline, cannot synthesize RNA hydrolase, and contains the white wax insect 302a1 gene fragment Therefore, the E. coli can be cultured and propagated in a SOC liquid medium (containing ampicillin and tetracycline) in a single-cell cloning method.
  • the propagation process can produce dsRNA after IPTG induction, and a large amount of dsRNA containing the white waxworm 302a1 gene segment can be obtained.
  • the recombinant Escherichia coli bacteria liquid is collected, and the bacteria liquid is diluted and sprayed directly on the larval body of the white wax worm.
  • the method of the present invention can clone E. coli containing the white wax insect 302a1 target gene by single-cell cloning, and through this Escherichia coli's own secondary metabolism and self-reproduction and replication, obtain a bacterial solution containing a large number of white wax insect gene fragments dsRNA, and combine the white wax insect gene fragments
  • the dsRNA bacterial solution is sprayed with white wax worm larvae and enters the body from the surface of the white wax worm, which can effectively interfere with the expression of the target gene.
  • RNAi transfection pathway that synthesizes a large amount of dsRNA and enters the body through the body surface provides a reference.
  • the present invention has the following beneficial effects:
  • the present invention is the first to verify the function of the white waxworm RNA gene by introducing a single-cell cloned microorganism Escherichia coli, so that its genetic material contains the white waxworm 302a1 gene fragment, and the secondary metabolism contains the dsRNA of the 302a1 gene fragment, and there is no RNA hydrolase. During the propagation and self-replication of Escherichia coli, the dsRNA containing the 302a1 gene fragment was enriched.
  • the conventional RNAi experiment is synthesized with a kit.
  • the amount of dsRNA synthesized in a single time is 20ul, and the concentration is 0 to 1000ug/ml. (The concentration is related to the proficiency of the operator).
  • dsRNA can not be stored at ultra-low temperature for more than 1 year; the cost of synthesizing 1ul dsRNA with the kit is between 3 yuan and 5 yuan per ul, that is, the cost of 1ml is between 3000-5000 yuan; taking one gene as an example, an in vitro spray experiment requires 60ml, then The synthesis of the kit is between 180,000 and 300,000 yuan, which is too costly and undesirable; and the cost of each gene experiment with a single-cell cloned microbial coliform is between 800 and 1,500 yuan, cloning the modified E. coli strain, It can be stored in ultra-low temperature for 2-3 years, and can also be expanded according to the requirements of the experiment. In the later stage of ultra-low temperature storage, it can still be activated and resuscitated for repeated use, which greatly reduces the cost of the experiment.
  • Figure 1 is a schematic diagram of the construction process of a system for obtaining a large amount of dsRNA in vitro;
  • Figure 2 is an electrophoresis diagram of PCR products; among them, a is PCR amplification product, the band is between 250bp and 500bp; M is mark; b ⁇ f different target gene control, because it does not affect the present invention, the present invention does not do this Excessive disclosure
  • Figure 3 is a comparison electrophoresis diagram of L4440 plasmid before and after double digestion; among them, a1: control, circular L4440 plasmid (before double digestion); b1: linear L4440 plasmid (after double digestion); M: mark; because the plasmid is in the double enzyme Before cutting, it is circular, so the moving speed of electrophoresis is very slow. After double digestion, the plasmid becomes linear between 2500bp and 2000bp;
  • Figure 4 is a picture of 302a1-L4440 recombinant plasmid transformed into E. coli competent cell HT115(DE3) strain on SOC solid medium containing 100 ⁇ g/mL ampicillin and 50 ⁇ g/mL tetracycline;
  • Figure 5 is an electrophoresis diagram before and after induction; where m: DNA molecule Mark; a: after induction; b: before induction.
  • a method for obtaining large amounts of white wax insect double-stranded RNA in vitro including the following steps:
  • Step (1) extract the total RNA of white wax worm and reverse transcribed into cDNA and then carry out PCR amplification.
  • the amplified product and the L4440 vector plasmid were digested with SacI and SalI respectively ( Figure 3), and the product after the double digestion was connected with T4 Enzyme ligation overnight at 4°C to obtain recombinant plasmid 302a1-L4440 containing the 302a1 gene fragment;
  • Step (2) Transform the 302a1-L4440 recombinant plasmid obtained in step (1) into the E. coli competent cell HT115 (DE3) strain, and apply a solid medium to screen a single colony to obtain 302a1-L4440-HT115 bacteria;
  • step (4) the dsRNA bacterial solution containing the white waxworm 302a1 gene fragment obtained in step (3) is concentrated, and the bacterial solution is used for the RNA interference experiment.
  • Step (1) extract the total RNA of white wax worm and reverse transcribed into cDNA and then carry out PCR amplification.
  • the amplified product and the L4440 vector plasmid were digested with SacI and SalI respectively ( Figure 3), and the product after the double digestion was connected with T4 Enzyme ligation overnight at 4°C to obtain recombinant plasmid 302a1-L4440 containing the 302a1 gene fragment;
  • step (2) the 302a1-L4440 recombinant plasmid obtained in step (1) was transformed into the E. coli competent cell HT115 (DE3) strain, and solid medium was coated to screen a single colony to obtain 302a1-L4440-HT115 strain ( Figure 4);
  • step (4) the dsRNA bacterial solution containing the white waxworm 302a1 gene fragment obtained in step (3) is concentrated, and the bacterial solution is used for the RNA interference experiment.
  • the white waxworm RNA includes the total RNA of the white waxworm 302a1 gene.
  • the primers used in the PCR amplification are ECEGT01-F and ECEGT01-R;
  • ECEGT01-F 5'-atgagctccgctctggtataaattcatttggac-3' (SEQ ID NO.6);
  • ECEGT01-R 5'-atgtcgacgcctgttgaatcattatcactccta-3' (SEQ ID NO.7);
  • the PCR amplification system is:
  • the PCR amplification procedure is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 5 min, and storage at 4°C.
  • step (1) the condition system for restriction digestion is:
  • the digestion time is 10 minutes and the temperature is 37°C.
  • connection system is:
  • the product was transformed into competent cells DH5 ⁇ , and then spread and screened on SOC solid medium containing 100 ⁇ g/mL ampicillin. The single colony grown was expanded and multiplied and the plasmid was extracted to obtain the 302a1-L4440 recombinant plasmid.
  • step (2) transform the 302a1-L4440 recombinant plasmid obtained in step (1) into the E. coli competent cell HT115 (DE3) strain, and apply it to a strain containing 100 ⁇ g/mL ampicillin and 50 ⁇ g/mL tetracycline. On the SOC solid medium, culture for 12 hours in the dark, and pick a single colony to obtain 302a1-L4440-HT115 bacteria;
  • Step (3) The obtained bacterial liquid is further centrifuged at 4°C to concentrate the bacterial cells.
  • SOC medium components peptone 20.0g; yeast powder 5.0g; sodium chloride 0.5g; magnesium sulfate heptahydrate 5.0g; D-glucose 3.6g.
  • SOC solid medium components SOC medium: 34g, agar powder: 12g, distilled water: 1L
  • SOC liquid medium components SOC medium: 34g, distilled water: 1L
  • SOC solid/liquid medium containing ampicillin 1ml of medium contains 100 ⁇ g of ampicillin.
  • SOC solid/liquid medium containing ampicillin and tetracycline 1ml medium contains 100 ⁇ g of ampicillin and 50 ⁇ g of tetracycline.
  • 2 ⁇ YT liquid medium containing ampicillin and tetracycline 75 ⁇ g of ampicillin and 12.5 ⁇ g of tetracycline are contained in each milliliter of medium.
  • the present invention is a method for obtaining a large amount of dsRNA containing the white wax insect gene fragment by using a single-cell cloned microorganism in the gene function verification of the white wax insect, and is used for the RNAi body surface spray transfection of the white wax insect.
  • the purpose of the present invention 1. To explore how to obtain a large amount of dsRNA containing white wax insect gene fragments in vitro; The plasmid containing the white wax insect gene can be stored at low temperature and can be used for downstream RNAi experiments repeatedly. Based on these three purposes, a construction system for obtaining dsRNA in vitro was established
  • RNA extraction kit EZ-10 Tatal RNA Minin-presps kit, Shenggong Bioengineering (Shanghai) Co., Ltd.
  • kit instructions for the extraction process and use the reverse transcription kit (PrimeScript TM RT reagent Kit with gDNA Eraser, Takara) reverse transcription into cDNA.
  • ECEGT01-R 5'-atgtcgacgcctgttgaatcattatcactccta-3' were designed.
  • PCR reaction system is shown in Table 1.
  • PCR reaction conditions were 94°C pre-denaturation for 5 minutes; 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 72°C extension for 1 minute, 30 cycles; 72°C extension for 5 minutes, and 4°C storage.
  • Taq PCR Master Mix 25.0 ⁇ l, cDNA 3.0 ⁇ l, ECEGT01-F 10 ⁇ M 2.0 ⁇ l, ECEGT01-R 10 ⁇ M 2.0 ⁇ l, dH 2 O 18.0 ⁇ l, total 50.0 ⁇ l.
  • connection system is:
  • the 302a1 and L4440 plasmids were digested with double enzymes and then ligated and transformed into 50 ⁇ L DH5 ⁇ competent cells, spread on SOC solid medium containing ampicillin, cultured for 12 hours, and single colonies were picked and cultured in SOC liquid medium.
  • Direct PCR amplification with primers ECEGT01-F, ECEGT01-R and bacterial liquid the reaction system is shown in Table 3.
  • PCR reaction conditions are 94°C pre-denaturation 5min; 94°C denaturation 30s, 60°C annealing 30s, 72 Extend at °C for 1 min, 30 cycles; extend at 72 °C for 5 min, store at 4 °C. Sent to the company for sequencing, and the result was consistent with the target fragment.
  • the 302a1-L4440 recombinant plasmid has been successfully constructed.
  • SEQ ID NO.1 to SEQ ID NO.5 is the alignment of the PCR product and the sequence of the 302a1 gene fragment; wherein SEQ ID NO.1 is the sequence of the PCR product sequenced with the primer ECEGT01-F as the template; SEQ ID NO.2 The PCR product sequence was sequenced with primer ECEGT01-R as the template; SEQ ID NO.3 ⁇ SEQ ID NO.5 are the 302a1 gene, and 3 gene fragments from transcriptome data (transcriptome data only exons, no introns) The components are >CL1378.Contig1_All, >CL1378.Contig2_All, >CL1378.Contig3_All.
  • SEQ ID NO.3 to SEQ ID NO.5 are 302a1 gene fragments, the underlined part of the sequence overlaps with SEQ ID NO.1 and SEQ ID NO.2, and the PCR product belongs to the 302a1 gene fragment, that is, the expansion band is the desired target gene.
  • RNA extracted from the 302a1-L4440-HT115 bacterial solution after induction was one more RNA band than before the induction, while the RNA extracted from the 302a1-L4440-HT115 bacterial solution before the induction did not contain this band.
  • the dsRNA of the 302a1 gene fragment was successfully induced and expressed.
  • a large amount of bacterial solution containing the dsRNA of the white waxworm 302a1 gene fragment is obtained by transforming the constructed 302a1-L4440 recombinant plasmid into HT115 competent cells, and spreading it on the SOC solid medium containing ampicillin and tetracycline.
  • the Escherichia coli 302a1-L4440-HT115 formed into the system was successfully cultured, and dsRNA containing the target 302a1 gene fragment was obtained by induction.
  • the culture of the bacterial solution can be expanded and multiplied according to the needs to obtain the required dsRNA bacterial solution.

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Abstract

提供一种体外大量获得白蜡虫dsRNA的体系构建方法，首先提取白蜡虫总RNA并反转录为cDNA后进行PCR扩增，扩增产物与L4440载体质粒分别双酶切，将双酶切后产物连接，得到的重组质粒转化进入大肠杆菌感受态细胞HT115(DE3)菌株中，涂布固体培养基筛选单菌落，然后将单菌落液体摇床培养至OD600＝0.38-0.42，加入IPTG诱导后浓缩菌液，用于RNA干扰实验。通过单细胞克隆大肠杆菌含有白蜡虫目标基因，又通过此大肠杆菌自身新陈代谢和大量的自我繁殖复制，获得大量含白蜡虫基因的dsRNA的菌液，将含dsRNA菌液喷施白蜡虫幼虫，从白蜡虫虫体表面进入体内，有效干扰目标基因表达量。

Description

一种体外大量获得白蜡虫dsRNA的体系构建方法 技术领域
本发明属于基因工程技术领域，具体涉及一种体外大量获得白蜡虫dsRNA的体系构建方法。
背景技术
白蜡虫，Ericerus pela(Chavannes)，俗称蜡虫，白蜡虫在分类上属于半翅目(Hemipetera)蚧总科(Coccoidea)白蜡蚧属(Ericerus)(Chavannes,1819)。白蜡虫是一种具有重大经济价值的介壳虫，白蜡虫2龄雄虫幼虫生活在寄主植物枝条上，分泌白色的蜡质覆盖物，这种昆虫分泌的蜡成为白蜡(Cerachinensis，也称虫白蜡)，即白蜡虫的分泌物，为中国特产。白蜡是一种天然的高分子化合物，熔点高，化学性稳定。具有防潮、润滑、着光等特点，是一种重要的化工原料，被广泛应用于化工、机械、食品、药业、化妆品、农业等行业。
RNA干扰(RNA interference，RNAi)是指在真核生物中，由双链RNA(double-stranded RNA，dsRNA)诱发同源mRNA降解，使靶基因表达沉默的现象。dsRNA转染主要有注射、喂食、浸泡、病毒感染和基因枪等方法。但是如果研究对象过小，不能移动的时候，喂食、浸泡、体表吸收的传染方式存在试剂盒制备的dsRNA用量不足的局面。
本研究的昆虫为白蜡虫，虫体个体较小，且是刺吸式口器，通过吸取寄主盐肤木的汁液生长繁殖，大部分时间不能移动，会采取体表皮肤吸收的方式进行传染。由于体表吸收，用量大，dsRNA在外界不稳定、易降解，需要多次对白蜡昆虫的体表皮肤进行喷施吸收，试剂盒合成dsRNA产量较低(ul计量)，不足以用于喷施的转染方式，大量的试剂盒合成使实验***格高昂，因此在一些RNA干扰实验中，如何制备高产量的dsRNA是基因工程需要解决的问题。
发明内容
在RNAi转染过程中，由于客观原因，研究对象不能使用基因枪等微剂量的转染方式，但采取体外转染时，都需要大剂量的dsRNA，因此本发明为了克服现有技术中的不足，提供一种体外大量获得白蜡虫dsRNA的体系构建方法，该方法可为如何低成本获得大量的dsRNA提供参考。
为实现上述目的，本发明采用的技术方案如下：
一种体外大量获得白蜡虫dsRNA的体系构建方法，包括如下步骤：
步骤(1)，提取白蜡虫总RNA并反转录为cDNA后进行PCR扩增，扩增 产物与L4440载体质粒分别经SacI和SalI双酶切，双酶切后产物利用T4连接酶4℃过夜连接，得到含302a1基因片段的重组质粒302a1-L4440；
步骤(2)，将步骤(1)得到的302a1-L4440重组质粒转化进入大肠杆菌感受态细胞HT115(DE3)菌株中，涂布固体培养基筛选单菌落，得到302a1-L4440-HT115菌体；
步骤(3)，将步骤(2)获得的302a1-L4440-HT115菌体液体摇床培养至OD 600＝0.38-0.42，加入IPTG诱导，获得含有白蜡虫302a1基因片段的dsRNA菌液；
步骤(4)，将步骤(3)得到含有白蜡虫302a1基因片段的dsRNA菌液，浓缩菌液，用于RNA干扰实验。
进一步，优选的是，所述的白蜡虫总RNA包含中白蜡虫302a1基因的总RNA。
进一步，优选的是，步骤(1)中，采用PCR扩增的方法，设计302a1基因引物(引物名称为ECEGT01)含SacⅠ和SalⅠ双酶切位点，其中
ECEGT01-F：5'-atgagctccgctctggtataaattcatttggac-3'(SEQ ID NO.6)；
ECEGT01-R：5'-atgtcgacgcctgttgaatcattatcactccta-3'(SEQ ID NO.7)。
进一步，优选的是，步骤(1)中，所述的PCR扩增体系为：
Figure PCTCN2020098184-appb-000001
PCR扩增程序为：94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸1min，30次循环；72℃延伸5min，4℃保存。
进一步，优选的是，步骤(1)中，酶切的条件体系为：
Figure PCTCN2020098184-appb-000002
Figure PCTCN2020098184-appb-000003
酶切的时间为10min，温度为37℃。
进一步，优选的是，连接体系为：
Figure PCTCN2020098184-appb-000004
进一步，优选的是，连接后产物转化入感受态细胞DH5ɑ中，在含100μg/mL氨苄青霉素的SOC固培养基涂布筛选培养，长出的单菌落扩大繁殖后提取质粒，即得到302a1-L4440重组质粒。其中，对于扩大繁殖的方法不做具体限制，优选为长出的单菌落后，用接种环挑取单菌落于含100μg/mL氨苄青霉素的SOC液体培养基的离心管中，摇床培养10小时提取质粒。
进一步，优选的是，步骤(2)的具体方法是：将步骤(1)得到的302a1-L4440重组质粒转化进入大肠杆菌感受态细胞HT115(DE3)菌株中，涂布于含100μg/mL氨苄青霉素和50μg/mL四环素的SOC固体培养基上，避光培养12h，挑取单菌落，得到302a1-L4440-HT115菌体；
进一步，优选的是，步骤(3)的具体方法是：将302a1-L4440-HT115菌体于含100μg/mL氨苄青霉素和50μg/mL四环素的SOC液体培养基中，37℃避光培养10-12h，然后于含75μg/mL氨苄青霉素和12.5μg/mL四环素的2×YT液体培养基中摇床培养至OD 600＝0.38～0.42，加入IPTG诱导培养3～4h，获得含有白蜡虫302a1基因片段的dsRNA菌液。
本发明提供一种单细胞克隆方式大量获得dsRNA的体系构建方法，介导白蜡虫的RNAi引起目的基因表达沉默。该方法以白蜡虫RNAi干扰为例，通过PCR的方式以白蜡虫cDNA为模板获得白蜡虫302a1目的基因片段，经过限制性内切酶修饰基因片段两端形成粘性末端，与相同方式经过限制性内切酶修饰形成线性的L4440质粒连接，形成含有白蜡虫目的基因片段的质粒302a1-L4440重组质粒。将302a1-L4440重组质粒转化进入大肠杆菌感受态HT115(DE3)菌株，形成302a1-L4440-HT1115菌株，该组合菌株同时具有氨苄和四环素抗性，不可合成RNA水解酶且含有白腊虫302a1基因片段，所以可通过单细胞克隆的 方式，SOC液体培养基(含氨苄和四环素)摇床培养繁殖该大肠杆菌，其繁殖过程经过IPTG诱导可产生dsRNA，可大量获得含白蜡虫302a1基因片段的dsRNA，收集该重组大肠杆菌菌液，将菌液稀释后直接喷施在白蜡虫幼虫期虫体上。本发明方法可通过单细胞克隆大肠杆菌含有白蜡虫302a1目标基因，又通过此大肠杆菌自身次生代谢以及自我繁殖复制，获得含大量白蜡虫基因片段dsRNA的菌液，将含白蜡虫基因片段的dsRNA菌液喷施白蜡虫幼虫，从白蜡虫虫体表面进入体内，有效干扰目标基因表达量，为RNAi转染过程中因昆虫体积过小、无咀嚼式口器，不能采用微量注射的昆虫类型，合成大量的dsRNA且通过体表进入体内的RNAi转染途径提供参考。
本发明与现有技术相比，其有益效果为：
本发明首次在白蜡虫RNA基因功能验证，引入单细胞克隆微生物大肠杆菌次，使其遗传物质中含有白蜡虫302a1基因片段，次生代谢中含有302a1基因片段的dsRNA，且没有RNA水解酶，通过大肠杆菌的繁殖和自我复制过程中使含有302a1基因片段的dsRNA得到富集。在研究对象过小，如本研究的白蜡虫体积小，不能移动的特点，RNAi转染过程中需要dsRNA喷施体表或者需要浸泡、喂食等dsRNA用量大的转染方式时，此方法可以提供大量的dsRNA，易于推广应用。
以白蜡虫喷施实验为例，常规的RNAi实验采用试剂盒合成，单次合成的dsRNA量在20ul，浓度在0至1000ug/ml，(浓度高低与操作人员的熟练程度有关系)，合成的dsRNA超低温保存不能超过1年；以试剂盒合成1ul dsRNA成本在3元至5元每ul，即1ml的成本在3000-5000元间；以一个基因为例，一次体外喷施实验需要60ml，那么试剂盒的合成就在18-30万元间，费用代价太大不可取；而采取单细胞克隆微生物大肠杆每个基因实验的费用是在800-1500元间，克隆改造过的大肠杆菌菌株，可以超低温保存2-3年，也可根据实验的要求扩大培养，超低温保存后期仍可以活化复苏重复使用，较大程度的降低实验成本。
附图说明
图1为体外大量获得dsRNA体系构建流程简图；
图2为PCR产物电泳图；其中，a为PCR扩增产物，条带处于250bp-500bp间；M为mark；b～f不同目的基因对照，由于对本发明不产生影响，对此本发明不做过多的披露；
图3为L4440质粒双酶切前后对比电泳图；其中，a1：对照，环形L4440质粒(双酶切前)；b1：线性L4440质粒(双酶切后)；M：mark；由于质粒在双酶切前为环形，故电泳的移动速度非常慢，质粒双酶切后成线性处于2500bp-2000bp间；
图4为302a1-L4440重组质粒转化进入大肠杆菌感受态细胞HT115(DE3)菌株中在含100μg/mL氨苄青霉素和50μg/mL四环素的SOC固体培养基上培养的图片；
图5为诱导前后电泳图；其中，m：DNA分子Mark；a:诱导后；b:诱导前。
具体实施方式
下面结合实施例对本发明作进一步的详细描述。
本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用材料或设备未注明生产厂商者，均为可以通过购买获得的常规产品。
实施例1
一种体外大量获得白蜡虫双链RNA白蜡虫的方法，包括如下步骤：
步骤(1)，提取白蜡虫总RNA并反转录为cDNA后进行PCR扩增，扩增产物与L4440载体质粒分别经SacI和SalI双酶切(图3)，双酶切后产物利用T4连接酶4℃过夜连接，得到含302a1基因片段的重组质粒302a1-L4440；
步骤(2)，将步骤(1)得到的302a1-L4440重组质粒转化进入大肠杆菌感受态细胞HT115(DE3)菌株中，涂布固体培养基筛选单菌落，得到302a1-L4440-HT115菌体；
步骤(3)，将步骤(2)获得的302a1-L4440-HT115菌体液体摇床培养至OD 600＝0.38，加入IPTG诱导，获得含有白蜡虫302a1基因片段的dsRNA菌液；
步骤(4)，将步骤(3)得到含有白蜡虫302a1基因片段的dsRNA菌液，浓缩菌液，用于RNA干扰实验。
实施例2
步骤(1)，提取白蜡虫总RNA并反转录为cDNA后进行PCR扩增，扩增产物与L4440载体质粒分别经SacI和SalI双酶切(图3)，双酶切后产物利用T4连接酶4℃过夜连接，得到含302a1基因片段的重组质粒302a1-L4440；
步骤(2)，将步骤(1)得到的302a1-L4440重组质粒转化进入大肠杆菌感受态细胞HT115(DE3)菌株中，涂布固体培养基筛选单菌落，得到302a1-L4440-HT115菌体(图4)；
步骤(3)，将步骤(2)获得的302a1-L4440-HT115菌体液体摇床培养至OD 600＝0.42，加入IPTG诱导3.5h，获得含有白蜡虫302a1基因片段的dsRNA菌液；
步骤(4)，将步骤(3)得到含有白蜡虫302a1基因片段的dsRNA菌液，浓缩菌液，用于RNA干扰实验。
步骤(1)中，所述的白蜡虫RNA包含白蜡虫302a1基因的总RNA。
所述的PCR扩增采用的引物为ECEGT01-F和ECEGT01-R；
ECEGT01-F：5'-atgagctccgctctggtataaattcatttggac-3'(SEQ ID NO.6)；
ECEGT01-R：5'-atgtcgacgcctgttgaatcattatcactccta-3'(SEQ ID NO.7)；
所述的PCR扩增体系为：
Figure PCTCN2020098184-appb-000005
PCR扩增程序为：94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸1min，30次循环；72℃延伸5min，4℃保存。
步骤(1)中，酶切的条件体系为：
Figure PCTCN2020098184-appb-000006
酶切的时间为10min，温度为37℃。
连接体系为：
Figure PCTCN2020098184-appb-000007
Figure PCTCN2020098184-appb-000008
连接后产物转化入感受态细胞DH5ɑ中，在含100μg/mL氨苄青霉素的SOC固培养基涂布筛选培养，长出的单菌落扩大繁殖后提取质粒，即得到302a1-L4440重组质粒。
步骤(2)的具体方法是：将步骤(1)得到的302a1-L4440重组质粒转化进入大肠杆菌感受态细胞HT115(DE3)菌株中，涂布于含100μg/mL氨苄青霉素和50μg/mL四环素的SOC固体培养基上，避光培养12h，挑取单菌落，得到302a1-L4440-HT115菌体；
步骤(3)的具体方法是：将302a1-L4440-HT115菌体于含100μg/mL氨苄青霉素和50μg/mL四环素的SOC液体培养基中，37℃避光培养10-12h，然后于含75μg/mL氨苄青霉素和12.5μg/mL四环素的2×YT液体培养基中摇床培养至OD 600＝0.42，加入IPTG诱导培养3h，获得含有白蜡虫302a1基因片段的dsRNA菌液。
步骤(3)将获得的菌液进一步4℃离心浓缩菌体。
应用实例
实验涉及的所有培养基购买于生工生物工程(上海)股份有限公司，其中：
SOC培养基组分：蛋白胨20.0g；酵母粉5.0g；氯化钠0.5g；七水硫酸镁5.0g；D-葡萄糖3.6g。
SOC固体培养基组分：SOC培养基：34g，琼脂粉：12g，蒸馏水：1L
SOC液体培养基组分：SOC培养基：34g，蒸馏水：1L
含有氨苄青霉素的SOC固/液体培养基：1ml培养基中含有100μg的氨苄青霉素。
含有氨苄青霉素和四环素的SOC固/液体培养基：1ml培养基中含有100μg的氨苄青霉素、含有50μg的四环素。
2×YT培养基组分：酵母粉10.0g；蛋白胨16.0g；氯化钠5.0g。
含有氨苄青霉素和四环素的2×YT液体培养基中：每毫升培养基中含有75μg的氨苄青霉素，含有12.5μg的四环素。
注：文中“/”代表“或”的意思。302a1基因是可以替换成任何想做干扰实验的基因。
本发明是白蜡虫的基因功能验证中采用单细胞克隆微生物获得大量含白蜡虫基因片段dsRNA的方法，用于白蜡虫的RNAi体表喷施转染。本发明的目的1.探索如何在体外获得大量含白蜡虫基因片段dsRNA的体系构建方法；2.降低在RNAi实验中，常规试剂盒合成dsRNA的成本；3.一旦制备成功，可将改造过且含有白蜡虫基因的质粒低温保存，可反复做下游RNAi实验。基于这三个目的出发建立体外获得dsRNA的构建体系
1重组载体的构建
利用RNA提取试剂盒(EZ-10 Tatal RNA Minin-presps kit，生工生物工程(上海)股份有限公司)提取白蜡虫总RNA，其提取过程参照试剂盒说明书，并用反转录试剂盒(PrimeScript TM RT reagent Kit with gDNA Eraser，Takara)反转录为cDNA。根据302a1基因片段序列设计引物ECEGT01-F：5'-atgagctccgctctggtataaattcatttggac-3'；ECEGT01-R：5'-atgtcgacgcctgttgaatcattatcactccta-3'。以cDNA为模板进行PCR扩增[Taq PCR Master Mix(2X,with Red Dye)，生工生物工程(上海)股份有限公司]，PCR反应体系如表1所示。PCR反应条件为94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸1min，30次循环；72℃延伸5min，4℃保存。
表1
Taq PCR Master Mix 25.0μl，
cDNA 3.0μl，
ECEGT01-F 10μM 2.0μl，
ECEGT01-R 10μM 2.0μl，
dH 2O 18.0μl，
总计 50.0μl。
扩增片段的电泳检测结果显示介于250-500bp之间(图2)，与预期相符。根据图1载体构建的流程，302a1基因片段胶回收产物和L4440载体经SacI和SalI(NEB(北京)有限公司)在37℃水浴中酶切10min，其反应体系如表2所示，分别电泳胶回收纯化酶切产物，酶切产物通过琼脂糖凝胶电泳检测(图3)。之后利用T4连接酶连接4℃过夜，得到302a1-L4440重组质粒。
表2
Sac Ⅰ 1.0μl，
Sal Ⅰ 1.0μl，
NE Buffer(10x) 5.0μl，
扩增产物或L4440 4.0μl，
dH 2O 39.0μl，
总计 50.0μl。
连接体系为：
Figure PCTCN2020098184-appb-000009
将302a1和L4440质粒双酶切后连接转化到50μL DH5α感受态细胞中，涂布于含有氨苄青霉素的SOC固体培养基上，培养12h，挑取单菌落于SOC液体培养基中培养。经引物ECEGT01-F、ECEGT01-R和菌液直接PCR扩增，反应体系如表3所示，PCR反应条件：PCR反应条件为94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸1min，30次循环；72℃延伸5min，4℃保存。送公司测序，结果比对与目的片段吻合，302a1-L4440重组质粒已成功构建。
序列表SEQ ID NO.1～SEQ ID NO.5为PCR产物与302a1基因片段序列的比对；其中SEQ ID NO.1是以引物ECEGT01-F为模板测序PCR产物的序列；SEQ ID NO.2是以引物ECEGT01-R为模板测序PCR产物序列；SEQ ID NO.3～SEQ ID NO.5为302a1基因，由转录组数据(转录组数据只有外显子，无内含子)3个基因片段组成分别是>CL1378.Contig1_All、>CL1378.Contig2_All、>CL1378.Contig3_All。其中，SEQ ID NO.3～SEQ ID NO.5为302a1基因片段，下划线部分序列与SEQ ID NO.1和SEQ ID NO.2重合，PCR产物属于302a1基因片段，即扩征条带为所要的目的基因。
2dsRNA的诱导表达
将步骤(1)得到的302a1-L4440重组质粒转化进入大肠杆菌感受态细胞HT115(DE3)菌株中，涂布于含100μg/mL氨苄青霉素和50μg/mL四环素的SOC固体培养基上，避光培养12h，挑取单菌落，得到302a1-L4440-HT115菌 体；将302a1-L4440-HT115菌体于含100μg/mL氨苄青霉素和50μg/mL四环素的SOC液体培养基中，37℃避光培养10-12h，然后于含75μg/mL氨苄青霉素和12.5μg/mL四环素的2×YT液体培养基中摇床培养至OD 600＝0.4，加入IPTG诱导4h，收集菌体。诱导后的菌液提总RNA检测，结果如图5所示。凝胶电泳结果显示，诱导后302a1-L4440-HT115菌液提取的RNA比诱导前多一条RNA条带，而诱导前302a1-L4440-HT115菌液提取的RNA不含此条带，据此可以说明302a1基因片段的dsRNA诱导表达成功。
其中，大量获得含白蜡虫302a1基因片段的dsRNA的菌液是：将构建好的302a1-L4440重组质粒转化到HT115感受态细胞中，涂布于含有氨苄青霉素和四环素的SOC固体培养基上，避光培养12h，挑取单菌至SOC液体培养基(含100μg/mL氨苄青霉素和50μg/mL四环素)37℃避光培养10-12h，然后置于2×YT液体培养基(含75μg/mL氨苄青霉素和12.5μg/mL四环素)培养至OD 600＝0.38～0.42。
结果：
所构建成体系形成的大肠杆菌302a1-L4440-HT115成功培养，经诱导得到含有目的302a1基因片段dsRNA，菌液的培养可根据需求扩大培养繁殖，获得所需的dsRNA菌液。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Figure PCTCN2020098184-appb-000010
Figure PCTCN2020098184-appb-000011
Figure PCTCN2020098184-appb-000012
Figure PCTCN2020098184-appb-000013
Figure PCTCN2020098184-appb-000014
Figure PCTCN2020098184-appb-000015

Claims (9)

  1. 一种体外大量获得白蜡虫dsRNA的体系构建方法，其特征在于，包括如下步骤：
    步骤(1)，提取白蜡虫总RNA并反转录为cDNA后进行PCR扩增，扩增产物与L4440载体质粒分别经SacI和SalI双酶切，双酶切后产物利用T4连接酶4℃过夜连接，得到含302a1基因片段的重组质粒302a1-L4440；
    步骤(2)，将步骤(1)得到的302a1-L4440重组质粒转化进入大肠杆菌感受态细胞HT115(DE3)菌株中，涂布固体培养基筛选单菌落，得到302a1-L4440-HT115菌体；
    步骤(3)，将步骤(2)获得的302a1-L4440-HT115菌体液体摇床培养至OD 600＝0.38-0.42，加入IPTG诱导，获得含有白蜡虫302a1基因片段的dsRN A菌液；
    步骤(4)，将步骤(3)得到含有白蜡虫302a1基因片段的dsRNA菌液，浓缩菌液，用于RNA干扰实验。
  2. 根据权利要求1所述的体外大量获得白蜡虫dsRNA的体系构建方法，其特征在于，所述的白蜡虫总RNA包含中白蜡虫302a1基因的总RNA。
  3. 根据权利要求1所述的体外大量获得白蜡虫dsRNA的体系构建方法，其特征在于，所述的PCR扩增采用的引物为ECEGT01-F和ECEGT01-R；
    ECEGT01-F：5'-atgagctccgctctggtataaattcatttggac-3'(SEQ ID NO.6)；
    ECEGT01-R：5'-atgtcgacgcctgttgaatcattatcactccta-3'(SEQ ID NO.7)。
  4. 根据权利要求1所述的体外大量获得白蜡虫dsRNA的体系构建方法，其特征在于，步骤(1)中，所述的PCR扩增体系为：
    Figure PCTCN2020098184-appb-100001
    PCR扩增程序为：94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸1min，30次循环；72℃延伸5min，4℃保存。
  5. 根据权利要求1所述的体外大量获得白蜡虫dsRNA的体系构建方法，其特征在于，步骤(1)中，酶切的条件体系为：
    Figure PCTCN2020098184-appb-100002
    酶切的时间为10min，温度为37℃。
  6. 根据权利要求1所述的体外大量获得白蜡虫dsRNA的体系构建方法，其特征在于，连接体系为：
    Figure PCTCN2020098184-appb-100003
  7. 根据权利要求1所述的体外大量获得白蜡虫dsRNA的体系构建方法，其特征在于，连接后产物转化入感受态细胞DH5ɑ中，在含100μg/mL氨苄青霉素的SOC固培养基涂布筛选培养，长出的单菌落扩大繁殖后提取质粒，即得到302a1-L4440重组质粒。
  8. 根据权利要求1所述的体外大量获得白蜡虫dsRNA的体系构建方法，其特征在于，步骤(2)的具体方法是：将步骤(1)得到的302a1-L4440重组质粒转化进入大肠杆菌感受态细胞HT115(DE3)菌株中，涂布于含100μg/mL氨苄青霉素和50μg/mL四环素的SOC固体培养基上，避光培养12h，挑取单菌落，得到302a1-L4440-HT115菌体；
  9. 根据权利要求1所述的体外大量获得白蜡虫dsRNA的体系构建方法，其特征在于，步骤(3)的具体方法是：将302a1-L4440-HT115菌体于含100μg/mL氨苄青霉素和50μg/mL四环素的SOC液体培养基中，37℃避光培养10-12h，然后于含75μg/mL氨苄青霉素和12.5μg/mL四环素的2×YT液体培养基中摇床培养至OD 600＝0.38～0.42，加入IPTG诱导培养3～4h，获得含有白蜡虫302a1基因片段的dsRNA菌液。
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